current applications of biotechnology

CURRENT APPLICATIONS OF BIOTECHNOLOGY Editor in Chief Munis Dündar

Editors Fabrizio Bruschi Kevan Gartland Mariapia Viola Magni Peter Gahan Yusuf Deeni

CURRENT APPLICATIONS OF BIOTECHNOLOGY

CURRENT APPLICATIONS OF BIOTECHNOLOGY Editor in Chief Munis Dündar, MD, PhD

Professor, Head of Medical Genetics Department, School of Medicine, Erciyes University, Kayseri, Turkey.

Editors Fabrizio Bruschi, MD

Professor, Department of Translational Research, School of Medicine, University of Pisa, Pisa, Italy.

Kevan Gartland, PhD

Professor, Special Advisor, Biological Science, Glasgow Caledonian University, Glasgow, United Kingdom.

Mariapia Viola Magni, PhD Professor, Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy. Peter Gahan, PhD

Professor, Cell biology, Anatomy & Human Sciences, King’s College London, London, United Kingdom.

Yusuf Deeni, PhD

Senior Lecturer, Engineering and Technology, Scotish Informatics, Mathematics, Biology and Statistics Centre, School of Science, University of Abertay, Dundee, Scotland, United Kingdom.

Assistant Editor Seher Polat, PhD

Medical Genetics, School of Medicine, Erciyes University, Kayseri, Turkey.

Cover by

Vahit Talib, ([email protected])

Designed by

Müge Yeğen

ISBN: First edition of this book run into 1000 editions, Erciyes University publication no 199. Copyright belongs to Erciyes University and it can't be published partly or totaly. Author/authors are responsible for quotations about scientific ideas or comments, every kind of chart, formation, graphic and picture. Erciyes University doesn't bear any legal responsibility about that kind of cases. The sale price must be deposited to the following bank: Bank Accounts Details The Bank Name and Adress : Ziraat Bank, Erciyes Univeristy Campus, Kayseri, Turkey. Bank Branch Code : 00159 Account Numbers : 400 63600 5001 IBAN : TR38 0001 0001 59400 63600 5001 SWIFT Code : TCZBTR2A "Book sales price" should be indicated in the receipt (Current Price can be learned from following information). For details please contact with Erciyes University Central Library Phone : 0090 352 437 52 53 • Fax : 0090 352 437 76 22

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This book is dedicated to: Mariapia Viola Magni, founder of EBTNA, for her contribution to Biotechnology in Europe, All EBTNA members and All Biotechnology lovers

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FOREWORD The town of Scholars “Kayseri” is a large and industrialized city in Central Anatolia. It is rich in historical monuments dating especially from the Roma and Seljuk periods. It was located on the intersecting roads of the caravans and was an important city in the XIII th century.

Gevher Nesibe Sultan Hospital (1206)

It is mentioned that almost fifteen madrasas had been known to exist during the Seljuk period in Kayseri and known as a centre of science and art, therefore Kayseri was mentioned as “Makarr-ı Ulema” (The town of Scholars). Twin Madrasa, the oldest medical centre in Anatolia, was built up as a medical madrasa by Gıyaseddin Keyhüsrev-I between 1205-1206 and dedicated to the daughter of Kılıçarslan II, Gevher Nesibe Sultan as a hospital (şifahane) to cure sick people with no charge and meanwhile to do research to find cure for incurable illnesses at that time.

The first faculty opened at Erciyes University in 1969 was Gevher Nesibe Medical faculty to take the mission from our history followed by the establishment of 17 more faculties including Faculty of Economics and Administrative Sciences, Faculty of Theology, Faculty of Engineering and different research centres. One of them is established in 2010 as Genome and Stem Cell Centre to combine basic science with technology mainly to investigate molecular pathology of genetic disease and for deeper understanding of biological developmental processes with cell culture, animal model and in silico modelling as well with the departments of Genome Research and Applications, Stem Cell and Gene Therapy Laboratories with Good Manufacturing Erciyes University, Genome and Stem Cell Centre VII


Practices standards, Proteomics, Molecular Microbiology, Transgenic Animal Facility and Bioinformatics. The centre not only provides research opportunities, but also serves as a place for education. Moreover, Erciyes University takes responsibility of developing internationally with the academic staff eager to carry out international collaborations or to give lectures in different European universities and/or of hiring international scientists to work as academic members under the roof of Erciyes University. Therefore, I would like to thank to Prof. Munis DÜNDAR, President of European Biotechnology Thematic Network Association for his international collaborations and preparing the book and also to the authors who work in different universities for their efforts in the preparation process of the book. Prof. Dr. H. Fahrettin KELEŞTEMUR President Erciyes University Kayseri, Turkey

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PREFACE This book provides a starting point for readers who wish to gain a basic knowledge in various areas of biotechnology and its application for future professional practices. Biotechnology is the science of integration and system technology. It is a multidisciplinary and interdisciplinary science systematically manipulating and integrating basic science advancements into biotechnological application such as genetics, (bio)chemistry, biology, microbiology, mathematics, engineering, and other fields.The resultant practical aims are to improve: crop yield, meat quality, human health, the production of vitamins, hormones, biofuels, gene or protein engineering, in vitro cell cultivation, disease diagnosis, food safety, biomaterials energy, environmental management and so on. To successfully achieve ultimate, specific goals through the application of biotechnology, it is particularly important to have a thorough understanding of physical and biological mechanism basics in addition to understanding the chemical processes that occur in various bioprocesses during the different development stages. Impressive milestones reached so far in whole genome sequencing, analytical instrumentation, computing power with user-friendly software tools as well as advancements seen in bioinformatics have led to irreversible changes in the practice of biology. After long studies conducted on individual component of living organisms, we are now able to study the same comprehensively and in deep molecular detail thanks to scientists devoting their lives to science technology. In the twentieth century, huge advances in the development of life science and biotechnology began. In addition the overall improvement of life in all respects with further exponential growth led to a vast development potential of the global economy. In this context, the development of training programmes and tools to close the technological and academic gap that existed between present and future highly qualified specialists in the various branches of biotechnology was not only useful but also became absolutely essential. This book was written and designed to comprehensively assist undergraduate, graduate or postgraduate students studying various aspects of biotechnology as well as researchers, engineers, teachers and commercial agents such as microbiologists, biochemists, biologists, bioengineers, chemists, veterinarians, physicians, and those in food engineering and pharmaceuticals chemists who need at least basic knowledge in this field. Moreover, the authors hope to help young researchers and students to choose their best career path in biotechnology as well. IX


The presentation of each chapter is simple and given systematically for a comprehensive understanding of the core principles of each area, the interrelationships between biotechnology and its application and other disciplines, and how biotechnology affects our everyday lives. The book highlights the collective inspiration and the hard work of the authors and editorial board, all of whom are members of the European Biotechnology Thematic Network Association (EBTNA) which was created in 2007 as part of the European Network (1996) of colleagues from different European Universities which have presented various European projects on European Biotechnology development. The EBTNA has a significant numbers of members from over 35 different countries and it continues to extend its reputation steadily with each passing day. The European Biotechnology Congresses under the aegis of Presidency of the Republic of Turkey in Istanbul in 2011 and following Kayseri in 2012, Bratislava in 2013 and Lecce in 2014 were organised under the auspices of EBTNA. All were successfully organised with international participants from all over the World. In addition the EBTNA also highlights the importance of biotechnology education by organising Open Distance Learning (ODLs) programmes to benefit all those interested in biotechnology regardless of location or age, together with workshops and now this biotechnology book to demonstrate the importance of high quality training, education and collaborations in fostering innovation and future discoveries. I would like to thank to all participants for their willingness to contribute their time and extensive knowledge and suggestions which made this work possible and last but not least, I would like to thank Erciyes University’s President, Prof. Dr. Fahrettin KELEŞTEMUR, for his sincere encouragement and support throughout. Prof. Dr. Munis DÜNDAR President of European Biotecnology Thematic Network Association February 2015

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CONTENTS

BOOK CONTENTS CHAPTER 1 HISTORY OF BIOTECHNOLOGY ................................................................. 1 Summary ................................................................................................................................ 3

1. Genetics and Biotechnology .......................................................................................... 3

2. Biotechnology and Medicine ......................................................................................... 4

3. Short History of Biotechnology .................................................................................... 8

Review Questions and Answers .............................................................................................. 8 Further Readings and References ........................................................................................... 8 CHAPTER 2 MOLECULAR TECHNIQUES IN FOOD MICROBIOLOGY

9

Summary ............................................................................................................................... 11

1. Molecular Biology ........................................................................................................ 11

2. DNA Extraction ............................................................................................................ 11

3. Polymerase Chain Reaction Based Methods .................................................................. 12

4. Quantitative PCR and Reverse Transcription qPCR .................................................... 13

4.1 Applications ................................................................................................................ 14

5. Electrophoresis ............................................................................................................. 15

5.1 Denaturing Gradient Gel Electrophoresis and Temperature Gradient Gel Electrophoresis 15 5.2 Applications ................................................................................................................ 16

6. Restriction Fragment Lenght Polymorphism ................................................................ 16

7. Blotting ......................................................................................................................... 16

7.1 Southern Blotting and Dot Blot .................................................................................. 17

7.2 Blotting Techniques Applied to Food Analysis ............................................................. 18

8. Microarrays ........................................................................................................................ 19

8.1 Applications ..................................................................................................................... 21

Review Questions and Answers .............................................................................................. 21 Further Readings .................................................................................................................... 22 References .............................................................................................................................. 23 CONTENTS

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CHAPTER 3 FOOD BIOTECHNOLOGY: DEVELOPMENTS AND PERSPECTIVES ........ 25 Summary ................................................................................................................................. 27

1. The Journey from Traditional Agriculture to Genetic Engineering ................................. 27

2. Transgenic Food and Crops ............................................................................................ 28

3. How are Transgenic Organisms Tested? .......................................................................... 30

4. Economic Impact of GMOs ............................................................................................ 31

5. What the Future Holds in Store: Genomics and Food Production .................................. 32

Review Questions and Answers ................................................................................................ 33 Further Readings ..................................................................................................................... 34 CHAPTER 4 BIOTECHNOLOGICAL PLANT BREEDING ................................................. 35 Summary ................................................................................................................................. 37

1. Plant Breeding ................................................................................................................ 38

1.1 Plant Domestication as a Breeding Process .................................................................... 38

1.2 The Beginning of Modern Plant Breeding ..................................................................... 39

2. Molecular Markers in Plant Breeding ............................................................................. 39

2.1 Marker Assisted Selection .............................................................................................. 40

2.2 Other Uses of Molecular Markers .................................................................................. 42

2.2.1 Plant Genetic Diversity and Germplasm Management ......................................... 42

2.2.2 Hybrid Vigour Prediction ..................................................................................... 43

2.2.3 Cultivar Identification and Protection .................................................................. 43

2.2.4 Genetic Maps ....................................................................................................... 44

3. Plant Breeding in the Genomics Era ............................................................................... 45

3.1 High-Throughput Genomic Techniques ....................................................................... 45

3.2 Some Plant Breeding Applications ............................................................................... 46

4. In Vitro Culture Techniques ........................................................................................... 47 4.1 In Vitro Techniques Used to Increase Diversity .............................................................. 48

4.1.1 In Vitro Pollination and Embryo Rescue ............................................................... 48

4.1.2 Somatic Hybridization .......................................................................................... 48 4.1.3 Somaclonal Variation ............................................................................................ 49 4.1.4 Induced Mutation ................................................................................................ 49

4.2 In Vitro Techniques Used to Improve The Breeding Process .......................................... 50

4.2.1 In Vitro Selection .................................................................................................. 50 4.2.2 Double-Haploid Production ................................................................................. 50

5. An Introduction to Transgenic Plants ............................................................................. 50

5.1 What is a ‘Transgenic’ or ‘Genetically Modified’ Plant? ................................................. 51

5.2 ‘Classical’ or ‘Traditional’ Plant Breeding vs. Genetic Engineering of Crops .................. 51

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CONTENTS

6. Generation of Transgenic Plants ...................................................................................

51

6.1 Steps to Produce a Transgenic Plant ............................................................................

51

6.2 Gene Transfer Methods ...............................................................................................

52

6.2.1 Agrobacterium Tumefaciens – Mediated Transformation .......................................

52

6.2.2 Agrobacterium Tumefaciens as a Vector for Gene Transfer to Plants .....................

53

6.2.3 Leaf-Disc transformation ....................................................................................

54

6.3 Particle Bombardment (Biolistics) ...............................................................................

54

6.4 Other Methods for Direct Gene Transfer Into Plants ..................................................

55

7. Biotech Crops ...............................................................................................................

56

7.1 Improvement of Agronomic Traits ..............................................................................

56

7.1.1 Herbicide Tolerance ............................................................................................

56

7.1.2 Insect Resistance ................................................................................................. 57

7.1.3 Other Crops, Other Traits ..................................................................................

57

7.2 Perspectives for The Near Future .................................................................................

57

Review Questions and Answers .............................................................................................

58

Further Readings ...................................................................................................................

62

CHAPTER 5 PRODUCTION OF THERAPEUTIC RECOMBINANT PROTEINS IN TRANSGENIC ANIMALS ....................................................................................................

65

Summary ...............................................................................................................................

67

68

1. Production of Transgenic Mice Expressing Human Interferon Gamma ........................

1.1 Construction of The Hybrid Human Interferon Gamma Genes ................................. 68

1.2 Generation of Transgenic Mice ..................................................................................

69

1.3 Identification of Transgenic Mice ...............................................................................

69

1.3.1 PCR Analysis of The Integrated Transgene ........................................................

69

1.4 Milk Collection for hIFN-g Analysis .........................................................................

70

1.5 Concluding Remarks .................................................................................................

70

Review Questions and Answers ..............................................................................................

71


71

CHAPTER 6 RECENT ADVANCES IN ANIMAL BIOTECHNOLOGY ..............................

73

Summary ............................................................................................................................... 75

1. Selective Breeding of Farm Animals ..............................................................................

75

1.1 BLUP Procedure (Sire model) .....................................................................................

76

1.2 Single Trait Animal Model ..........................................................................................

77

CONTENTS

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2. Molecular Methods ......................................................................................................... 78

2.1 Genetic Markers ............................................................................................................ 78

2.2 High-Throughput Marker Genotyping .......................................................................... 79

2.3 DNA Chips .................................................................................................................. 79

3. Identifying Markers for Specific Traits ........................................................................... 79

3.1 Quantitative Trait Loci Mapping in Livestock .............................................................. 80

3.2 Genetic Mapping and Linkage Analysis ......................................................................... 80

3.3 Identification and Analysis of a QTL ............................................................................. 81

4. Genomic Selection .......................................................................................................... 83

Review Questions and Answers ................................................................................................ 84 Further Readings ..................................................................................................................... 85 CHAPTER 7 REMOVAL OF HEAVY METALS FROM AQUEOUS SOLUTIONS THROUGH BIOSORPTION ........................................................ 87 Summary ................................................................................................................................. 89

1. Toxic Metallic Elements .................................................................................................. 89

2. Achievements over the Past Decade ................................................................................ 91

3. Biosorption .................................................................................................................... 91

3.1 Sorption ........................................................................................................................ 91

4. Activated Charcoal/Carbon ............................................................................................ 92

5. Mechanism of Heavy Metals Removal ............................................................................ 93

6. Factors Affecting Biosorption ......................................................................................... 94

6.1 Influence of pH ........................................................................................................... 94

6.2 Influence of Temperature ............................................................................................. 95

6.3 Effect of Initial Metal Concentration ........................................................................... 95

6.4 Effect of Adsorbent Dosage .......................................................................................... 95

7. Biosorption Equilibrium Models and Isotherms-Assessment of Sorption Performance .. 96

7.1 Langmuir Isotherm ....................................................................................................... 96

7.2 Freundlich Isotherm ..................................................................................................... 96

7.3 Dubinin-Radushkevich Isotherm ................................................................................. 97

7.4 Adsorption Kinetics ..................................................................................................... 97

7.5 Pseudo-First-Order Kinetic Equation ........................................................................... 97

7.6 Pseudo-Second-Order Kinetic Equation ....................................................................... 97

Review Questions and Answers ................................................................................................ 99 Further Readings ..................................................................................................................... 102

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CHAPTER 8 INDUSTRIAL ENGINEERING ...................................................................... 103 Summary ............................................................................................................................... 105

1. Production of Enzymes by Microorganisms ................................................................. 106

1.1 Enzymes ..................................................................................................................... 106

1.2 Strains of Microorganisms Producing Enzymes ........................................................... 108

1.3 Fermentation Process .................................................................................................. 111

1.4 Extremozymes ............................................................................................................ 113

1.4.1 Enzymes From Extremophiles ............................................................................ 114

1.4.2 Immobilized Enzymes ........................................................................................ 116

1.4.3 Genetic and Protein Engineering of Enzymes ..................................................... 116

2. Ecological Potential of Plants ....................................................................................... 117

2.1 Higher Plants Organic Contaminants Detoxifiers ....................................................... 117

2.2 Organic Contaminants Transformation in Plant Cells ................................................. 119

2.3 Enzymes ..................................................................................................................... 121

2.4 Contaminants Action on Plant Ultrastructure ............................................................. 124

2.5 General Considerations ............................................................................................... 125

3. Bacterial Viruses Against Crop Pathogens .................................................................... 127

3.1 Bacterial Diseases of Plants ......................................................................................... 3.2 Plant Disease Control ................................................................................................. 3.3 Bacteriophages as Biological Control Agents .............................................................. 3.4 Challenges in Using Phages for Disease Control ......................................................... 3.5 Strategies for Optimization in Using Phages for Disease Control ................................ 3.6 Phages as Part of integrated Disease Management Strategies ....................................... 3.7 Other Application of Bacteriophages ........................................................................... 3.8 Engineered Bacteriophages .......................................................................................... 3.9 Bacteriophages as Potential Bioterrorism Agents and Anti-Terrorism Tools ...............

127 128 129 132 133 135 136 136 136

Review Questions and Answers .............................................................................................. 137 Further Readings ................................................................................................................... 139 CHAPTER 9 BIOREFINERY AND BIOENERGY APPLICATIONS .................................... 141 Summary ............................................................................................................................... 143

1. Structure and Recalcitrance of Biomass ........................................................................ 144

2. Biorefinery Technologies .............................................................................................. 146

2.1 Biochemical Processes ................................................................................................. 146

2.1.1 Pre-treatment Under Alkaline Conditions .......................................................... 2.1.2 Acid-Catalyzed Pre-treatment And Steam Pre-treatment ..................................... 2.1.3 Ionic Liquid Pre-treatment ................................................................................. 2.1.4 Biological Pre-treatment ..................................................................................... 2.1.5 Alternative Pre-treatments .................................................................................. CONTENTS

146 147 149 149 149 XV


2.2 Thermal Chemical Processes ........................................................................................ 150

2.2.1 Pyrolysis .............................................................................................................. 150 2.2.2 Liquefaction ........................................................................................................ 151 2.2.3 Gasification ......................................................................................................... 151 2.2.4 Combustion ........................................................................................................ 152

3. Conclusions .................................................................................................................. 152

Acknowledgements ................................................................................................................ 152 Summary Box ........................................................................................................................ 152 Review Questions and Answers .............................................................................................. 153 References .............................................................................................................................. 153 CHAPTER 10 NANOBIOTECHNOLOGY .......................................................................... 157 Summary ............................................................................................................................... 159

1. Introduction to Nanobiotechnology ............................................................................. 159

2. Nanomaterials Used in Nanotechnology: A Nanomedicine Perspective ........................ 160

2.1 Quantum Dots ........................................................................................................... 160

2.2 Gold Nanoparticles ..................................................................................................... 163

2.2.1 Synthesis and Modification of AuNPs ................................................................. 163

2.3 Superparamagnetic Iron Oxide Nanoparticles ............................................................. 164

2.1.1 Synthesis and Modification of ODs .................................................................... 161

2.3.1 Synthesis and Modification of SPION ................................................................ 165

3. Challenges of Nanomedicine ........................................................................................ 166

3.1 Requirements for Efficient Diagnostic and Therapeutic Nanoparticle ......................... 166

4. Nanobiosensors ............................................................................................................ 168

5. Self-Assembly ................................................................................................................ 169

5.1 Evaporation Induced Self-Assembly ............................................................................ 169

5.2 Programmed Self-Assembly ......................................................................................... 170

5.3 Geometry (Shape) Driven Self-Assembly ..................................................................... 170

6. Conclusions .................................................................................................................. 170

Review Questions and Answers .............................................................................................. 171 Further Readings ................................................................................................................... 172

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CONTENTS

CHAPTER 11 PRINCIPLES OF TISSUE ENGINEERING ................................................. 173 Summary ............................................................................................................................... 175

1. Stem Cells ..................................................................................................................... 175

1.1 Brief History of Stem Cell Research ............................................................................ 175

1.2 Definition and Basic Biological Properties of Stem Cells ............................................. 176

1.3 Types of Stem Cells ..................................................................................................... 177

1.3.1 Embryonic Stem Cells ........................................................................................ 177

1.3.2 Primordial Stem Cells ......................................................................................... 178

1.3.3 Induced Pluripotent Stem Cells .......................................................................... 178

1.3.4 Foetal Stem Cells ................................................................................................ 178

1.3.5 Adult Stem Cells ................................................................................................ 179

2. Bioactive Molecules ...................................................................................................... 180

2.1 Hormones ................................................................................................................... 180 2.2 Cytokines .................................................................................................................... 181

2.3 Growth Factors ........................................................................................................... 181

3. Scaffolds ....................................................................................................................... 182

3.1 Natural Polymers for Scaffold Fabrication ................................................................... 182

3.2 Synthetic Polymers for Scaffold Fabrication ................................................................ 183

4. Conclusions ................................................................................................................. 184

Acknowledgements ................................................................................................................ 184 Summary Box ........................................................................................................................ 184 Review Questions and Answers .............................................................................................. 184 Further Readings ................................................................................................................... 185 CHAPTER 12 BACTERIOPHAGES AND THEIR APPLICATIONS ................................... 187 Summary ............................................................................................................................... 189

1. Bacteriophages ............................................................................................................. 189

1.1 Lytic and Temperate Phages ....................................................................................... 190

1.2 Classification of Bacteriophages ................................................................................. 191

2. Phage Typing ................................................................................................................ 192

3. Phage-Based Detection Systems of Bacterial Targets .................................................... 193

3.1 Reporter Phages ......................................................................................................... 194

3.2 Quantum Dots .......................................................................................................... 194

3.3 Phage Amplification ................................................................................................... 194

3.4 Phage-Based Biosensors .............................................................................................. 195

CONTENTS

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4. Phage-Based Controlling of Bacteria in Agriculture and Food Processing .................... 195

5. Phage Therapy ............................................................................................................. 198

5.1 Animal Experiments .................................................................................................. 199

5.2 Phages in Medicine .................................................................................................... 202

6. Conclusions .................................................................................................................. 203

Review Questions and Answers ............................................................................................. 204 Further Readings ................................................................................................................... 204 CHAPTER 13 OMICS SCIENCES ....................................................................................... 209 Summary ............................................................................................................................... 211

1. Genomics, Metabolomics, Lipidomics, Epigenomics .................................................... 211

2. The Post-Genome Era: Proteomics ............................................................................... 212

2.1 Methods for Protein Separation .................................................................................. 213

2.1.1 Gel-Based Proteomics: 2-DE .............................................................................. 213

2.1.2 Gel Free-Based Approaches ................................................................................ 214

3. Limitations of Current Proteomics Approach ............................................................... 215

3.1 Protein Abundance ..................................................................................................... 215

4. Clinical Applications of Proteomics ............................................................................. 217

5. Omics Science in 2013: Toward Omic Personalized Medicine ...................................... 219

Acknowledgement ................................................................................................................. 219 Summary Box ........................................................................................................................ 219 Review Questions and Answers .............................................................................................. 220 Further Readings ................................................................................................................... 220 CHAPTER 14 ENZYMES AND PROTEINS A BIOTECHNOLOGY TAILORING POINT OF VIEW ...................................................... 223 Summary ............................................................................................................................... 225

1. Enzymes ....................................................................................................................... 225

2. Enzyme Engineering .................................................................................................... 225

2.1 Directed Evolution .................................................................................................... 226

2.2 Semi-Rational Design ................................................................................................ 226

2.3 Rational Design ......................................................................................................... 226

2.4 De Novo Design ........................................................................................................ 226

3. Drug Metabolising Enzymes ........................................................................................ 227

3.1 Cytochrome P450 Enzymes: Structure, Evolution And Function ............................... 228

XVIII

3.1.1 Engineering of Cytochrome P450s ..................................................................... 229

CONTENTS

CONTENTS

4. P450 Enzyme Engineering and Methodology Tools ..................................................... 229

4.1 Polymerase Chain Reaction ........................................................................................ 230

4.2 Transformation of Cells .............................................................................................. 230

4.3 Purification of Protein ................................................................................................ 230

4.4 SDS Polyacrylamide Electrophoresis and Western Blot .............................................. 231

4.5 Lysate Assay ............................................................................................................... 231

5. Case Study .................................................................................................................... 232

5.1 Cytochrome P450 CYP2D6-NADPH Reductase Fusion Protein ................................ 232

5.2 Change of Selectivity (Regioselectivity and Enantioselectivity) .................................... 233

5.3 Change of Protein Stability (Thermostability) ............................................................. 234

5.4 Designing and Tailoring (Redesigning) Enzyme for New Substrates/Reactions ............ 234

5.5 Sequence-Based Enzyme Redesign .............................................................................. 235

5.6 Structure-Based Enzyme Redesign ............................................................................... 235

5.7 Computational Enzyme Redesign ............................................................................... 236

5.8 De Novo Enzyme Redesign (Rosetta) ........................................................................... 236

6. Conclusions .................................................................................................................. 236

Review Questions and Answers .............................................................................................. 237 Further Readings ................................................................................................................... 237 CHAPTER 15 DNA SEQUENCING TECHNOLOGY: THE CLINICAL POTENTIAL ...... 241 Summary ............................................................................................................................... 243

1. History ......................................................................................................................... 243

2. First Generation DNA Sequencing ............................................................................... 244

3. Second Generation Sequencing .................................................................................... 244

4. Sequencing By Synthesis ............................................................................................... 246

4.1 Roche GS-FLX 454 .................................................................................................... 246

4.2 Illumina MiSeq ........................................................................................................... 247

4.3 Ion Torrent ................................................................................................................ 248

5. Sequencing By Ligation ................................................................................................ 249

5.1 Life Technologies’ SOLiD ........................................................................................... 249

6. Third Generation Sequencing Technologies .................................................................. 250

7. Single-Molecule Sequencing ......................................................................................... 250

7.1 Heliscope™ Single Molecule Sequencer ....................................................................... 250

7.2 Pacific Bioscience ........................................................................................................ 250

7.3 Nanopore DNA Sequencer ......................................................................................... 251

8. Complete Genomics ..................................................................................................... 251

9. Single-Cell Sequencing ................................................................................................. 252 CONTENTS

XIX


10. Clinical Application of HT-NGS ................................................................................ 253

10.1 Genetic mutations in Mendelian Disorders ............................................................... 253

10.2 Epigenetics ................................................................................................................ 253

10.3 Genetics in Common Diseases .................................................................................. 254

10.4 Cancer Research and Biomarkers .............................................................................. 254

10.5 Discovering Noncoding RNAs .................................................................................. 255

10.6 Pathology-Important Cells ........................................................................................ 255

11. Conclusions ................................................................................................................ 255

Review Questions and Answers ............................................................................................. 256 Further Readings ................................................................................................................... 257 CHAPTER 16 RECOMBINANT DNA TECHNOLOGY AND GENETIC ENGINEERING ......................................................................................... 259 Summary ............................................................................................................................... 261

1. History ......................................................................................................................... 261

2. Techniques Used in Genetic Engineering ...................................................................... 262

2.1 Enzymes ...................................................................................................................... 262

2.2 Host/Vector Systems ................................................................................................... 262

2.2.1 Host Cells .......................................................................................................... 262 2.3 Cloning Vectors .......................................................................................................... 263

2.4 Insertion of Recombinant DNA into Cells ................................................................. 264

2.5 Cloning Strategies ....................................................................................................... 264

3.Applications .................................................................................................................. 265

3.1 Applications in Animal and Plant Sciences ................................................................. 265

3.2 Marker Assisted Selection ............................................................................................ 265

3.3 Genetic Modification .................................................................................................. 266

3.4 Cloning ...................................................................................................................... 268

4. Ethical, Legal and Social Issues of Recombinant

DNA Technology and Genetic Engineering .................................................................. 268

4.1 Historical Events in the Recombinant DNA/Genetic Engineering Debate .................. 268

4.2 Web of Regulatory Issues ............................................................................................ 269

4.3 Legal Controversies ..................................................................................................... 269

4.4 Ethical and Social Debates .......................................................................................... 270


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CONTENTS

CHAPTER 17 DRUG DISCOVERY AND DEVELOPMENT .............................................. 273 Summary ............................................................................................................................... 275

1. Stages of The Pharmaceutical R & D ............................................................................ 277

2. Approaches to Drug Discovery ..................................................................................... 277

3. Drug Targets ................................................................................................................. 278

4. High Throughput Screening ......................................................................................... 279

5. Biochemical Assays, Cellular Assays & Animal Models ................................................ 279

6. Drug Manufacturing .................................................................................................... 280

7. ADME, Toxicology, Pre-Clinical Studies ...................................................................... 281

8. Clinical Studies ............................................................................................................. 282

Review Questions and Answers .............................................................................................. 283 Further Readings ................................................................................................................... 285 References .............................................................................................................................. 285 CHAPTER 18 VACCINES .................................................................................................... 287 Summary ............................................................................................................................... 289

1. Principles of Vaccinology .............................................................................................. 289

2. Genetic Vaccines ........................................................................................................... 290

2.1 Recombinant Vaccines ................................................................................................ 290 2.1.1 Recombinant Subunit Vaccines ........................................................................... 290 2.1.2 Recombinant Whole Cell Vaccines ..................................................................... 291 2.1.3 Bacterial Carriers ................................................................................................ 292 2.2 OMV-Based Vaccines ................................................................................................. 292 2.3 DNA Vaccines ............................................................................................................ 292

3. Routes of Vaccine Administration ................................................................................. 293

4. Adjuvants ..................................................................................................................... 293

4.1 Bacterial-Derived Adjuvants ........................................................................................ 4.2 Adjuvant Emulsions .................................................................................................... 4.3 Saponins ..................................................................................................................... 4.4 Polymer-Based Vaccines .............................................................................................. 4.5 Liposome Adjuvants .................................................................................................... 4.6 ISCOM-Based Adjuvants ........................................................................................... 4.7 Virus-Like Particles ..................................................................................................... 4.8 Other Adjuvants .........................................................................................................

294 294 295 295 295 295 296 296

5. Cancer Vaccines ............................................................................................................ 296

6. Plant-Based Vaccines .................................................................................................... 296

7. Nanovaccines ................................................................................................................ 298

Review Questions and Answers .............................................................................................. 298 Further Readings ................................................................................................................... 298 CONTENTS

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CHAPTER 19 MEDICAL GENETICS ................................................................................. 299 Summary ............................................................................................................................... 301

1. Principles of Medical Genetics ..................................................................................... 302

1.1 Genetics: Historical Background ................................................................................. 302

1.2 Medical Genetics ........................................................................................................ 303

1.3 Personalized Medicine and Genetic Counseling .......................................................... 304

2. Chromosomal Disorders .............................................................................................. 305

2.1 Numerical Chromosomal Abnormalities ..................................................................... 305

2.1.1 Autosomal Chromosome Number Abnormalities ............................................... 306

2.1.2 Sex Chromosome Number Abnormalities .......................................................... 306

2.2 Structural Chromosomal Abnormalities ...................................................................... 307

3. Single-Gene Inheritance ............................................................................................... 310

3.1 Basic Concepts of Mendelian Inheritance ................................................................ 310

3.2 Patterns of Mendelian Inheritance ............................................................................. 310

3.2.1 Autosomal Inheritance ....................................................................................... 310 3.2.2 X-Linked Inheritance ......................................................................................... 311

3.3 Penetrance and Expressivity ........................................................................................ 311

3.4 New Advances in Mendelian Inheritance Research ..................................................... 313

Review Questions and Answers ............................................................................................. 314 Further Readings ................................................................................................................... 316 CHAPTER 20 COMPLEX DISORDERS, GENETIC COUNSELING AND GENETIC DIAGNOSTICS INCLUDING PRENATAL DIAGNOSIS ................................................... 319 Summary ............................................................................................................................... 321

1. Complex Disorders ...................................................................................................... 321

1.1 Determination of Genetic and Environmental Factors in the Etiology of Diseases ...... 322

1.2 Threshold Model in Multifactorial Inheritance ............................................................ 322

1.2.1 Factors Influencing the Recurrence Risk ............................................................. 323

1.3 Benefits of Human Genome Project to Gene Searching for Complex Diseases ............ 323

1.4 Examples of Complex Disorders ................................................................................. 324

1.4.1 Isolated Congenital Anomalies ........................................................................... 324

1.4.1.1 Neural Tube Defects .................................................................................. 324

1.4.1.2 Cleft lip and Palate .................................................................................... 324

1.4.1.3 Congenital Cardiac Anomalies ................................................................... 325

1.4.2. Complex Disorders of Adulthood ...................................................................... 325

1.4.2.1 Diabetes Mellitus ....................................................................................... 325 1.4.2.2 Coronary Artery Disease ............................................................................ 325 1.4.2.3 Obesity ...................................................................................................... 325 XXII

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2. Genetic Counseling ...................................................................................................... 326

2.1 Genetic Counseling in Single-Gene Disorders ............................................................. 326

2.1.1 Autosomal Dominant Inheritance ...................................................................... 327

2.1.2 Autosomal Recessive Inheritance ........................................................................ 327

2.1.3 X-linked Recessive and X-linked Dominant Inheritance ..................................... 328

2.2 Genetic Counseling in Mitochondrial Inheritance ...................................................... 328

2.3 Genetic Counseling in Multifactorial Diseases ............................................................ 328

3. Genetic Diagnostics Including Prenatal Diagnosis ....................................................... 329

3.1 Methods of Genetic Diagnosis .................................................................................... 329

3.1.1 Methods for Cytogenetic Diagnosis .................................................................... 329

3.1.1.1 Structure of Chromosomes ......................................................................... 329

3.1.1.2 Morphologic Features of Chromosomes ..................................................... 330

3.1.1.3 Banding Techniques Used in Cytogenetic ................................................... 330

3.1.2 Molecular Cytogenetic Methods ......................................................................... 332

3.1.3 Molecular Diagnostic Methods ........................................................................... 333

3.1.3.1 The Organization of Genome ..................................................................... 333

3.1.3.2 The Structure of DNA ............................................................................... 333

3.1.3.3 Gene Structure ........................................................................................... 334 3.1.3.4 Types of Mutations ..................................................................................... 334

3.2 Methods for Prenatal Diagnosis .................................................................................. 336

3.1.3.5 Diagnostic Techniques in Molecular Genetics ............................................ 334

3.2.1 Techniques for Prenatal Diagnosis ...................................................................... 336

3.3 Preimplantation Genetic Diagnosis ............................................................................. 337

Review Questions and Answers .............................................................................................. 337 Further Readings ................................................................................................................... 337 CHAPTER 21 CANCER, HIGH THROUGHPUT GENOME ANALYSIS AND GENETIC DISORDERS ........................................................................................................................ 341 Summary ............................................................................................................................... 343

1. Cancer .......................................................................................................................... 343

1.1 Basic Properties of Cancer Cells .................................................................................. 344

1.2 What are The Causes and Risk Factors of Cancer? ...................................................... 345

1.3 Molecular Genetics of Cancer ..................................................................................... 346

1.3.1 Oncogenes .......................................................................................................... 347 1.3.2 Tumour Suppressor Genes .................................................................................. 349

1.3.3 DNA Repair Genes and Genetic Instability ........................................................ 349

1.3.4 MicroRNAs ........................................................................................................ 349

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1.4 Tumour Initiation, Promotion and Progression ........................................................... 349

1.5 Metastasis .................................................................................................................... 350

2. High Throughput Genome Analysis and Genetic Disorders ........................................ 350

2.1. DNA Sequencing ...................................................................................................... 350

2.2. The Search for Disease-Causing Variants ................................................................... 351

2.3. Applications of Next-Generation Sequencing ............................................................ 351

2.3.1 Whole-Transcriptome Sequencing ..................................................................... 352 2.3.2 Whole-Exome Sequencing ................................................................................. 352 2.3.3 Whole-Genome Sequencing ............................................................................... 352 Review Questions and Answers ............................................................................................. 354 Further Readings ................................................................................................................... 355 CHAPTER 22 CANCER SYSTEMS BIOLOGY: INTEGRATING EXPERIMENTAL AND THEORETICAL SYSTEMS AT CELLULAR AND TISSUE SCALES .................................. 357 Summary .............................................................................................................................. 359

1. Cancer .......................................................................................................................... 359

2. Systems Biology ........................................................................................................... 360

3. Intracellular Signalling Dynamics ................................................................................ 363

3.1 Experimental Systems ................................................................................................. 364

3.1.1 Genomic Data Generation Systems .................................................................... 364

3.1.2 Proteomic Data Generation Systems .................................................................. 366

3.2 Theoretical Approaches ............................................................................................... 368

3.2.1 Process-Based Models ......................................................................................... 368 3.2.2 Data-Driven Models .......................................................................................... 371

4. Tissue-Scale Systems and Analysis ................................................................................ 373

4.1 Tissue-Scale Data Generation ..................................................................................... 373

4.2 Morphometrics: Beyond Volumetric Analysis ............................................................. 374

5. Linking Cell and Tissue Scales ..................................................................................... 377

6. Summary: Towards a Cancer Ecology ........................................................................... 382

Review Questions and Answers ............................................................................................. 382 Further Readings .................................................................................................................. 385 References ............................................................................................................................. 386

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CHAPTER 23 BIOTECHNOLOGY AND ETHICS ............................................................. 387 Summary ............................................................................................................................... 389

1. From Life Sciences to Biotechnology and Information Technology .............................. 389

1.1 Genomic Era and Biotechnology ................................................................................ 389

1.1.1 From National Library of Medicine to NCBI ..................................................... 389

1.2 Biotechnology And Ethical Implications .................................................................... 390

1.2.1 A Brief History of Bioethics ................................................................................ 390

1.2.2 Ethical, Legal and Social Implications ................................................................. 390

2. Ethical Implications of Recombinant DNA Technology in Biotechnology ................... 391

2.1 Recombinant DNA Technology in Biotechnology ...................................................... 391

2.1.1 Red Biotechnology ............................................................................................. 392

2.1.2 Blue Biotechnology: Research Related to Marine and Aquatic Processes ............. 393

2.1.3 Black Biotechnology: Research Related to Bioterrorism ...................................... 393

2.2 Bioprospecting and Biopiracy ..................................................................................... 394

3. Ethical Regulations and Code of Conducts in Biotechnology Research and Industry .. 394


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CONTRIBUTERS

CONTRIBUTERS CHAPTER 1 HISTORY OF BIOTECHNOLOGY Munis Dündar, MD, PhD Professor, Head of Medical Genetics Department, Medical Faculty, Erciyes University, Kayseri, Turkey. Müge Gülcihan Önal, PhD Researcher, Medical Genetics Department, Medical Faculty, Erciyes University, Kayseri, Turkey. Yagut Erdem, PhD Assistant Professor, Genetics Department, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey.

CHAPTER 2 MOLECULAR TECHNIQUES IN FOOD MICROBIOLOGY Marisa Manzano, PhD Associate Professor, Department of Food Science, Faculty of Agriculture, University of Udine, Udine, Italy. Lucilla Iacumin, PhD Postdoctoral Researcher, Department of Food Science, Faculty of Agriculture, University of Udine, Udine, Italy.

CHAPTER 3 FOOD BIOTECHNOLOGY: DEVELOPMENTS AND PERSPECTIVES Daniel Ramón, PhD Professor, CEO of Biopolis S.L., University of Valencia Science Park, Valencia, Spain.

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CHAPTER 4 BIOTECHNOLOGICAL PLANT BREEDING Ana Fita, PhD Associate Professor, Institute for Conservation and Breeding of Valencian Agrodiversity, Polytechnic University of Valencia, Valencia, Spain. Óscar Vicente, PhD Professor, Institute of Plant Molecular and Cellular Biology, Polytechnic University of Valencia, Valencia, Spain. Mónica Boscaiu, PhD Associate Professor, Mediterranean Agroforestal Institute, Polytechnic University of Valencia, Valencia, Spain. Adrián Rodriguez-Burruezo, PhD Associate Professor, Institute for Conservation and Breeding of Valencian Agrodiversity, Polytechnic University of Valencia, Valencia, Spain.

CHAPTER 5 PRODUCTION OF THERAPEUTIC RECOMBINANT PROTEINS IN TRANSGENIC ANIMALS Haydar Bağış, PhD Professor, Head of Medical Genetic Department, Vice Dean of Medical Faculty, Adıyaman University, Adıyaman, Turkey.

CHAPTER 6 RECENT ADVANCES IN ANIMAL BIOTECHNOLOGY Edo D’Agaro, PhD Senior Lecturer, Department of Food Science, University of Udine, Udine, Italy.

CHAPTER 7 REMOVAL OF HEAVY METALS FROM AQUEOUS SOLUTIONS THROUGH BIOSORPTION H. Lalhruaitluanga, PhD Assistant Professor, Department of Biotechnology, Mizoram University, Mizoram, India. M.N.V. Prasad, PhD Professor, Department of Plant Sciences, New Life Science Building (South Campus), University of Hyderabad, Telangana, India.

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CHAPTER 8 INDUSTRIAL ENGINEERING Edisher Kvesitadze, PhD Professor, Department of Biochemistry and Biotechnology, Georgian Technical University, Tbilisi, Georgia. Teo Urushadze, PhD Professor, Agronomy Faculty, Agricultural University of Georgia, Tbilisi, Georgia. Tinatin Sadunishvili, PhD Professor, Durmishidze Institute of Biochemistry and Biotechnology, Agricultural University of Georgia, Tbilisi, Georgia. Giorgi Kvesitadze, PhD Professor, Georgian National Academy of Sciences, Tbilisi, Georgia.

CHAPTER 9 BIOREFINERY AND BIOENERGY APPLICATIONS Haiyan Yang, PhD Researher, Beijing Key Laboratory of Lignocellulosic Chemistry, College of Material Science and Technology, Beijing Forestry University, Beijing, China. Kun Wang, PhD Lecturer, Beijing Key Laboratory of Lignocellulosic Chemistry, College of Material Science and Technology, Beijing Forestry University, Beijing, China. Run-Cang Sun, PhD Professsor, Beijing Key Laboratory of Lignocellulosic Chemistry, College of Material Science and Technology, Beijing Forestry University, Beijing, China.

CHAPTER 10 NANOBIOTECHNOLOGY Mine Altunbek, PhD Researcher, Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey. Ertuğ Avcı, PhD Researcher, Department of Genetics and Bioengineering, Faculty of Engineering,Yeditepe University, Istanbul, Turkey. Mustafa Çulha, PhD Professor, Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey.

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CHAPTER 11 PRINCIPLES OF TISSUE ENGINEERING Lubos Danisovic, PhD Postdoctoral Researcher, Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University in Bratislava, Bratislava, Slovakia.

CHAPTER 12 BACTERIOPHAGES AND THEIR APPLICATIONS Ľubomíra Tóthová, PhD Research Scientist, Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia.

CHAPTER 13 OMICS SCIENCES Daniele Vergara, PhD Postdoctoral Researcher, Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy. Pasquale Simeone, PhD Postdoctoral Researcher, Department of Neurosciences, Imaging and Clinical Sciences, Unit of Cancer Pathology, Foundation University, Chieti, Italy. Claudia Toto, MSc Post-Graduate Fellow, Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy. Michele Maffia, PhD Professor, Laboratory of General Physiology, Department of Biological and Environmental Sciences and Tecnologies, University of Salento, Lecce, Italy.

CHAPTER 14 ENZYMES AND PROTEINS - A BIOTECHNOLOGY TAILORING POINT OF VIEW Yusuf Deeni, PhD Senior Lecturer, Engineering and Technology, Scotish Informatics, Mathematics, Biology and Statistics Centre, School of Science, University of Abertay, Dundee, Scotland, United Kingdom. Nuruddeen Sojimade, PhD Research Scientist, Engineering and Technology, Scotish Informatics, Mathematics, Biology and Statistics Centre, School of Science, Abertay University, Dundee, Scotland, United Kingdom.

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CHAPTER 15 DNA SEQUENCING TECHNOLOGY: THE CLINICAL POTENTIAL Tommaso Beccari, PhD Professor, Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy. Maria Rachele Ceccarini, PhD Resercher, Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy. Michela Codini, BSc Researcher, Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy. Mariapia Viola Magni, PhD Professor, Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.

CHAPTER 16 RECOMBINANT DNA TECHNOLOGY AND GENETIC ENGINEERING Dijana Plaseska-Karanfilska, MD, PhD Professor, Research Centre for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Sciences and Arts, Ss. Cyril and Methodius University of Skopje, Skopje, Macedonia. Zoran Popovski, PhD Professor, Department for Biochemistry and Genetic Engineering, Faculty of Agricultural Sciences and Food, Ss. Cyril and Methodius University of Skopje, Skopje, Macedonia. Bratislav Stankovic, PhD Professor, University for Information Science and Technology “St Paul the Apostle”, Ohrid, Macedonia.

CHAPTER 17 DRUG DISCOVERY AND DEVELOPMENT Dimitar Trifonov, BSc Researcher, Center for Molecular and Cellular Biosensor Research, Abertay University, Dundee, Scotland, United Kingdom. Nikolai Zhelev, PhD Professor, Center for Molecular and Cellular Biosensor Research, Abertay University, Dundee, Scotland, United Kingdom.

CHAPTER 18 VACCINES Fabrizio Bruschi, MD Professor, Department of Translational Research, School of Medicine, University of Pisa, Pisa, Italy.

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CHAPTER 19 MEDICAL GENETICS Yavuz Oktay, PhD Assistant Professor, Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Acıbadem University, Istanbul, Turkey. Deniz Ağırbaşlı, MD, PhD Postdoctoral Researcher, Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Acıbadem University, Istanbul, Turkey. Sevim Dalva-Aydemir, PhD Researcher, Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Acıbadem University, Istanbul, Turkey. Cemaliye Akyerli Boylu, PhD Assistant Professor, Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Acıbadem University, Istanbul, Turkey. Meltem Müftüoğlu, PhD Associate Professor, Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Acıbadem University, Istanbul, Turkey. M. Cengiz Yakıcıer, MD, PhD Professor, Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Acıbadem University, Istanbul, Turkey.

CHAPTER 20 COMPLEX DISORDERS, GENETIC COUNSELING AND GENETIC DIAGNOSTICS INCLUDING PRENATAL DIAGNOSIS Cavidan Nur Semerci, MD Professor, Department of Medical Genetics, School of Medicine, Pamukkale University, Denizli, Turkey.

CHAPTER 21 CANCER, HIGH THROUGHPUT GENOME ANALYSIS AND GENETIC DISORDERS Naciye Lale Şatıroğlu Tufan, MD, PhD Professor, Deptartment of Pediatric Genetics, School of Medicine, Ankara University, Ankara, Turkey.

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CHAPTER 22 CANCER SYSTEMS BIOLOGY: INTEGRATING EXPERIMENTAL AND THEORETICAL SYSTEMS AT CELLULAR AND TISSUE SCALES James Bown, PhD Professor, Scotish Informatics, Mathematics, Biology and Statistics Centre, University of Abertay, Dundee, Scotland, United Kingdom. Yusuf Deeni, PhD Senior Lecturer, Scotish Informatics, Mathematics, Biology and Statistics Centre, School of Contemporary Science, University of Abertay, Dundee, Scotland, United Kingdom. Alexey Goltsov, PhD Senior Lecturer, SICSA Reader, Scotish Informatics, Mathematics, Biology and Statistics Centre, University of Abertay, Dundee, Scotland, United Kingdom. Hilal Khalil, PhD Postdoctoral Researcher, Scotish Informatics, Mathematics, Biology and Statistics Centre, University of Abertay, Dundee, Scotland, United Kingdom. Michael Idowu, PhD Researcher, Scotish Informatics, Mathematics, Biology and Statistics Centre, University of Abertay, Dundee, Scotland, United Kingdom. John Isaacs, PhD Lecturer, Computing and Applied Mathematics, School of Engineering, University of Abertay, Dundee, Scotland,United Kingdom. Ye Li, PhD Researcher, Scotish Informatics, Mathematics, Biology and Statistics Centre, University of Abertay Dundee, Scotland, United Kingdom. Adam T. Sampson, PhD Lecturer, Scotish Informatics, Mathematics, Biology and Statistics Centre, University of Abertay, Dundee, Scotland, United Kingdom. Anne Savage, PhD Postdoctoral Researcher, Scotish Informatics, Mathematics, Biology and Statistics Centre, University of Abertay, Dundee, Scotland, United Kingdom. Nikolai Zhelev, PhD Professor, Scotish Informatics, Mathematics, Biology and Statistics Centre, University of Abertay, Dundee, Scotland, United Kingdom.

CHAPTER 23 BIOTECHNOLOGY AND ETHICS Nilgün Tekin, PhD Postdoctoral Researcher, Central Laboratory, Biotechnology Institute, Ankara University, Ankara, Turkey. Özge Cumaoğulları, PhD Researcher, Central Laboratory, Biotechnology Institute, Ankara University, Ankara, Turkey. Hilal Özdağ, PhD Professor, Central Laboratory, Biotechnology Institute, Ankara University, Ankara, Turkey.

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HISTORY OF BIOTECHNOLOGY

CHAPTER 1 HISTORY OF BIOTECHNOLOGY Munis Dündar, Müge Gülcihan Önal, Yagut Erdem

CONTENTS Summary ................................................................................................................................ 3

1. Genetics and Biotechnology .......................................................................................... 3

2. Biotechnology and Medicine ......................................................................................... 4

3. Short History of Biotechnology .................................................................................... 8

Review Questions and Answers .............................................................................................. 8 Further Readings and References ........................................................................................... 8 HISTORY OF BIOTECHNOLOGY

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CHAPTER 1 / M. Dündar, M. G. Önal, Y. Erdem


Summary

I

t includes practices for the implementation of advanced and modern techniques to biological systems. Mutations or mutants with new phenotypic characters created with the aid of recombinant DNA technology or transgenic organisms have been begun to use extensively in industry and in all areas. Biotechnology increasingly comprises the purpose of medical, agricultural and industrial biological materials production with the help of applications of genetic engineering. Therefore, in the last decade of 20th century, Biotechnology has been defined as an applied and interdisciplinary field. From now on, this technology enables changing all the information and codes contained in an organism’s genome, transferring DNA sequences or genes to identical or different organism of genus, extracting of desired DNA sequences or genes, transferring to other organisms or integration, determining of DNA and RNA sequences, gene mapping, generating transgenic animals, plants, microorganisms, genetic arrangements on embryonic levels, creating living beings with new phenotype and genotype, producing materials and chemicals like proteins, enzymes, antibiotics, hormones used in diagnostics, therapy, protecting and researches.

1. Genetics and Biotechnology Biotechnology has become very popular in the last quarter of the 20th century in the science world. Biotechnology is concerned with biological agents and their components to generate useful products, better health, and recycleing. The importance and significance behind this huge attention for biotechnology may be due to its unlimited potential to both serve and benefit mankind. So far, biotechnology has touched our lives in all aspects, such as, food, health and animal life. Biotechnology draws on the pure biological sciences such as, genetics, microbiology, animal cell culture, molecular biology, biochemistry, and embryology and cell HISTORY OF BIOTECHNOLOGY

biology. In the two last two decades biotechnology has resulted in new and different sciences e.g. genomics, recombinant gene technologies and applied immunology. According to the historical development, biotechnology can be divided into two categories: traditional biotechnology and modern biotechnology. In traditional biotechnology, biological systems (usually bacteria, yeasts and fungi) are used without any modifications as in the production of bread, cheese, alcohol, various alcoholic beverages, vinegar, yogurt. ”Fermentation Technology” is mainly intended to cover this form of production. Modern biotechnology allows us to obtain human blood proteins, hormones, insulin and biotechnological vaccines by using recombinant DNA technologies and mutagenesis. Today, traditional biotechnology and modern biotechnology are used at every point of life. Before their discovery, microorganisms were being used for human needs to preserve milk, fruit and vegetables and to improve the quality of life with the resultant beverages, cheeses, bread and vinegar. The oldest biotechnology usages were in Mesopotamia and Babylonia where yeast was used to make beer and the conversion of sugar to alcohol. By 4000 BC, the Egyptian culture was the first to produce leavened bread. In 3500 BC in Assyria, wine was produced and milk was converted to lactic acid to make yoghurt and kefir for the first time. Later, other civilizations started the process of lactic acid fermentation and this allowed fermentation and preservation of foods like soy sauce. Louis Pasteur (1822-1895) was interested in the mechanisms of these processes and his investigations have led to the development of vaccines and concepts of hygiene. In 1917, Chaim Weizmann first used a pure microbiological culture in an industrial process using Clostridium acetobutylicum to produce acetone. Penicillin was discovered as an antibiotic by Alexander Fleming in 1929. All commercial antibiotics in 1940s were natural, but today most of them are semisynthetic. Manipulating the genetic character of plants and animals to improve crop yields is far from new. Cross-breeding for desired traits such as tallness, greater milk yield or sweeter CHAPTER 1

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fruits, has been practiced ever since humans began farming. However classical breeding methods have drawbacks, especially the length of time required to get the desired quality. Biotechnology can dramatically reduce the time and efforts required to improve crops and livestock. These methods allow scientists to modify plants and animals in a more controlled way, choosing selected genes for cross-breeding to obtain the desired characteristic. The basis of modern biotechnology depends on the genetic material of all living cells, “DNA”. All chromosomes contain DNA inside the cell nucleus. Understanding the molecular structure of DNA led to rapid advances in all areas of biotechnology. Instead of mixing all the hundreds of genes within a plant or animal in back-crossing, now scientists can select a particular gene which is responsible for a particular character. After the discovery of recombinant DNA technology by the 1970s, it has been applied to the production of antibiotics. In 1971, Paul Berg had a success in gene splicing experiments. This success can count as the birth of modern biotechnology. Restriction enzymes are the most important part of this technology. Many genes encoding enzymes of antibiotic biosynthesis have been cloned and expressed at high levels in different microorganisms. Efforts in the application of recombinant DNA technology to bioengineering have led to overproduction of limiting enzymes of important biosynthetic pathways, thereby increasing the production of the final products. Also, modern usage of biotechnology includes genetic engineering and cell and tissue culture technologies. More recently, rising demand for biofuels is expected to be good news for biotechnology which plays a crucial role by enriching farm productivity of biofuel. Modern biotechnology also can be used to produce existing drugs more cheaply and easily. The application of biotechnology to basic sciences has rapidly improved our knowledge through the Human Genome Project. Nanotechnology is defined as the understanding and control of matter at dimensions between 1 to 100 nanometers approximately. As a result, novel applications become feasible when working with 4


bulk materials, or with single atoms or molecules. Although nanoscale materials were used by craftsmen since ancient times, nanotechnology as known now began about 30 years ago. Examples of ancient applications include the usage of nanoscale gold in stained glasses and the finding of nanotubes used in blades of swords. Since the invention of the high-powered microscopes reserachers in various fields such as physicists, biologists, chemists, electrical and optical engineers, and material scientists, were able to see things at nanoscale and work with materials at nanoscale. Nanotechnology has several commercial applications. For example, nanomaterials are used to manufacture lighweight, strong materials for applications in different industries. In healthcare, nanoceramics are used in some dental implants or to fill holes in diseased bones while some pharmaceutical products are reformulated with nanosized particles to improve their absorption. In clinical trials, nanotechnologybased medicines are used and in the near future may be used to treat patients. Nanoparticles are used to deliver toxic anti-cancer drugs directly to tumors, resulting in minimum damage to other body parts. Also, nanotechnology is being used in imaging tools, like Magnetic resonance imaging (MRI) and Computed Axial Tomography (CAT) scans, work better and more safely. Moreover, nanotechnology is helping scientists to find better ways to purify drinking water and to clean up environmental waste and damage. Nanotechnology seems to have so many applications in different fields and offers a great potential in future.

2. Biotechnology and Medicine Medicine has existed in one form or another since prehistoric times. Each culture had its healers. In our time medicine has spread based on a scientific approach. It is necessary to start writing about the history of medicine by beginning from the prehistoric times. Since the writing started in Mesopotamia, it is fair to assume that prehistoric times cover all periods before that. There are no written materials about


prehistoric times. As a result, scientists in the fields of archaeology and palaeontology need to interpret the findings related to prehistoric times. Medical history can be traced back to methods of treatment that are used in the Indian city of Ladakh, which is located in the region of northwest India in Jammu and Kashmir. Ladakh has a Tibetan Buddhist culture. A patient in Ladakh may apply to the shaman, herbalist, traditional Tibetan medicine practitioner, or to a modern doctor. He/ she can even see them all in turn. Materials found in cuneiform scripture from the Sumerian period circa 3000 B.C. give us valuable information about the history of medical applications. Among historical materials found there are two important seals, one belonging to a Sumerian doctor displayed in the Wellcome Museum in London and the other displayed in the Louvre Museum in Paris belonging to a Babylonian doctor. Vernon Coleman has written the Story of Medicine in 1985, indicating that Mesopotamia was an important geographical area for the history of medicine since the first steps to eliminate the beliefs regarding the formation of diseases caused by evil spirits and demons and healed by interfering with these spirits and bewitchery.

The first steps toward the establishment of the medical profession have been taken neither by a clergyman nor a doctor but by the Babylonian King Hammurabi. He detailed the articles to protect the doctors and the patients in his famous laws. Over the centuries medical knowledge has developed substantially. It can be seen that broken bones are bonded by bandages and certain medicines are prepared using different weighing instruments. Also, historical records indicated that doctors started to specialize in different areas of diseases. A few thousand years before Freud, psychiatrists existed in Babylon and the effects of fear, crime, and sorrow on human health was well known. A small portion of documents written on papyrus during ancient Egypt give valuable information about the history of medicine. Among these important records, Ebers Papyrus, which was found in Thebes and is kept in Leipzig University and thought to be written in 1500 B.C., contains 900 prescriptions and consists of 110 pages and is 10 meters long. China is an important country for the development of medicine in the orient. The Chinese em-

Gevher Nesibe Sultan Hospital (1206) HISTORY OF BIOTECHNOLOGY

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peror Sheng Nung, who lived in 3000 B.C., developed new methods about animal husbandry and horticulture and contributed to medicine by experimenting with different drugs and poisons on himself. He wrote a book called Pen Tsao, which means great book of plants. Pen Tsao was translated into English at the beginning of the twentieth century and considered as an important book for the history of medicine. 500 B.C. is considered as the great century. Hippocrates made significant contribution in the area of medicine. People believed until Hippocrates that diseases were caused by evil spirits and demons and were sent to humans by Gods who were angered. Hippocrates claimed that diseases were results of natural causes. He observed several symptoms in the progress of a disease and was considered as the founder of clinical medicine by stating the history of cases and by the bedside instruction of patients. He is famous for the phrase ‘’nature heals, the physician is nature’s assistant’’. Avicenna (İbn-i Sina) was born in 980 in the Middle East, became a famous physician at the age of 18 and authored 276 books of which 43 were in the area of medicine. He described meningitis and gave information about epidemics. He also recommended the use of forceps in problematic birth delivery. It is interesting to note that forceps started to be used after the 17th century in the Western World. He emphasized that some diseases could be passed through the placenta. He was best known for understanding the psychosomatic diseases and for using various food and drinks, drugs, hot and cold showers, sun bathing and physical therapy. Gevher Nesibe Darussifa is the first known medical school and hospital in Seljuk Anatolia in Kayseri, built in A.D. 1206. This Darussifa was the first and the oldest where medical education was instructed. The medical complex of Gevher Nesibe is supposed to be the first actual medical institution in the world, because of its architectural structure and medical education. In the medical school, doctors, surgeons, eye experts and assistants had worked. In addition to this, there was even a pharmacy for the School. 6


We can trace the history of medicine at the progress made in this field in different cultures. Nevertheless, it is important to remember that despite the development of knowledge about the human body and diseases, it was a long time before scientific progress has been integrated into a real medical practice. This was due to the fact that for a long time practitioners were separated from scientists. In addition, many of the scientific advances that have really brought about a revolution in medicine appeared not earlier than the nineteenth or twentieth century. Prior to that, medical care in the world mainly consisted of the recommendations regarding diet, hygiene, exercise and lifestyle. Surgery was performed in simplest form and some effective herbal medicines were prescribed. Doctors could alleviate a number of chronic diseases and heal some light ailments, but they lacked the capacity to deal with most of the major killer diseases. Nevertheless, some of the treatments have been fairly effective for reasons that became understood only much later. For example, the ritual cleansing that is part of many religious Timeline of Biotechnology BC 3000 Leaven of Bread, Alcoholic fermentation, Learning of making vinegar BC 2000 Wine production in Mesopotamia BC 500 Moldy soybean curds are used by the Chinese to treat boils as antibiotic. BC 300 The production of beer by Sumerians, the Babylonians and the Egyptians 1150 Ethanol production 14th Century industrial vinegar production 1650 The production of cultivated mushrooms 1857 Michael Faraday discovered colloidal “ruby” gold, demonstrating that nanostructured gold under certain lighting conditions produces different-colored solutions 1866 Discovery of Mendelian inheritance 1881 Microbial production of lactic acid 1900 Rediscovery of Mendelian inheritance by deVries and Correns 1909 The first identification of inborn errors of metabolism 1928 The discovery of penicillin by Alexander Fleming 1938 The studies with DNA and protein. The usage of the term ‘Molecular Biology’


1941 The hypothesis of one gene-one enzyme

1986 First recombinant vaccine (Hepatitis B)

1943 The first plant reclamation in biotechnology history

1988 the first patent for a genetically altered animal, a mouse that is highly susceptible to breast cancer

1949 The definition of “Molecular disease”: sickle cell anemia by Pauling 1953 Cortisone is the first product which produced on a large scale 1953 Discovery of the DNA structure by Watson and Crick 1956 Determination of human chromosome number (46) by Tjio and Levan 1956 Identification of the first mutation in a single gene disease: sickle cell anemia by Ingram 1959 Discovery of trisomy 21 by Lejeune 1953–1976 The studies with DNA 1974 Tokyo Science University Professor Norio Taniguchi coined the term nanotechnology to describe precision machining of materials to within atomic-scale dimensional tolerances

1989 The gene identification of Cystic fibrosis on chromosome number 7 with linkage analysis 1990 The first successful gene therapy takes place, on a four-year-old girl with an immune-system disorder called ADA deficiency, The human genome project is formally launched 1990 The first BRCA1 gene is discovered. 1991 Sumio Iijima of NEC is credited with discovering the carbon nanotube (CNT), although there were early observations of tubular carbon structures by others as well. Iijima shared the Kavli Prize in Nanoscience in 2008 for this advance and other advances in the field 1991 Mary-Claire King found evidence that a gene on chromosome 17 which causes the inherited form of breast cancer and also increases the risk of ovarian cancer

1977 DNA sequencing method by Sanger

1991 The first transgenic sheep is born

1977 Somatostatin- a human growth hormone-releasing inhibitory factor, the first human protein manufactured in bacteria. A synthetic, recombinant gene was used to clone a protein for the first time

1992 The first liver xenotransplant from one type of animal to another is carried out successfully

1978 The first application of molecular diagnosis: sickle cell anemia by Kan and Dozy 1978 Hutchinson and Edgell show that it is possible to introduce specific mutations at specific sites in the DNA molecule 1979 John Baxter reports cloning the gene for human growth hormone 1980 The prokaryote model, E. coli, is used to produce insulin and other medicine, in human form. Researchers successfully introduce a human gene - one that codes for the protein interferon- into a bacterium 1981 Scientists from Ohio University produce the first transgenic animals by transferring genes from other animals into the mice. The first gene-synthesizing machines are developed. Chinese scientists successfully cloned a golden carp fish

1994 The first BRCA2 gene is discovered. 1994 The first genetically modified tomatoes and acceptance by the world food organization 1995 Researchers from Duke University transplanted hearts from genetically altered pigs into baboons, proving that cross-species operations are possible. The whole genome of bacterium Haemophilus influenzae is sequenced 1995 Development of a rapid and accurate diagnosis system of mad cow disease 1996 The discovery of a gene associated with Parkinson’s disease 1996 The usage of biosensors 1997 Cloning of Dolly 1997 The FDA approves Rituxan, the first antibody-based therapy for cancer

1982 Food and Drug Administration approved to market genetically engineered human insulin

1998 The first complete animal genome (C.elegans) is sequenced

1982 Production of genetically engineered recombinant human insulin protein in bacteria

1998 The production of embryonic stem cell 1998 Discovery of RNA interference

1984 The first cloning and sequencing of the entire human immunodeficiency virus (HIV) genome. Alec Jeffreys introduces technique for DNA fingerprinting to identify individuals.

2000 The first cloned pigs are born

1985 Plant growing studies resistant to Insects, bacteria and viruses in soil

2003 The completion of Human Genome Project

1985 The discovery of Polymerase Chain Reaction by Mullis


2001 The first draft of the human genome 2002 The first sequenced crop 2004-2005 Next generation DNA sequencing technologies 2009 Whole exome sequencing studies

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traditions, in some cases turned into a desire to maintain hygiene. In particular hand washing was common for centuries, even among those people who generally cared little about cleanliness. The importance of hand washing to prevent disease was realized only in 1847 when an Austrian physician, Ignaz Semmelweis (1818-65) found that high levels of illness among pregnant women in hospitals was associated with contamination of the hands of doctors who took delivery. These doctors proceeded to leave, returning from other patients or autopsy and not washing their hands. After he introduced the rule of mandatory thorough hand washing, the mortality rate declined sharply. However, even Semmelweis did not understand what was involved. Only a few decades later, it was found that germs cause many diseases. Today, health care is associated with modern and high-tech medicine. Although in many parts of the world ancient traditions are still used along with modern medicine. In some countries, the ancient techniques have become part of the overall approach to treatment.

3. Short History of Biotechnology Humankind has been using the biotechnology since they discovered farming, with controlling plant growth and crop production. Actually, the cultivation of plants can be counted as the earliest biotechnological application. Another form of biotechnology in farming is animal breeding. Recently, cross-pollination of plants and cross-breeding of animals which are used for enhancing product quality are macro-biological techniques in biotechnology. Today, pioneers of biotechnology are presenting new solutions for better feeding, food and consumer products. They are building on the knowledge we gained through the scientific innovations of earlier pioneers, civilizations such as the Sumerians, Assyrians, Babylonians, Egyptians and people like Christopher Columbus, Louis Pasteur, Gregor Mendel, James Watson and Francis Crick. 8


Review Questions and Answers Q1. Who did use the biotechnology for the first time and when? A1. The oldest biotechnology usages were in Mesopotamia and Babylonia where yeast was used to make beer and the conversion of sugar to alcohol. By 4000 BC, the Egyptians could leaven bread. In 3500 BC in Assyria, wine was produced and milk was converted to lactic acid to make yoghurt and kefir for the first time. Q2. Who was Avicenna? A2. Avicenna was born in 980 in the Middle East, became a famous physician at the age of 18 and authored 276 books of which 43 were in the area of medicine. He described meningitis and gave information about epidemics.

Further Readings and References 1. Verma AS, Agrahari S, Rastogi S, Singh A. Biotechnology in the Realm of History. Journal of Pharmacy & Bioallied Sciences 2011;3:321-3. 2. Dundar M, Emirogullari EF, Biotechnology, Cloning and Ethics. Global Bioethics 2012;27:179182. 3. Scheper T. Advances In Biochemical Engineering Biotechnology. Springer 2000;69. 4. Bademci G, Tekin M.Genetic Basis of Human Disorders and New Molecular Tests. Turkish Journal of Pediatrics 2011;7:7-12. 5. PDF adı eksik http://www.bioentrepreneur.net/ Advance-definition-biotech.pdf 6. Kenneth G, Zysk. Asceticism and Healing in Ancient India: Medicine in the Buddhist Monastery. Oxford University Press 1998. 7. Aksoy S. Tıp Tarihi. Harran Universitesi 2010. 8. http://www.nano.gov/

MOLECULAR TECHNIQUES IN FOOD MICROBIOLOGY

CHAPTER 2 MOLECULAR TECHNIQUES IN FOOD MICROBIOLOGY Marisa Manzano, Lucilla Iacumin

CONTENTS Summary ............................................................................................................................... 11

1. Molecular Biology ........................................................................................................ 11

2. DNA Extraction ............................................................................................................ 11

3. Polymerase Chain Reaction Based Methods .................................................................. 12

4. Quantitative PCR and Reverse Transcription qPCR .................................................... 13

4.1 Applications ................................................................................................................ 14

5. Electrophoresis ............................................................................................................. 15

5.1 Denaturing Gradient Gel Electrophoresis and Temperature Gradient Gel Electrophoresis 15 5.2 Applications ................................................................................................................ 16

6. Restriction Fragment Lenght Polymorphism ................................................................ 16

7. Blotting ......................................................................................................................... 16

7.1 Southern Blotting and Dot Blot .................................................................................. 17

7.2 Blotting Techniques Applied to Food Analysis ............................................................. 18

8. Microarrays ........................................................................................................................ 19

8.1 Applications ..................................................................................................................... 21

Review Questions and Answers .............................................................................................. 21 Further Readings .................................................................................................................... 22 References .............................................................................................................................. 23 MOLECULAR TECHNIQUES IN FOOD MICROBIOLOGY

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10

CHAPTER 2 / M. Manzano, L. Iacumin


Summary

T

he chapter is focused on the description of molecular biology techniques used in various fields including food microbiology; it describes methods used to extract DNA from microorganisms and from food samples; polymerase chain reaction (PCR), Quantitative PCR (qPCR) and reverse transcription (RT) qPCR; the basic concepts of the electrophoresis, and denaturant gel electophoresis with regard to the denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TTGE); utilization and concepts of restriction fragment lenght polymorphism (RFLP); DNA based hybridization methods such as Southern blot, dot blot and microarrays.

1. Molecular Biology The name Molecular Biology was coined by Warren Weaver of the Rockefeller Foundation in 1983. It originated by the convergence of work by geneticists, physicists, and chemists that had a common objective, the understanding of the structure and function of the gene, rather then by a coherent discipline. A lot of studies have been developed on cell, on macromolecules that are at the basis of life, then on how DNA, RNA and protein are interrelated. Molecular biologists began to use the knowledge about the properties and interactions of biology to their topics of investigation. Synthesis and manipulation of macromolecules accelerated scientific progress. Several enzymes working on DNA were discovered, that enabled molecular biologists to cut and paste DNA, leading to recombinant DNA technology and to multiplying DNA. Knowledge about DNA synthesis has been also used to arrange thousands of DNA fragments in an array, and fixed on a chip for experiments. Molecular biology techniques enabled numerous other fields to go molecular. Now Molecular biology is applied also in food microbiology to detect quickly pathogens in food, to study fermentation processes in food, and to study ecology of microorganisms. Depending of the topic, various molecular techniques developed

in the past few years, can be succesfully used both for qualitative and quantitative analyses. Sensitivity and specificity make molecular biology approach advantageous. The increased knowledge in computer science in bioinformatic and computational biology last decade helps the obtainment of the important results shown by molecular biology applications in the field of food microbiology.

2. DNA Extraction One important early step in molecular biology techniques is the DNA extraction from cells. Various methods can be used to break cells, from chemical, enzymatic, physical to mechanical. Most used are sonication, lysozime, temperature shock, and bead beating using glass beads. Utilization of phenol, sodium dodecylsulfate (SDS) and ammonium or sodium acetate are effective for breaking down proteinaceous cellular walls, remove lipid membranes, and precipitate proteins. DNA associated proteins, as well as other cellular proteins, may be degraded with the addition of a protease. Vortexing the sample with phenol-chloroform and centrifuging, the proteins will remain in the organic phase and the DNA will be found at the interface between the two phases. DNA is then precipitated by mixing with cold ethanol or isopropanol (DNA is insoluble in the alcohol) and centrifuging. DNA will precipitate. The resultant DNA pellet will be washed with cold alcohol, dried, and re-suspended in a buffer (Tris-EDTA) before using. Presence and integrity of DNA can be checked by electrophoresing on an agarose gel containing a fluorescent dye (ethidium bromide (EtBr), gelred, SYBR Green, etc.) and visualized under UV light. Similar protocols could be used also for the extraction of DNA from food samples, even if some specific pre-treatment could be used to improve extraction. Quality of DNA is important for the results of various techniques, as eventual presence of contaminants could inhibit hybridization or PCR reactions.


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3. Polymerase Chain Reaction Based Methods PCR has become a standard method in various research fields, from biomedical to environmental due to its extreme sensitivity, which makes possible the detection and analysis of limited amounts of DNA molecules. PCR can be used for the identification of unknown DNA, for cloning DNA, and it can be used both on DNA and complementary DNA (cDNA). PCR has been used for a plethora of applications (Table 1). The most critical parameter is the correct designing of PCR primers, as the correct choice of PCR primers often dictates the success or failure of the PCR technique. Computer programs are useful in the assessment of the basic parameters governing primer design. PCR rely on the unique annealing of the two primers to template with high efficiency and high specificity to obtain unique, specific products as dictated by the selected primers. When the target DNA sequence is known, a couple of primers (forward and reverse) can be designed to allow amplification of the target region, which will be defined at both ends by the primer sequences. This higly specific PCR is often used for the detection of pathogens, and the expected product is a single DNA band of known lenght (Figure 1). Techniques based on PCR became fundamental to study microbial communities last 30 years. Universal primers (annealing a conserved sequence, i.e. 16S rRNA (ribosomal RNA)) are designed to amplify all target sequences of a given population and produce amplicons of similar size that are differentiated by the nucleotide sequence differences eventually present in the PCR products. The conserved sequences, which are sites for the annealing of universal primers, flank variable domains, that allow discrimination over a wide variety of organisms.

PCR

utilization

Amplified Fragment Lenght Polymorphism

based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA

Colony PCR

to quickly screen for plasmid inserts

Alu-PCR

rapid and easy-to-perform “DNA fingerprinting” technique based on the simultaneous analysis of many genomic loci flanked by Alu repetitive elements

Degenerate PCR

unknow exact gene sequence

Long PCR

easy setup of sensitive and efficient long PCR amplifications

Nested PCR

sensitivity improvement specificity

PCR-RFLP

technique for genotyping

Random Amplification of Polymorphic DNA

unknow DNA fragment amplification

Real Time PCR (RT-PCR)

enables the quantification of DNA sequences as the PCR reaction proceeds

Vectorette PCR

employed to efficiently sequence orthologous gene regions

Asymmetric PCR

probe production

Differential Display -PCR

to amplify and display many cDNAs derived from the mRNAs of a given cell

In situ PCR

detection of non-genomic material such as RNA

Multiplex PCR

multiple primer sets within a single PCR mixture to produce amplicons specific to various genes

PCR-ELISA

PCR products hybridized to an immobilized capture probe

PCR-SSCP

used for mutation identification

Repetitive extragenic palindromic PCR (Rep-PCR)

method to classify bacteria on the basis of their genomic fingerprint patterns

RT-PCR

enables reliable detection and measurement of products generated during each cycle of PCR process

Competitive -RT-PCR

quantitation of RNA

TAIL-PCR

recovery of DNA fragments adjacent to known sequences

emulsion PCR (ePCR)

applied to next-generation DNA sequencing

Rapid Amplification of cDNA Ends (RACE)

amplification of nucleic acid sequences from a messenger RNA template between a defined internal site and unknown sequences at either the 3’ or the 5’ -end

Table 1. Various PCR methods.

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4. Quantitative PCR and Reverse Transcription qPCR PCR is hailed as one of the “monumental” scientific techniques of the twentieth century. In fact, its ability to amplify millions-fold a known DNA fragment has opened a wide range of possibilities in all areas of the life science, not leaving unharmed the field of food microbiology. Since its first application, its high-potential was promptly understood and the first attempts to transform the PCR in a quantitative tool date back to the 90s.

Figure 1. Step 1: PCR steps and reagents needed for the reaction, a) reaction buffer; b) dNTPs (dATP, dGTP, dCTP, dTTP); c) forward primer and reverse primer; d) MgCl2; e) DNA polymerase; f ) DNA template. Reagents concentration must be optimized based on the amplification protocol. Step 2: Agarose gel electrophoresis, in the well number 1, molecular weight marker; in the well number 2, amplicon.

When the target DNA sequence is unknown it is possible to amplify randomly DNA sequences of the genome using a single primer which will anneal as forward and reverse primer. When the target DNA sequence is partially known it is possible to amplify unknown regions at 5’ or 3’ ends of the known sequences (rep-PCR, RACE). PCR technique is used for sequencing of short DNA regions after amplification product purification. PCR is also used to measure gene expression (mRNA quantification) using RT-PCR, which developments competitor-PCR and real time quantitative PCR make this technique a quantitative technique.

The approaches primarily used were based on the quantification of the amplicons obtained by enzyme linked immune-sorbent assay and electrochemiluminescence. However, these approaches didn’t take into consideration an important aspect of PCR: amplification, after many cycles of effective exponential amplification, always reaches a yield plateau. During these last amplification cycles, the product is no longer being doubled per each cycle, due to the shortage of reagents and the accumulation of end products, which produce inhibition. The consequence is that, independently from the initial amount of DNA target, the final amount of amplicons is similar. This limitation has been overcome only in 1992, when Higuchi et al. had the brilliant idea of using a fluorescent dye to monitor the fluorescence emitted by the amplicons produced per each amplification cycle. Thanks to this intuition, as well as to the introduction of novel reagents, robust detection chemistries and instrumentation platforms (combining thermal cycler/fluorimeters), it was possible to exceed the misleading end-point quantification methods and upgrade the PCR to Quantitative PCR (qPCR) method, sometimes also called Real-Time PCR. The developed technique allowed quantifying microbial populations through measurement of gene numbers. Moreover, combined with RT, qPCR can also be used to quantify transcript amounts, providing data on microbial activity. Since the first paper using real-time monitoring of PCR amplification by the means of fluorescence


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probes in food microbiology was published, the interest in the application of this method has followed the same exponential trend, proved by the loads of research papers published since 1995, and has become the method of choice to quantify genes and gene expression, using qPCR and RTqPCR, respectively. The universal success is due to its speed, convenience, simplicity, sensitivity, specificity, robustness, high throughput, quantification, familiarity, and cost. The literature is reach of reviews regarding qPCR and RT-qPCR, as well as specific books that extensively explained the theoretical aspects of these reactions. Simply, thanks to the use of a fluorescent reporter, the exponential increase of amplicons, cycle by cycle, can be monitored in “real time”. The increase in fluorescence is plotted against the cycle number to generate the amplification curve, from which a cycle threshold (Ct) value can be determined, and corresponds to the point where the instrument first detect fluorescence above the background noise. This is the crucial conceptual innovation and the key for quantification. On this basis, the Ct can be linked to the initial concentration of target nucleic acid, following the different methods for quantification: absolute quantification and relative quantification. In the first case, quantification is based on a standard curve generated from amplification of known amounts of the target gene. It is used for the majority of quantification methods used in food microbiology to detect and quantify pathogens (but also useful microorganisms) in food. On the other hand, relative quantification is normally used to estimate changes in gene expression. The results are expressed as a target/reference gene ratio, and no standard curve is needed, but only a housekeeping gene to be used as a reference. The choice of the correct housekeeping gene is under debate, as well as the choice among using the total RNA, 16S rRNA or other “stable” expressed genes. Each one of these has its own pro and cons, but the use of at least three reference genes seem to be the best choice. 14


Other than the quantification method, there are aspects that have to be taken into consideration, such as: quality of nucleic acids extracts, detection chemistries, experimental variations of RT-qPCR, controls and normalization, and mode of expression of RT-qPCR data.

4.1 Applications As far as the quantification of microorganisms is concerned, in the last six years qPCR and RTqPCR methods were exponentially developed for the detection of pathogens, but also spoilers and useful microorganisms. The detection limit of these developed protocols is often the crucial point, because for some important food related pathogens (e.g. Listeria monocytogenes, Salmonella spp.) enrichment step is yet needed, excluding the opportunity to quantify the exact number of cells in the samples. On the other hand, it is possible to detect and quantify also microorganism in a viable but not culturable state, otherwise not detectable using culture dependent methods. Postollec et al. (2011) and Cocolin and Rantsiou (2012) follow out the existing literature creating exhaustive resuming tables of the published papers. Discrimination between viable and dead cells in a sample is possible only using RNA as a target instead of DNA. In this case, a RT step is essential, and crucial to remember that severe and numerous complications remain associated with RT. For example, the resolving power of qPCR is limited by the efficiency of RNA to cDNA conversion, which depends on the enzyme used. However, the conversion efficiency is significantly (more than 3-fold) lower when target templates are rare, and it is negatively affected by nonspecific or background RNA in the RT reaction. One of the last developments in this technique are the use the DNA-intercalating agents, such as propidium monoazide used in conjunction with qPCR to selectively detect live cells of pathogenic, spoilage bacteria, and useful bacteria. This compound selectively penetrates the membranes of dead cells and forms stable DNA mono adducts upon pho-


tolysis, resulting in DNA that cannot be amplified by PCR. In this manner, discrimination between live and dead cells is possible while avoiding the RT step and all of its complications. Finally, in the future the use of emulsion-PCR or droplet-PCR, combined with qPCR, could be useful to avoid the pre-enrichment and enrichment steps to detect microorganism at low concentration, but the study of up-to-date chemistries is needed. Regarding gene expression, few papers were published compared to the high number referring to microbial quantification. The first dates back to the 2001, studying the response of Staphylococcus epidermidis to various stresses. Other studies stressed out the stress response in Oenococcus oeni, but the major number of articles focuses on lactic acid bacteria. On the contest of the new trends in the study of metagenomics and metabolomics, but also risk assessment, qPCR and RT-qPCR are going to continue to be techniques of choice still for many years.

5. Electrophoresis Electrophoresis is a technique based on the the mobility of ions in an electric field. Positively charged ions migrate towards a negative electrode whereas negatively charged ions migrate toward a positive electrode. Positive and negative ions can be separated as their migration rates depend on their total charge, size, and shape. Thus electrophoresis is used to identify, quantify, and purify nucleic acid fragments. DNA samples are loaded into wells of the gel (usually agarose or acrylamide) and subjected to an electric field. Due to its negative charge DNA molecules migrate toward the anode (positive electrode) with a mobility that is inversely proportional to the log10 of the molecular weight. As the smallest fragments move the most quickly, they will migrate the farthest during the time the current is on, resulting in separation based on molecular size. Agarose gels are the most frequently used gels as they are nontoxic, easy to use, and offer a wide

separation range. Polyacrylamide (a cross-linked polymer) gels provide higher resolution of DNA molecules in smaller size range, moreover DNA molecules differing by a single base pair (bp) can be resolved under specific conditions. Various sophisticated techniques such as capillary electrophoresis, pulsed-field electrophoresis are also used for specific aims. DNA visualization is obtained by the addition of Ethidium bromide (an intercalating agent between the base pairs) to running buffer during the separation of DNA fragments by agarose gel electrophoresis. After the exposition of the gel to UV light the EtBr emits fluorescence allowing the visualization of DNA bands. Electrophoresis can be used after a molecular technique such as PCR, Restriction Fragment Lenght polymorphism to obtain visualization, identification and differentiation of target genes. Some examples of such electrophoresis are Denaturing Gradient Gel Electrophoresis (and the close Temporal Temperature Gradient Gel Electrophoreis), Southern blotting, associated to RFLP.

5.1 Denaturing Gradient Gel Electrophoresis and Temperature Gradient Gel Electrophoresis As described firstly by Muyzer et al. in 1993 and subsequently by Muyzer and Smalla in 1998, both DGGE and TGGE are useful tool to evaluate microbial diversity. These techniques base upon a denaturant polyacrylamide gel electrophoresis of DNA samples. The high resolution of the polyacrylamide gels allow the differentiation of DNA fragments based on the sequence: DNA fragments of similar length but with different sequences can be separated according to their melting properties that are due to their sequence. DNA fragments will change their migration mobility according to the denaturant conditions encountered during the run. DGGE uses chemical denaturation (formamide and urea), whereas TGGE uses a temperature gradient, which increases during the electrophoresis. The result will be the formation of branched molecules (usually PCR-amplified DNA fragments of


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200–700 bp) obtained by the addition of a GC clamp (30–40 bp) to one of the primers. The clamp holds together the strands partially denatured, leading to a slow molecule migration in comparison to the double stranded DNA molecule. The melting process proceeds through the gel depending on the melting domain of each molecule. Single point mutations present in the molecule can be detected by this method when the conditions are optimazed. Juste´ et al. 2008 reported some applications of DGGE/TTGE techniques. Various conserved regions of the 16S rRNA gene, mRNA and gyrB gene have been used for microbial differentiation. A drawback associated with DGGE/TTGE applied to community studies is the possible loss of bands representing less abundant community members.

6. Restriction Fragment Lenght Polymorphism RFLP analysis is a technique for genetic analysis that bases on the utilization of endonucleases to cut genomic DNA or PCR products. The number and size of the fragments can allow the comparison of different genomic samples (Figure 2). Point mutations can be detected using a simple technique which also involve gel electrophoresis for the visualization of DNA. RFLP is used to fragment genomic DNAs that can be subsequently loaded into a polyacrylmide gel and transfer to a membrane. RFLP allows he identification of point mutation in DNA sequences.

5.2 Applications After the first application of DGGE in food microbiology by Ampe et al. in 1999 (on the spatial distribution of microorganisms in pozol balls, a Mexican fermented maize dough) several food or associated products have been analyzed with DGGE including mineral water, wine, dairy products sourdough and fermenting cassava dough. TTGE has been used for the evaluation of biodiveristy between Bacillus cereus and Bacillus thuringiensis strains isolated from different sources (foods, pesticide and hmans) by Manzano et al. (2009). Strains from the same origin are present in specific levels. Iacumin et al. (2009) applied culture-dependent and independent methods at both the DNA and RNA levels to study the ecology of sourdoughs produced in different plants in the north of Italy. LAB (Lactic Acid Bacteria) and yeast strains were isolated and identified through molecular methods. The total DNA and RNA were directly extracted from the sourdoughs and PCR-DGGE was performed using universal primers for bacteria and yeast allowing the understanding of their ecology. Bester et al. (2010) results indicate that PCR-DGGE can be used for the detection of the wine spoilage microorganims.

16


Figure 2. Example of various DNA bands obtained by the utilization of different endonucleases onto a genomic DNA.

7. Blotting Blotting is a process used to transfer biomolecules (e.g. nucleic acids or proteins) onto a membrane substrate. Membranes are made from highly adsorptive polymers, an ideal substrate for the binding and retention of biomolecules, and easier to handle and manipulate. Different types of membranes, nitrocellulose membranes (electrostatic), and nylon membranes are


mainly used for DNA or RNA probes, although nylon membranes have improved the performance and capabilities of hybridization-based assays. The nucleic acid can be blotted onto the membrane using different techniques mainly two systems are most widely used: the dot blot and the Southern blot. Dot blot bases on the direct blotting of colonies (bacteria) or plaques (viruses) grown on agar plates and transferred by laying the membrane on the plate. The nucleic acid, i.e. the whole genomic DNA are extracted from the colony, whereas Southern blotting requires a previous electrophoresis step to separate the DNA fragments obtained by an endonuclease restriction reaction of the genomic DNA. Agarose gels or polyacrylamide gels can be placed onto the membrane for the trasfer of the nucleic acids. Nylon membranes have a greater mechanical strength, a higher binding capacity, a stronger retention of bound nucleic acids than nitrocellulose membranes. Visualization and identification of the target DNA/ RNA can be achieved using specific short sequences of DNA called DNA probes that are labelled using radioactive or non radioactive molecules. The probe sequence is complementary to the target DNA sequence to which it will bind. This method bases on a fundamental tool in molecular genetics, nucleic acid hybridization capability. Individual single-stranded nucleic acid molecules with sufficiently high degree of base complementarity, anneal each other to form double-stranded molecules. The presence of the target DNA sequence can be detected using the specific labelled probe. Various kind of labelling can be done, one is the end-labeling, that consists in the addition of a labeled group to one end of the probe (5’-end or 3’-end). Hybridization of the probe to the target is possible after denaturation of the double strand DNA (at 95°C for few min), usually obtained by heating of the sample at the melting temperature. Reassociation is obtained by decreasing the temperature to room temperature. The utilization of radioactive phosphorus or sulphur, the utilization of biotin or

fluorophore allows the detection of the target sequence annealed to the labelled probe, by the development of radioactivity, chemiliminescence or fluorescence. Southern blot and dot blot are techniques widely used for this purpose.

7.1 Southern Blotting and Dot Blot Southern Blotting is one of the central techniques in molecular biology. This technique was named after Edward M. Southern developed this procedure at Edinburgh University in 1975. Southern blotting combines transfer of electrophoresis separated DNA fragments to a filter membrane (usually nitrocellulose or nylon) (Figure 3) and subsequent fragment detection by probe hybridization. This technique bases on DNA hybridization, allows the identification and the isolation of specific DNA sequences, including specific genes. Principal steps include: DNA (genomic or other source) digestion with a Restriction Endonuclease DNA fragments separation by gel electrophoresis (agarose or polyacrylamide); DNA denaturation into single strands by incubation with NaOH; DNA transfer to a membrane; incubation of the membrane containing all the DNA fragments with many copies of a probe (which is single-stranded DNA, or RNA up to 100 nt), usually tagged with radioactivity or an enzyme so that the probe can be detected. The probe sequence is complementary to the target sequence, thus when a probe base pairs to its target, the investigator can detect this binding and know where the target sequence is. The amount of DNA needed for this technique depends on the size and specific activity of the probe. Short probes tend to be more specific. It is possible to detect 0.1 picogram (pg) of the DNA. Southern blotting can be used to detect the presence of a gene into an entire genome. Various protocols can be set up to optimize Southern blotting, depending on DNA used, fragment separation method, transfer and detection systems utilized. Southern blotting has been used in RFLP, to detect


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DNA sequence variation between individuals of a species. Various applications of Southern blotting (detection of gene mutations, identification of a specific DNA sequence, identification of a single gene among thousands of fragments of DNA, DNA fingerprinting, cloning) made this technique useful in molecular biology field, especially for the detection of unculturable or not yet culturable microorganisms both in foods and in humans and/or animals.

extraction methods allow the utilization of DNA samples extracted directly from foods in molecular biology techniques. As described by Cecchini et al. (2012) Southern blot can be used for the detection of not yet culturable or nonculturable microorganisms. The DNA sample was obtained by the amplification of a mixed DNA extracted from the gut of a fish. The utilization of universal primers allowed the amplification of the target organism (Candidatus arthromitus) responsible for the gastroenteritis of trout. The amplicons were subjected to DGGE and the gel was blotted for Southern blotting assay to obtain the identification of the pathogen. A positive reaction is developed by the utilization of the labelled DNA probe which anneals to the target DNA from Candidatus arthromitus.

Figure 3. Principle of DNA transfer in Southern blotting, DNA migration from the gel to the membrane.

Dot blot was used by Rodríguez-Romo et al. (1998) to detect Enterotoxigenic Clostridium perfringens, a spore-forming foodborne pathogen responsible for gastrointestinal disease, in Spices. Samadpour et al. 1990 dscribed the application of DNA probes for SLT-I and -II for detection of SLTEC (Shiga-LikeToxin- Producing Escherichia coli) in food by either colony hybridization or dot blot in overnight enrichment cultures. Wiwat and Boonchaisuk (2009) used colony blot, dot blot and Southern blot to detect a enterotoxic B. cereus in food. Vegetables, cooked rice, spice mixes and cereal powders purchased from local markets were tested by using a 584 bp DNA probe obtained by a PCR reaction. Cecchini et al. (2013) described the application of dot blot to wine samples for the detection the spoiler Brettanomyces bruxellensis, well-known for producing unpleasant aromas caused by compounds such as 4-ethylphenol (4-EP), 4-ethylguaiacol (4-EG) and volatile tetrahydropyridines. A DNA probe labelled with digoxigenin on both 5’ and 3’ end was used to increase the sensitivity. The possibility to use various kind of DNA as targets, i. e. DNA extracted from a pure colony, DNA directly extracted from a food sample, DNA produced by amplification, make it a useful method for microbial identification. The design of a specific DNA probe is the crucial point of the method, the hybridization temperature as well.

Also dot blot is a technique which uses DNA hybridization onto a membrane. Denatured genomic DNA (or DNA from other sources) is spotted onto a membrane (nitrocellulose or nylon) without previous digestion, hybridized with a labelled probe and visualized. Common tags are enymes (horseradish peroxidase and alkaline phosphatase) that catalyze a substrate to produce either light (detected with radiography film) or color (visualized on the membrane) when an appropriate substrate is added to the reaction mixture.

7.2 Blotting Techniques Applied to Food Analysis Important requirements for reliable analysis include: specificity, sensitivity, robustness, repeteability, speed, and low cost. Last decades food microbiology changed the approach to food microorganisms due to the developments obtained by DNA techniques. The presence of unculturable pathogens can affect foods, thus the possibility to detect these microorganisms is an important tool for food safety. The unculturable nature of such organisms, due to the absence of appropriate culture media make them a real risk for human health. Current technologies in DNA 18



8. Microarrays Molecular based methods, such as PCR, reduced the time needed for the detection and identification of pathogens, and increased the information about them, but maintained the number of the analysis limited to a small number of pathogens or genes. DNA microarray technology overcomes this limit as it is a high-throughput technology used for parallel gene expression analysis for thousands of genes in both prokaryotic and eukaryotic cells as enables the analysis of multiple DNA target sequences. In 1997 Lashkari et al., published the complete genome of Saccharomyces cerevisiae by using microarray technology. Microarray can be also used in various fields such as medical analysis, food safety, agricultural, environmental, and industrial microbiology for the analysis of pathogens. One additional application concerns the analysis of microbial comunities; Zhou (2003) proposed the microarray technology for the characterization of microorganisms from natural samples. Microarray is a technique based on the hyridization of a nucleic acid (the sample) to a set of oligonucleotide (probes), which are attached to a solid support, to determine the sequence or to detect variations in a gene sequence or expression developed in the early 1990s. It is formed by an orderly arrangement of thousands of identified sequenced genes printed or synthetised on an impermeable solid support, usually glass, silicon chips or nylon membrane. Glass is an inexpensive support medium, easily available, that has a chemical surface that provides chemical modification to ensure appropriate attachment of the probes. Silanization is one of the most widely modification used, as chemistry of this reaction is well known. We can recognise three components: the inert support, the coating (which is the intermediary substance between the biologic molecule and the support) and the biological probes. The design of the probes is the first step for a successful microarray experiment, thus it takes into account multiple parameters such as the oligonucleotide sequence and its binding capacity in order to

ensure high specificity and sensitivity. DNA/RNA probes should be highly specific for the target DNA and/or cDNA, to enable a specific annealing to target sequence in order to obtain the identification of the specific target. After the selection of the type of probe (oligo or DNA amplicon), the selection of the sequence of the probe, and the selection of the solid substrate for the attachment of the probes, the next step is the modification of the surface to allow the attachmnent of the probe to the substrate. The method of attachment of the probe to the solid substrate is an important parameter to obtain good results. Based on the solid support composition, 5’ or 3’ end of the oligo can be modified by the introduction of a specific group. The amino group is used to bind covalently to glass slides treated with silan or isothiocyanate, a disulphur group to bind covalently to mercaptosilanised glass supports, succinylated probes for aminophenyl or aminopropyl derivatised glasses. Oligo (50-120 bases) (at up to 500.000 oligo/ cm2) or cDNA amplicons (0.6-2.4 kb) (at 10.000 cDNA/cm2 density) can be used as probes (Table 2). Size Density Spacing Macro 2 80 x 120 mm 10-100/cm 1–2 mm Array Micro Array 27 x 18 mm 500-5000/cm2 to 300 µm (Nylon) Micro Array < 300 µm 18 x 18 mm 10000/cm2 (Vetro) Chips (Oligo)

12.8 x 12.8 mm

300.000 oligo

20-30 µm to 30-40 Angstrom

Table 2. Size, density and spacing in the classification of the array.

The density of the oligonucleotide probe bound to the surface is a parameter of an array. A low surface density will lead to a light signal whereas high probe densities may hindrance the binding between the probe and the target DNA strand. Usually a single DNA microarray slide/chip may contain thousands of spots each representing a single gene and collectively the entire genome of an organism, thus experiments using oligonucleotides microrarrays are often referred as single channel hybridization.


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In oligonucleotide microarray each gene is represented by more than one probe, and the different probes serves to map various regions of the gene. In choosing a support and consequently the chemistry for the attachment some characteristics such as the level of scattering and fluorescence background of the support material has to be considered in association with the stability of the construct, and the loading capacity. Glass is a substrate frequently choosen for microarray applications because of its properties: flatness, low fluorescence, chemical inertness, and low cost. A flatness of < 50 μm is required to obtain a good immobilization of the probes by both printing and in situ synthesis. Spots can have a diameter of 80200 μm when produced by printing (by a contact or noncontact print head). This process is repeated thousands of times for microarrays with 100 -10.000 spots per array. Spots can have a diameter < 20 μm when using in situ synthesis, a technique by which the probes are synthesized directly on the coated surface. DNA fragments (usually created by PCR) or oligos are stuck to glass slides. These arrays can be created in individual labs. The two most commercially successful DNA microarray companies use printing (Agilent uses an ink jet printing) and photolitography (Affymetrix uses photochemistry). The probes on these arrays are synthesized using a light mask technology. Photo-sensitive reactions are used to remove a blocking group and then extend. The characteristic of in situ synthesis is the high density obtained reducing the spot size. Grows another base at the end of a molecule by deprotecting the end and attaching a new base with a protected end to avoid duplication. DNA micorarrays allow the analysis of thousands of genes. Each spot contains multiple copies of identical strands of DNA. Thousands of spots are arrayed in orderly rows and columns on a solid support. Each spot has a specific sequence located at a specific position on the array (Figure 4), and can represents one gene. Image detection is obtained by a scanning or direct imaging system. Scanning uses a laser excitation source (when fluorescently-labeled DNA are used) (on each spot) coupled with a detector which 20


measures the light. Direct imaging uses an excitation source that illuminates the whole array, and a camera is used to capture the image (for radioactive or colorimetry labelling). Scanning has higher resolution and sensitivity than direct imaging. Using dyes, the resulting image files require special software to determine the intensity of light at each of the wavelengths measured to be analyzed.

Figure 4. Array organization, probes and final image (*) Image:http://www.har.mrc.ac.uk/services/MPC/microarray/ Ashleigh Thorogood and Krishna Ingram, James Madison University).

DNA microarray enables the quantification of gene expression, thus it is possible to analyse cellular behaviour in various environments, including the interaction between host and microbial pathogens. By following the pattern of gene expression, it is possible to clarify which genes are up or downregulated. cDNA-based microarrays is the method for analyzing the simultaneous expression of up to 15.000. Microarrays have been applied to human cancer research. All the data are recorded in a computer database. The analysis of the data obtained by a microarray experiment requires adequate bioinformatic skill to be able to distinguish specific gene expression values. Different points should be used considered during the analysis step of the data: the


microarray image processing, the determination of true spots, data normalization, and statistical analysis. The discrepancies in microarray results are a consequence of differences in microarray measures, such as accuracy. Quality, sensitivity, specificity and reproducibility are sensitive points in microarrays. Microarrays provide the ability to examine thousands of genes with a single test and overcome the limitations of other culture-independent approaches.

8.1 Applications Oligonucleotide microarrays can be used for gene detection, quantification and expression, and for profiling microbial communities as it is possible to analyse both the microbial diversity and the metabolic capacity of complex communities in one experiment. Wang et al. (2007) used microarrays to detect bacteria from food samples. They applied the microarray technology to isolates from foods and obtained a 97.4% of correct identification. The method based on the utilisation of a previous PCR using as target the 16S rRNA gene, and reached the sensitivity of 102 CFU (Colony Forming Units) bacteria. Feng et al. (2013) used microarry for the detection of toxigenic food-borne toxigenic microorganisms. Specific probes reahed the sensitivity of 7.1 x 102 CFU / ml allowing the detection of toxinogenic microorganisms from foods. Liu et al. (2011) described an application of microarrays to monitor the gene expression of L. monocytogences strain F2365 in Ultra-High Temperature (UHT) processed skim milk. Microarrays have been also used to study probiotic microorganisms in food (Lactobacillis acidophilus in skim milk, Lactobacillus helveticu in milk). Cho and Tiedje (2002) demonstrated the difficulty in obtaining positive results using RNA extracted from environmental samples, in fact they were unable to detect any positive nirS (nitrate reductase) sequences from either sediment or soil samples. DNA hybridization on microarrays can be affected by the presence of humic acids and organic materials in soil, water and sediments. DNA microarray technology can be used to compare between sequenced and unsequenced genomes. Behr

et al. (1999) used comparative hybridization experiments on a DNA microarray to evaluate similarity and differences among attenuated strains of Mycobacterium bovis (used for the Calmette-Guérin (BCG) vaccines) and Mycobacterium tuberculosis. They obtained a good understanding of the differences present in the genomes. Microarray have a great potential. They can be applied to various scenario, from pathogen detection to gene expression and pharmacogenomics. Although the positive results obtained they still need implementation in some critical points such as DNA array instruments, DNA chip production, probes, and bioinformatics are fairly expensive before being optimized.

Review Questions and Answers Q1. Which are the utilizations of polymerase chain reaction (PCR)? A1. Polymerase chain reaction is a technique that have various applications including isolation, microbes detection, mutation detection, sequencing, cloning, microarrays, genotyping, forensics, and paternity testing. Q2. Which is the advantage in using qPCR instead of end point PCR? A2. The advantage is the possibility to quantify the target, allowing pathogen detection, and gene expression. Q3. Which the basic difference between agarose electrophoresis and DGGE/TTGE? A3. The migration of DNA in an agarose gel bases on the lenght of the fragment, whereas the migration in DGGE/TTGE bases on the sequence Q4. What is RFLP? A4. RFLP is a technique utilised for the detection of point mutation in genomes, PCR products, allowing the identification of genetic polymorphism Q5. Describe the bases of dot blot compared with Southern blot. A5. Dot blot bases on the direct blotting of colonies, plaques, genomic DNA whereas Southern blot


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requires a previous RFLP followed by an electrophoresis for the separation of the DNA fragments. Q6. When microarrays can be useful? A6. Microarrays can be used for the evaluation of gene expression as they allow the analysis of thousand of genes on the same glass.

Further Readings 1. Ampe F, Omar NB, Moizan C, Wacher C, Guyot JP. Polyphasic study of the spatial distribution of microorganisms in mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations. Applied and Environmental Microbiology 1999;65:5464-5473.

8. Desfossés-Foucault E, Lapointe G, Roy D. Transcription profiling of interactions between Lactococcus lactis subsp. Cremoris SK11 and Lactobacillus paracasei ATCC 334 during Cheddar cheese simulation. International Journal of Food Microbiology 2014;16;178:76-86. 9. Dewettinck T, Hulsbosch W, Hege K, Top E, Verstraete W. Molecular fingerprinting of bacterial populations in groundwater and bottled mineral water. Applied Microbiology and Biotechnology 2001;57:412- 418. 10. Elizaquìvel P, Sànchez G, Aznar R. Application of propidium monoazide quantitative PCR for selective detection of live Escherichia coli O157:H7 in vegetables after inactivation by essential oils. International Journal of Food Microbiology 2012;159:115-121.

2. Beltramo C, Desroche N, Tourdot-Marechal R, Grandvalet C, Guzzo J. Real-time PCR for characterizing the stress response of Oenococcus oeni in a wine-like medium. Research in Microbiology 2006;157:267- 274.

11. Fuka MM, Viviane Radl V, Matijašic BB, Schloter M. Evaluation of denaturing gradient gel electrophoresis (DGGE) used to describe structure of bacterial communities in Istrian cheese. African Journal of Biotechnology 2012;11:16650-16654.

3. Bester L, Cameron M, Du Toit MD, Witthuhn RC. PCR and DGGE detection limits for wine spoilage microbes. South African Journal for Enology and Viticulture 2010;3:26-33

12. Guo Z, Guilfoyle RA, Thiel AJ, Wang R, Smith LM. Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Research 1994;22:5456-5465.

4. Bustin AS. A-Z of quantitative PCR. Iul Biotechnology Series 2006. 5. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical Chemistry 2009;55:611622. 6. Carvalho AL, Turner DL, Fonseca LL, Solopova A, Catarino T, Kuipers OP, Voit EO, Neves AR, Santos H. Metabolic and transcriptional analysis of acid stress in Lactococcus lactis, with a focus on the kinetics of lactic acid pools. PLoS ONE 2013;8. 7. Curry J, McHale C, Smith MT. Low efficiency of the Moloney murine leukemia virus reverse transcriptase during reverse transcription of rare t(8;21) fusion gene transcripts. Biotechniques 2002;32:755-772. 22


13. Kostić T, Sessitsch A. Microbial Diagnostic Microarrays for the Detection and Typing of Foodand Water-Borne (Bacterial) Pathogens. Microarrays 2012;1:3-24. 14. Mamlouk K, Macé S, Guilbaud M, Jaffrès E, Ferchichi M, Prévost H, Pilet MF, Dousset X. Quantification of viable Brochotrix thermosphacta in cooked shrimp and salmon by real-time PCR. Food Microbiology 2012;30:173-179. 15. Manzano M, Cocolin L, Cantoni C, Comi G. Detection and identification of Listeria monocytogene in food by PCR and oligonucleotide-specific capture plate hybridization. Food Microbiology 1998;15:651-657. 16. McKillip JL, Drake M. Molecular beacon polymerase chain reaction detection of Escherichia coli 0157:H7 in milk. Journal of Food Protection 2000;63:855-859.


17. Miambi E, Guyot JP, Ampe F. Identification, isolation and quantification of representative bacteria from fermented cassava dough using and integrated approach of culture-dependent and cultureindependent methods. International Journal of Food Microbiology 2003;82:111-120. 18. Nogva HK, DrØmtorp SM, Nissen H, Rudi K. Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5’-nuclease PCR. BioTechniques 2003;4:804-813. 19. Parisot N, Denonfoux J, Dugat-Bony E, Peyretaillade E and Peyret P. Software Tools for the Selection of Oligonucleotide Probes for Microarrays, Microarrays: Current Technology, Innovations and Applications. Caister Academic Press 2014. 20. Rasooly A, Herold KE. Food Microbial Pathogen Detection and Analysis Using DNA Microarray Technologies. Foodborne Pathogens and Disease 2008;5:531-550. 21. Rodríguez-Romo LA, Heredia NL, Labbé RG, García-Alvarado JS. Detection of Enterotoxigenic Clostridium perfringens in Spices Used in Mexico by Dot Blotting Using a DNA Probe. Journal of Food Protectection 1998;2:141-256. 22. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985;230:1350-1354. 23. Vandecasteele SJ, Peetermans WE, Merckx R, Van Eldere J. Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. Journal of Bacteriology 2001;183:7094-7101. 24. VanGuilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene expression analysis. BioTechniques 2008;44:619-626. 25. Vendrame M, Iacumin L, Manzano M, Comi G. Use of propidium monoazide for the enumeration of viable Oenococcus oeni in must and wine by quantitative PCR. Food Microbiology 2013;35:49- 57. 26. Vendrame M, Manzano M, Comi G, Bertrand J, Iacumin L. Use of propidium monoazide for the

enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR. Food Micro-biology 2014. 27. Venkitanarayanan KS, Faustman C, Crivello JF, Khan MI, Hoagland TA, Berry B. Rapid estimation of spoilage bacterial load in aerobically stored meat by a quantitative polymerase chain reaction. Applied Microbiology and Biotechnology 1997;82:359-364. 28. Wang XW, Zangh L, Jin LQ, Jin M, Shen ZQ, An S, Chao FH, Li JW. Development and application of an oligonucleotide microarray for the detection of food-borne bacterial pathgens. Applied Microbiology and Biotechnology 2007;76:225-233. 29. Wong ML, Medrano JF. Real-time PCR for mRNA quantitation. BioTechniques 2005;39:75- 85.

References 1. Behr MA, Wilson MA, Gill WP, Salamon H, Schoolnik GK, Rane S, Small PM. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 1999; 28:284:1520-3. 2. Cecchini F, Iacumin L, Fontanot M, Comi G, Manzano M. Identification of the unculturable bacteria Candidatus arthromitus in the intestinal content of trouts using Dot blot and Southern blot techniques. Veterinery Microbiology 2012;156:384-394. 3. Cecchini F, Iacumin L, Fontanot M, Comuzzo P, Comi G, Manzano M. Dot Blot and PCR for Brettanomyces bruxellensis detection in red wine. Food Control 2013;34:40-46. 4. Cho JC, Tiedje JM. Quantitative detection of microbial genes by using DNA microarrays. Applied Microbiology and Biotechnology 2002;68:14251430. 5. Cocolin L, Rantsiou K. Quantitative polymerase chain reaction in food microbiology. In: Quantitative Real Time PCR in applied microbiology. Edited by Filion M, 2012. 6. Feng J, Wang X, Cao G, Hu S, Kuang X, Tang S, You S, Liu L. Establishment and preliminary application of oligonucleotide microarray assay


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for detection of food-borne toxigenic microorganisms. European Food Research and Technology 2013;236:1073-1083. 7. Higuci R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology 1992;10:413417. 8. Iacumin L, Cecchini F, Manzano M, Osualdini M, Boscolo D, Orlic S, Comi G. Description of the Microflora of Sourdoughs by Culture-Dependent and Culture-Independent Methods. Food Microbiology 2009;26:128-135. 9. Juste´ A, Thomma BPHJ, Lievens B. Recent advances in molecular techniques to study microbial communities in food-associated matrices and processes. Food Microbiology 2008;25:745-761. 10. Lashkari DA, DeRisi JL, McCusker JH, Namath AF, Gentile C, Hwang SY, Brown PO, Davis R W. Yeast microarrays for genome wide parallel genetic and gene expression analysis. Proceedings of the National Academy of Sciences 1997;94:13057. 11. Liu Y, Wang Y, Gang J. Challenges of microarray applications for microbial detection and gene expression profiling in food. Journal of Microbial & Biochemical Technology 2011;2:001 12. Manzano M, Giusto C, Iacumin L, Cantoni C, Comi G. Molecular methods to evaluate biodiversity in Bacillus cereus and Bacillus thuringiensis strains from different origins. Food Microbiology 2009;26:259-264.

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13. Muyzer G, Dewaal EC, Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electroforesis (DGGE) and temperature gradient gel electroforese (TGGE) in microbial ecology. Antonıe Van Leeuwenhoek Internatıonal Journal Of General And Molecular Microbiology 1993;73:127-141. 14. Muyzer G, Smalla K. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 1998;73:127-141. 15. Postollec F, Falentin H, Pavan S, Combrisson J, Sohier D. Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food Microbiology 2011;28:848-861. 16. Samadpour M, Liston J, Ongerth JE, Tarr PI. Evaluation of DNA Probes forDetection of ShigaLike-Toxin- Producing Escherichia coli in Food and Calf Fecal Samples. Applied and Environmental Microbiology 1990;56:1212-1215. 17. Wiwat C and Boonchaisuk R. Development of a DNA-Probe for Detection of Enterotoxic Bacillus cereus Isolated from Foods in Thailand. Mahidol University science of Pharmacology 2009. 18. Zhou J. Microarrays for bacterial detection and microbial community analysis. Current Opinion in Micro-biology 2003;6:288–294.

FOOD BIOTECHNOLOGY: DEVELOPMENTS AND PERSPECTIVES

CHAPTER 3 FOOD BIOTECHNOLOGY: DEVELOPMENTS AND PERSPECTIVES Daniel Ramón

CONTENTS Summary ................................................................................................................................. 27

1. The Journey from Traditional Agriculture to Genetic Engineering ................................. 27

2. Transgenic Food and Crops ............................................................................................ 28

3. How are Transgenic Organisms Tested? .......................................................................... 30

4. Economic Impact of GMOs ............................................................................................ 31

5. What the Future Holds in Store: Genomics and Food Production .................................. 32

Review Questions and Answers ................................................................................................ 33 Further Readings ..................................................................................................................... 34 FOOD BIOTECHNOLOGY: DEVELOPMENTS AND PERSPECTIVES

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26

CHAPTER 3 / D. Ramón


Summary

F

or thousands of years man has been applying genetics to achieve the improvement of raw materials and final food products. Using selective breeding and/or mutagenesis, a large number of plant varieties, animal races and microbial starters has been produced. In fact, food biotechnology is the oldest biotechnology. Recently, recombinant DNA techniques have been applied in food technology creating the so-called ‘genetically modified foods’ (GM foods), a class of novel foods. Transgenic potatoes useful as an oral vaccine against cholera, recombinant wine yeasts able to produce wine of increased fruity aroma and transgenic cows or ewes producing milk with high levels of pharmaceutical proteins are some of the results of the new food biotechnology. In any case, the starting date for the future of food biotechnology was the publication in 2001 of the human genome sequence, shedding light on which genes are activated or deactivated in response to specific nutrient intake. It is also possible nowadays to determine the genetic differences between individuals that lead to different nutritional responses. Furthermore, each new day brings freshly sequenced genomes of animals, plants or microorganisms of common components of our diet like rice, bread yeast, the probiotic bacterium Bifidobacterium bifidum or pathogens responsible for food poisoning, like Escherichia coli. This provides information about key genes, making it possible to delineate classical and genetic engineering improvement strategies alike, demarcate defence mechanisms to combat pathogenicity or define new physiological functions. Application to food and nutrition is more advanced than many people imagine.

1. The Journey from Traditional Agriculture to Genetic Engineering Contrary to popular belief, tinkering with genetics in food and nutrition is nothing new. As Charles Darwin pointed out in his book “On the origin of FOOD BIOTECHNOLOGY: DEVELOPMENTS AND PERSPECTIVES

species”, since the dawn of agriculture and livestock farming, some 12.000 years ago, mankind has been improving farm-animal breeds and edible plant varieties, implementing genetic techniques. Indeed, humans began domesticating these organisms and ended up improving them through genetic selection. To do so, several techniques have been used, of which the most common include hybridization, known as “sexual crossing”, and the emergence of spontaneous mutants also called “natural genetic variability”. In the first hybridization, two parental organisms, each possessing an agriculturally relevant trait, are crossed with the expectation that the resulting hybrid will inherit the positive traits of both its parents. For example, a variety with good organoleptic properties but low productivity can be crossed with another variety that is highly productive in the field but deficient in aroma and taste. Thus breeders seek high productivity and a good organoleptic profile in the hybrid offspring. However, each parent harbours a genome with tens of thousands of genes, so what actually occurs at the molecular level in these crosses is the random mix of thousands of genes from each parent; therefore, the likelihood of giving rise to a combination of the right genes is extremely low. Notwithstanding, breeders can then select the best hybrid from among the offspring. Although it may sound complicated, this technique has been very successful. In fact, a high percentage of plant varieties and animal breeds we currently consume in our diet are products of these crossing and selection processes. For instance, this is how we have obtained the wheat varieties used to produce baking flour today. The genomes of these varieties hold a real chromosome puzzle, and can have up to six pairs of each chromosome whereas ancient varieties grown in Southeast Asia some 8.000 years ago had just two. In point of fact, wheat is a true Stone Age genetically modified organism (GMO). Egg-laying hens provide another example of improvement by sexual crossing. In the 1950s the most productive poultry breeds laid seventy eggs a year, but by applying sexual crossing techniques there are now breeds laying three hundred eggs a year. In fact, all data published over the last four years on genome sequencing would indicate that the CHAPTER 3

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majority of plant varieties and to a lesser extent animal breeds to be found in our diet are palaeolithic GMOs having undergone a long history of genetic modification empirically wrought by man. In the second of the two aforementioned techniques, novel mutant individuals are selected, which have randomly lost or undergone a modification in one or some of the tens of thousands of genes in their genome and –by chance– give rise to a new, much more effective, combination from the agrifood viewpoint. The cabbage is a good example. These vegetables did not exist five thousand years ago. They are the result of a mutation in a gene controlling flower-bud development in the genome of an extinct evolutionary ancestor. Other mutations in genes controlling the development of terminal buds, lateral buds or flowers and stems explain the appearance of cabbages, Brussels sprouts or broccoli, respectively. Sometimes, these mutants have been produced artificially by forced mutagenesis. This is the case of Star Ruby, the pink grapefruit variety, obtained by subjecting a variety of conventional grapefruit to X-ray irradiation. Despite certain success, all the genetic techniques mentioned so far have two important limitations: lack of precision and inability to jump the species barrier. With respect to the former, it is impossible to selectively group the desired genes from each parent in the hybrid offspring. Likewise, the selective mutation of a single gene in a given genome is impossible. As to the species barrier, a carrot cannot be mutated to obtain a new variety with the resveratrol content of grapes, nor is it possible to sexually cross these two plant species. Just over thirty years ago, some American researchers working in the field of basic biology discovered how to make hybrid (or recombinant) molecules with DNA taken from two different organisms. These techniques, generally known as genetic engineering, make it possible to control genetic improvement by selecting the genome fragment containing the desired gene, and the species barrier can also be crossed. Fundamentally, it involves taking the gene of choice from the genome of a donor organism and inserting it into the genome of a host, giving rise to a GMO. Need28


less to say, these techniques can be used in agriculture and food production and provide so-called transgenic food or crops. It is noteworthy that there are three significant differences between conventional genetic techniques and genetic engineering. With genetic engineering, one can control the genetic modification introduced, it is also quick and efficient and, as previously mentioned, the species barrier can be crossed. The last point may be of relevance to certain consumer groups, especially if the transferred genes are subject to ethical reservation. For example, genes taken from an animal genome and expressed in a plant genome, or genes from the genomes of animals whose consumption is forbidden by certain religions or ethnic groups.

2. Transgenic Food and Crops A transgenic food is easy to define. In the European Union, people are wary of the sale of transgenic food for reasons that have more to do with an ideological standpoint than with scientific debate (see below). By contrast, in other parts of the world, consumption of GMOs increases year after year. In fact, no other new technology has been adopted at the same speed in the history of food and agriculture, and one only has to look at the rise in the global extension of transgenic crops to realise this. According to the data in the International Service for the Acquisition of Agri-biotech Applications (http://www.isaaa.org/) 175.2 million hectares (ha) of transgenic plants were being grown on the planet in 2013, representing 10% of the world’s acreage. In total, 27 countries grew biotech crops. It is noteworthy in the same period 90% of the 18 million farmers growing transgenic crops live in poor countries, and they planted more than 50% of the global area corresponding to transgenic crops. On a global scale, since 1996 when the first transgenic crops were mass planted, the surface area planted with these crops has increased 95 fold, and more than 100 million positive decisions have been taken to reuse transgenic seeds. All these data should call reflection by those who consistently oppose this technology.


The first GM foods to be sold were edible transgenic plants with resistance to pests or herbicide treatments. They are known as “first-generation GMOs” and include almost all those currently marketed. They were the first to be developed because their transformation involves a single gene and they are, therefore, relatively simple to produce; furthermore, they provide unquestionable commercial benefit to farmers, guaranteeing harvests. Most herbicideresistant transgenic plants are resistant to the weedkiller glyphosate (Figure 1). This compound inhibits the phosphoenolpyruvate condensing enzyme thereby suppressing a key step in aromatic amino acid synthesis. Two strategies have been followed to construct glyphosate-resistant transgenic plants. In the first, the gene dosage of the target gene is increased, unbalancing the relative amount of herbicide to the number of targets. In the second, mutations in the gene encoding the condensing enzyme have been made, so that the active binding site to the herbicide is modified and the enzyme is not inhibited. The use of these transgenic crops is combined with direct seeding, a farming practice in which the seed is sprinkled on an unploughed field and treated with herbicide immediately. In Argentina, yields of more than 6 tons of beans/ha have been obtained using this dual technology, with a significant reduction in energy consumption and erosion, coupled with an increase in biodiversity. In the 1994-95 season, the last season without genetically modified soybeans, Argentinean farmers spent an average of $78/ha on herbicides whereas nowadays they spend $37/ ha. Moreover, there has been a 90% drop in overall consumption of pesticides. With respect to pestresistant plants, edible varieties have been produced with resistance to viroids, viruses, bacteria, fungi or insects. The best-known example is the Bt insecticidal protein from the bacterium Bacillus thuringiensis expressed in different plants, including cotton, corn, and bestowing resistance to pest attacks. These are known as “Bt crops” and their productivity is higher than that of conventional crops in the field in the event of pest outbreaks. Besides, cultivation of Bt crops has led to a drastic reduction in insecticide use, as demonstrated by the fact that the use of Bt cotton in India has reduced insecticide use by 50%. FOOD BIOTECHNOLOGY: DEVELOPMENTS AND PERSPECTIVES

In the European Union (EU), the sale of transgenic glyphosate-resistant soybeans and transgenic Bt corn is authorized. However, neither of these crops is used directly for human consumption, but rather as the basis to prepare animal feed or to obtain starch or glucose syrup from corn and lecithin or phytosterols from soybean. These ingredients are used to formulate thousands of food products, which must state their GM source on the label.

Figure 1. Construction of glyphosate-resistant soybean. Glyphosate is a competitive inhibitor of the enzyme involved in the synthesis of the aromatic amino acids tyrosine, tryptophan and phenylalanine from phospho-enol pyruvate (PEP) and sikimate-3-phosphate. The top of the figure shows a scheme of this reaction including the molecular structure of glyphosate and the three aromatic amino acids. At the bottom of the slide, photographs of petunia, soil bacteria and soybean (from left to right) are shown. Construction of transgenic soybean varieties were obtained from donor genes isolated from mutant strains/varieties of petunia and soil bacteria. This mutated versions of the enzyme are not inhibited by glyphosate.

There are other kinds of GM crops and food, the so-called second generation. In these genetic modifications are introduced that affect the physicochemical, organoleptic or nutritional properties. Such GMOs include edible plants, farm animals and microorganisms used to ferment food. Naturally, such modifications imply higher technological complexity as they tend to involve multiple genes thus they have been achieved later. In some of these certain physicochemical properties are altered like the rotting process. For instance, transgenic tomatoes have been obtained by reducing the expression of the gene encoding the polygalacturonase enzyme CHAPTER 3

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diminishing its activity by up to 80% and considerably delaying the onset of fruit rots. In other cases, organoleptic properties are improved, as in the case of transgenic wine yeasts, constructed to impart a fruitier aroma. Improved vitamin content

Increased b-carotene in rice and tomato Increased vitamin A content in corn Increase carotenoid content in rapeseed Increased folic acid in rice, lettuce, tomatoes Folic acid overproduction by lactic acid bacteria Increased vitamin C in lettuce Increased a-tocopherol and tocotrienols in corn Improved protein Increased lysine in rice, corn, content potato, beet and soybean Methionine increase in rice, sunflower and corn Overall increase in amino acid content in rice and potato Modified fat and oil Reduced linolenic acid and content increased oleic acid in soybean Increased stearic acid in cotton and rapeseed Increased arachidonic and eicosapentaenoic acid in mustard Increased iron in rice, peas and Mineral biofortification corn Increase in flavonoids

Increased genistein in alfalfa Increased flavonoids in tomato Increased anthocyanins and flavonoids in rice Resveratrol production in potatoes Resveratrol overproduction in wine yeasts

Other developments of Linamarin reduction in cassava nutritional interest Fructans increase in beet Design of anti-allergenic plants and microorganisms Design of oral vaccines

Table 1. GMOs with enhanced nutritional properties.

30


However, the use of genetic engineering is most appealing when implemented to tackle problems related to nutritionally deficient foods. There are already many GM foods with improved nutritional composition (Table 1). A couple of these are worth mentioning. The first example is the so-called golden rice, a transgenic rice variety in which three genes have been inserted to make it produce and accumulate b-carotene. Its use, scheduled for 2014, will alleviate the chronic problem of vitamin A deficiency in poor countries in Southeast Asia and Latin America where rice is the staple diet. According to the World Health Organization (WHO), this nutritional problem is responsible for the death of 2 million children each year, and condemns another 250.000 children to blindness yearly. The second example is a transgenic tomato which expresses two genes from the plant Antirrhimum majus, encoding two transcription factors. The result is a tomato that accumulates anthocyanins at levels comparable to those found in blueberries or blackberries. These tomatoes have a purple hue and have been used in preclinical trials using Trp53 (-/-) mutant mice, prone to developing cancer. Results show that the group of mice fed with these transgenic tomatoes remained tumour-free, whereas the group of mutant mice fed with conventional tomatoes developed tumours.

3. How are Transgenic Organisms Tested? As previously stated, in the EU, there is ongoing controversy over the commercialization of GM foods. This is an ideological debate, which is highly politicized and lacking in technical data. It is sufficient to recall that in the EU, foods derived from classical biotechnology techniques (including mutation with mutagenic agents) are not subject to health assessment regulation. By contrast, GM foods require mandatory pre-market assessment, which is done following the WHO and Food and Agriculture Organization (FAO) guidelines. These organizations have established their own working groups on consumer safety of novel GM foods over


the years, giving priority to scientific principles guiding such assessment. These guidelines involve evaluating nutritional content, the possible presence of allergens and toxicity levels. As for assessment of the nutritional composition, the substantial equivalence approach is adhered to by European regulations on GM food marketing. The substantial equivalence category is awarded to those novel foods whose nutritional composition and organoleptic characteristics are the same as those of the conventional food from which they were developed save the new characteristic introduced by genetic engineering. All GM foods marketed to date meet this requirement. To assess allergenicity guidelines follow the criteria set by the FAO, WHO and the Codex ad hoc Intergovernmental Task Force on Foods Derived from Biotechnology. This means that for each transgenic food analyses are run on the homology and structural similarity between the transgenic protein and all known allergens. Moreover, tests check for epitopes which, due to their amino acid sequence, may interact with immunoglobulin E or T-cell epitopes or significant structural motifs. In some cases, assessment also includes a study of transgenic protein digestibility in simulated gastric and intestinal fluid systems, as well as occupational exposure studies. All these trials are particularly relevant if the gene donor is known to have allergenicity. Finally, if substantial equivalence is found then toxicological studies focus on the transgenic protein itself. This requires the assessment of information on carcinogenicity, genotoxicity, metabolism, subchronic and chronic toxicity and toxicokinetics. In the event substantial equivalence is not found or there are indications of potential unintended effects then the whole food must be studied in depth. In these cases, ninety-day toxicity studies are conducted in rodents administered the maximum doses that do not cause nutritional imbalances. All GM foods marketed to date have undergone all these trials demonstrating there is not a shred of scientific evidence that these foods, albeit GM, pose a risk to consumer health greater than the risk posed by ingesting the corresponding conventional food. Indeed, this view is advocated FOOD BIOTECHNOLOGY: DEVELOPMENTS AND PERSPECTIVES

by the WHO itself (http://www.who.int/fsf/GMfood/). So all this goes to show that throughout the history of food, GMOs are the most thoroughly evaluated foods ever, and there is no scientific evidence whatsoever indicating they pose an unacceptable hazard to consumer health. The issue of assessing the environmental impact of transgenic crops is somewhat more complex, as there is a lack of knowledge and a need for methodologies to analyze the environmental hazards posed by transgenic and conventional plants alike. Even so, we must remember that hundreds of transgenic plants must be evaluated in the greenhouse before they can be planted in the field. This process is called controlled environmental release and plants must be tested in different ecosystems and during different seasons before permission to grow them is granted. These experiments have shown that there are no new risks associated with the cultivation of transgenic plants and they are, in fact, the same as with conventional plants. Such risks involve the potential transfer of foreign genes from transgenic to wild varieties, a decline in the biodiversity of the surrounding area and, in the case of pest-resistant plants, higher incidence of them attacking non-target organisms. So the key question that needs answering is whether growing transgenic plants could speed up the incidence of these hazards. Clearly the answer is no, as long as the standards of evaluation that are currently employed to assess transgenic plants are maintained and/or improved.

4. Economic Impact of GMOs Now let us look at the potential economic risks and benefits, an issue that varies depending on which part of the planet we look at. For example, in the Republic of China there has been a firm commitment to transgenic crops since the eighties, to the extent that the Chinese government’s National Biotechnology Program has funded projects to develop over 130 transgenic varieties and 100 specific genes. Meanwhile, the Indian government has funded 48 projects involving transgenic plants belonging to 15 different crops. Important transgenic CHAPTER 3

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plants have been obtained such as transgenic rice varieties resistant to drought and salinity, markerfree transgenic rice with provitamin A, potatoes and rice with higher protein content or Bt-transgenic potatoes. Looking at Latin America, we see that 98% of soybeans grown in Argentina today are genetically modified. In the first half of 2002, at the height of the financial crisis due to the corralito, 60% of Argentina’s revenue came from GM soya exports. In point of fact, it is estimated that GM soy directly or indirectly provides employment for a million Argentineans. By contrast, in Brazil the planting of GM soybeans was unauthorized; however, there was illegal trafficking of GM soy from Argentinean to Brazilian farmers. In his first election campaign, former President Lula advocated the ban on GMOs but, after coming to power, he discovered that 40% of soybeans in Brazil were, in fact, illegally planted GMOs. Faced with this scenario, he took the precautionary measure in 2003 of granting the transient commercialization of illegally produced transgenic soybeans. Since then, the marketing of genetically modified soybeans has been legalized and production has soared, making Brazil the world’s second producer of transgenic crops today. Meanwhile, Africa has witnessed some complicated cases. For instance, Zambia rejected humanitarian aid in the form of GM corn on the strength of environmentalists’ reports of its supposed carcinogenic potential; claims that lacked supporting scientific data. Notwithstanding, South Africa’s firm commitment to transgenics, with Burkina Faso and Egypt more recently following suit, provide a hopeful outlook. By contrast, in the EU, the state of affairs on transgenics and the legislation are different, and progress is slow.

5. What The Future Holds in Store: Genomics and Food Production The year 2001 witnessed the publication of our genome sequence, after great public and private research endeavour, shedding light on which genes 32


are activated or deactivated in response to specific nutrient intake. This discipline is called nutrigenomics. It is also possible nowadays to determine the genetic differences between individuals that lead to different nutritional responses. This is known as nutrigenetics. Furthermore, each new day brings freshly sequenced genomes of animals, plants or microorganisms of common components of our diet like rice, bread yeast, the probiotic bacterium Bifidobacterium bifidum or pathogens responsible for food poisoning like Escherichia coli (Figure 2). This provides information about key genes making it possible to delineate classical and genetic engineering improvement strategies alike, demarcate defence mechanisms to combat pathogenicity or define new physiological functions. Not long ago, genome sequencing techniques were costly both in terms of time and money, but recent developments have bought us new massive sequencing technologies providing speedy and lowcost sequencing. Application to food and nutrition is more advanced than many people imagine. For example, mass sequencing projects have recently been carried out in human volunteers, revealing that several thousand different strains of bacteria inhabit our digestive tract, with differences between the bacterial populations of lean and obese individuals. Likewise, epidemiological studies can be conducted to define the genes involved in metabolic disorders of interest. For example, the methylenetetrahydrofolate reductase enzyme is essential for controlling blood levels of homocysteine which in excess greatly increases the risk of cardiovascular disease. Some individuals with a genotype known as TT have a gene mutation which leads to a less active enzyme and consequently carriers of this genotype have an increased risk of developing cardiovascular disease. So, if this mutation is detected by sequencing, it is possible to draw up a suitable diet that can help to partially alleviate the genetic disorder. In this particular case, a diet rich in folic acid can counteract the problem of excess homocysteine in blood and therefore, simply prescribing this type of diet to people with this


genotype puts their risk of cardiovascular disease back to normal. Similarly, transgenic animals can be used to study the effect of food ingredients or even functional foods. Our research team uses the worm Caenorhabditis elegans to analyse the influence of certain functional ingredients on oxidative stress, ageing, obesity, infection by enteric pathogens (bacteria and viruses) and even Alzheimer’s disease. We use a multidisciplinary approach implementing transgenic worms, transcriptomics and/or metabolomics. Thus we can identify which metabolic pathways the ingredients target and then confirm this by using mutants. Following this strategy we have recently shown that the polyphenols in cocoa target sirtuin which acts in the insulin-like signalling pathway known to affect longevity.

ed systems or nanotechnologies that will bring new improvements to these scientific disciplines. However, a fundamental question we must ask ourselves is the following: is a scientific community as conservative as the one comprising food technologists and nutritionists prepared to welcome these new professionals on board? Let us hope so.

Review Questions and Answers Q1. What are the more important differences between classical genetic techniques and genetic engineering? A1. With genetic engineering, one can control the genetic modification introduced, it is also quick and efficient and the species barrier can be crossed. Q2. What is the second generation of GM crops and food and their implications? A2. In this second generation genetic modifications are introduced that affect the physico-chemical, organoleptic or nutritional properties. Such modifications imply higher technological complexity as they tend to involve multiple genes. Q3. What are the results of the food safety trials conducted with all GM foods marketed to date? A3. There is not a shred of scientific evidence that these foods, albeit GM, pose a risk to consumer health greater than the risk posed by ingesting the corresponding conventional food.

Figure 2. Map of the genome of a probiotic strain.

In summary, for all the reasons discussed here, we can conclude that the future of genetics has an important bearing on food-related issues. Each day takes us further away from the time when food technologists were experts in industrial processes. Nowadays, new professionals are needed with an understanding of the key role molecular biology, cell biology and genetics play in the search for new improved foods, and how certain foods keep us healthy. No doubt these developments also call for new food technologists with expertise in managing automatFOOD BIOTECHNOLOGY: DEVELOPMENTS AND PERSPECTIVES

Q4. What are the environmental risks of transgenic plants? Are new risks associated with transgenic plants? A4. The potential transfer of foreign genes from transgenic to wild varieties, a decline in the biodiversity of the surrounding area and in the case of pest-resistant plants, higher incidence of them attacking non-target organisms. There are no new risks associated with transgenic plants. Q5. What is nutrigenomics and nutrigenetics? A5. Nutrigenomics is the scientific discipline that identifies which genes are activated or deactivated in response to specific nutrient intake. It is also posCHAPTER 3

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sible nowadays to determine the genetic differences between individuals that lead to different nutritional responses. This is known as nutrigenetics.

4. Dill GM. Glyphosate-resistant crops: history, status and future. Pest Management Science 2005;61:219-224.

Further Readings

5. Jank B, Gaugitsch H. Assessing the environmental impacts of transgenic plants. Trends in Biotechnology 2001;19:371-372.

1. Beyer P, Al-Babili S, Ye X, Lucca P, et al,. Golden Rice: introducing the b-carotene biosynthesis pathway into rice endosperm by genetic engineering to defeat vitamin A deficiency. Journal of Nutrition 2002;132:506–510. 2. Bouchard C, Ordovas JM. Fundamentals of nutrigenetics and nutrigenomics. Progress in Molecular Biology and Translational Science 2012;108:115. 3. Butelli E, Titta L, Giorgio M, et al,. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nature Biotechnology 2008;26:1301-1308.

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6. Kuiper HA, Kleter GA, Noteborn HPJM, et al,. Assessment of the food safety issues related to genetically modified foods. The Plant Journal 2001;27:503- 528. 7. Quaim M. Benefits of genetically modified crops for the poor: household income, nutrition, and Elath. New Biotechnology 2010;27:552-557. 8. Ramón D, MacCabe AP, Gil JV. Questions linger over European GM food regulations. Nature Biotechnology 2004;22:149.

BIOTECHNOLOGICAL PLANT BREEDING

CHAPTER 4 BIOTECHNOLOGICAL PLANT BREEDING Ana Fita, Óscar Vicente, Mónica Boscaiu, Adrián Rodriguez-Burruezo


1. Plant Breeding ................................................................................................................ 38

1.1 Plant Domestication as a Breeding Process .................................................................... 38

1.2 The Beginning of Modern Plant Breeding ..................................................................... 39

2. Molecular Markers in Plant Breeding ............................................................................. 39

2.1 Marker Assisted Selection .............................................................................................. 40

2.2 Other Uses of Molecular Markers .................................................................................. 42

2.2.1 Plant Genetic Diversity and Germplasm Management ......................................... 42

2.2.2 Hybrid Vigour Prediction ..................................................................................... 43

2.2.3 Cultivar Identification and Protection .................................................................. 43

2.2.4 Genetic Maps ....................................................................................................... 44

3. Plant Breeding in the Genomics Era ............................................................................... 45

3.1 High-Throughput Genomic Techniques ....................................................................... 45

3.2 Some Plant Breeding Applications ............................................................................... 46

4. In Vitro Culture Techniques ........................................................................................... 47 4.1 In Vitro Techniques Used to Increase Diversity .............................................................. 48

4.1.1 In Vitro Pollination and Embryo Rescue ............................................................... 48

4.1.2 Somatic Hybridization .......................................................................................... 48 4.1.3 Somaclonal Variation ............................................................................................ 49 4.1.4 Induced Mutation ................................................................................................ 49

4.2 In Vitro Techniques Used to Improve The Breeding Process .......................................... 50

4.2.1 In Vitro Selection .................................................................................................. 50 4.2.2 Double-Haploid Production ................................................................................. 50 BIOTECHNOLOGICAL PLANT BREEDING

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35


5. An Introduction to Transgenic Plants ............................................................................. 50

5.1 What is a ‘Transgenic’ or ‘Genetically Modified’ Plant? ................................................. 51

5.2 ‘Classical’ or ‘Traditional’ Plant Breeding vs. Genetic Engineering of Crops .................. 51

6. Generation of Transgenic Plants ...................................................................................

51

6.1 Steps to Produce a Transgenic Plant ............................................................................

51

6.2 Gene Transfer Methods ...............................................................................................

52

6.2.1 Agrobacterium Tumefaciens – Mediated Transformation .......................................

52

6.2.2 Agrobacterium Tumefaciens as a Vector for Gene Transfer to Plants .....................

53

6.2.3 Leaf-Disc transformation ....................................................................................

54

6.3 Particle Bombardment (Biolistics) ...............................................................................

54

6.4 Other Methods for Direct Gene Transfer Into Plants ..................................................

55

7. Biotech Crops ...............................................................................................................

56

7.1 Improvement of Agronomic Traits ..............................................................................

56

7.1.1 Herbicide Tolerance ............................................................................................

56

7.1.2 Insect Resistance ................................................................................................. 57

7.1.3 Other Crops, Other Traits ..................................................................................

57

7.2 Perspectives for The Near Future .................................................................................

57

Review Questions and Answers .............................................................................................

58


62

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CHAPTER 4 / A. Fita, Ó. Vicente, M. Boscaiu, A. Rodriguez-Burruezo


Summary

P

lant breeding, the exploitation of plants’ genetic potential for the benefit of mankind, started empirically with the domestication of plants more than 10000 years ago, marking the beginning of agriculture. Nevertheless, plant breeding was born as a science only at the beginning of the 20th century, as a practical application of Mendel´s discoveries on the inheritance of traits and the subsequent development of Genetics. Modern plant breeding methods allowed cropimprovement remarkably in a few generations leading to the development of high-yield varieties of major crops, such as wheat, rice or corn, which were the basis of the so-called ‘Green Revolution’ of the mid-1960s. In the last few decades, scientific advances and innovative biotechnological tools – recombinant DNA techniques, plant transformation, DNA markers, genomics, in vitro culture methods – have revolutionized plant breeding, making the process much more efficient. The use of molecular markers in ‘marked assisted selection’ (MAS) is one of the most useful applications of molecular techniques in plant breeding: when a specific molecular marker is linked to an allele of interest it is possible to simplify selection of individuals with the desired phenotypes, saving much time, space and money in breeding programs. Molecular markers also have other applications in plant breeding: plant genetic diversity studies and germplasm management, prediction of hybrid vigour, identification and legal protection of cultivars and the development of genetic maps. Plant breeding has also profited from the development of genomics – especially high-throughput DNA sequencing technologies (‘next generation sequencing’, NGS) – which opened the possibility to assay many markers and individuals at the same time so accelerating the development of introgression lines. Other breeding applications of genomics include, among others, ‘association mapping’ through Genome-Wide Association (GWA) studies, the ‘breeding by design’ strategy, genotyping by sequencing or genomic selection (GS). In vitro culture techniques are also an essential component BIOTECHNOLOGICAL PLANT BREEDING

of modern plant breeding, used either to increase genetic diversity (in vitro pollination and embryo rescue, somatic hybridization, somaclonal variation or induced mutation) or to improve the breeding process itself (in vitro selection and double-haploid production). Despite the unquestionable importance of these biotechnological tools in modern plant breeding in terms of commercial activities and economic profits, at present ‘plant biotechnology’ almost exclusively refers to the exploitation of transgenic or ‘genetically modified’ (GM) crop plants. A GM plant contains one or a few foreign genes, stably integrated into its genome, the expression of which will confer a specific and pre-determined phenotype to the transformed plant. Generation of transgenic plants through genetic engineering shares the same objective (to introduce a desired trait into a specific plant variety) and is complementary to ‘classical’ breeding methods but also represents a qualitative step forward in the genetic improvement of crops, as it allows to overcome the main limitation of traditional breeding which requires genes to be transferred by natural (or forced) sexual crosses. Generation of a transgenic plant usually involves three successive steps: i) transfer of the foreign DNA construct to plant cells, generally in vitro (in cell cultures or plant explants); ii) stable integration of the exogenous DNA into the plant genome; iii) regeneration of full plants from the transformed cells, following standard in vitro culture protocols. Two main methods are routinely used to transfer DNA to plant cells. The first one, Agrobacteriummediated transformation, has been adapted from the natural mechanism used by the soil bacterium A. tumefaciens to transfer part of its genetic material to wounded plant cells causing the so-called crown-gall disease in the infected plants. The second method, particle bombardment or ‘biolistics’, uses especially designed devices to shoot tungsten or gold microprojectiles coated with DNA to be transferred into the target plant material. At present, over 160 million hectare (ha) are used worldwide to grow commercial ‘biotech’ (GM) crops in about 30 different countries, an impresCHAPTER 4

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sive ca. 100-fold increase over the figure of 1996 when they were first introduced. This makes GM plants the fastest growing technology in modern agriculture. Although now these transgenic crops are limited, practically, to only four species (soybean, maize, cotton and rapeseed), and only two traits (herbicide tolerance and insect resistance), other crops with these and/or new characteristics are grown at a smaller scale or are being developed, so that biotech crops will continue their steady increase in the future.

1. Plant Breeding 1.1 Plant Domestication as a Breeding Process Farmers have bred, unconsciously, plants with remarkable results since the birth of the agriculture more than 10.000 years ago. Agriculture was an extension of gathering. Firstly, hunter-gatherers merely took from the nature the fruits, seeds, tubers, fiber and other useful parts of wild plants. Gradually, they selected those individuals that showed desirable traits for human uses. Actually, those primitive breeders chose plants affected by spontaneous mutations, which caused their “desirable” and distinctive behavior, from wild populations. Later, this selection pressure combined with growing conditions controlled by humans (instead of wild conditions) as well as the accumulation of new mutations, crosspollination, continuous selection, etc., led to a dramatic transformation from wild plants to the current crop species (Figure 1) following the same process as the domestication and breeding of animals. This modification of the genetic basis of plant populations, from wild to cultivated genotypes is called domestication and was done by mankind without any knowledge of either the basis of inheritance or the reproductive system,or the population structure of plant species. It was done by intuitive selection and adaptation through thousands of years of farming. Domestication made possible the evolution from wild traits (more competitive under nature conditions) to man-useful traits, which is called the do38

mestication syndrome. Thus, for example, crop species are more productive and show edible and much bigger and fleshier fruits in comparison to their wild relatives, which on the contrary may have higher seed/flesh ratios, spines in the whole plant, toxic or bitter compounds and higher acidity, among other traits, (e.g. melons, watermelons, eggplants, tomatoes, Capsicum peppers). Other traits developed during plant domestication are those related to harvesting, particularly in grain species such as cereals or beans. Thus, wild relatives of wheat or corn (teosinte) show fragile ears/ cobs when ripe and the spikes disarticulate upon ripening which facilitates the dispersion of seeds by means of wind or animal hits, while the domesticated species keep the seeds in their rachis until harvesting. Similarly, many wild relatives of grain legumes (e.g. peas, chickpeas, lentils) are dehiscent to release their seeds, while domestication involved the development of partially dehiscent or even indehiscent genotypes.

Figure 1. Top: Cucumis zehygeri (left and center) wild relative of cultivated melons (Cucumis melo L.) (on the right). C. zehygeri retains many traits of wild species: high seed content, spines, low proportion of flesh, bitter and toxic compounds (e.g. saponins). Bottom (on the left): Cultivated Capsicum baccatum var. pendulum and wild Capsicums (at the bottom of the picture) from South America. Fruits from the cultivated species are considerably bigger, with higher fruit flesh/seeds ratio, and firmly attached to the peduncle at the ripe stage, while fruits from wild Capsicum peppers are much smaller, with very low flesh and easily detached from the peduncle when ripe. Bottom (center and right): Domestication in eggplants: evolution from prickly wild eggplants to the domesticated S. melongena (courtesy of J. Prohens).



1.2 The Beginning of Modern Plant Breeding Plant Breeding was born as a science at the beginning of the 20th century as a practical application of Mendel´s discoveries on the inheritance of traits and the subsequent development of Genetics. As a result, modern plant breeding offers the opportunity to increase the response to selection and accelerate the process started by farmers thousands of years ago improving crops in few generations (or even to produce or domesticate new species). In fact, one of the causes of the enormous productivity increase in crops like wheat, potatoes or corn (from 5 to 7-fold) in the last decades is the use of new improved varieties. Moreover, the use of hybrid varieties (firstly in corn and gradually in many other crop species) enabled the exploitation of heterosis, which has been a key factor in the increase of yield, plant vigour or earliness among other traits as well as the combination of desired traits in the hybrid offspring of two different parent lines. This fact, along with the use of fertilizers, pesticides and a better water management led to the so-called Green Revolution in the mid-1960s, which firstly supposed the breakthrough in wheat and rice production in Asia and then was expanded to other crops in other parts of the world. The classic plant breeding objectives are the improvement of: i) yield, ii) resistance/tolerance to pest, diseases and abiotic agents, iii) flavor, iv) shelflife, v) harvest ease. In addition, there are a myriad of specific breeding objectives depending on the crop and its use. For instance, a high accumulation of heavy metals could be a breeding objective for plants used in phytoremediation or a high accumulation of a specific terpenoid could be a breeding objective for a medicinal plant. Therefore, as a whole, Plant Breeding can be defined as the scientific exploitation of the plant’s genetic potential to benefit mankind’s demands whatever are these demands. Plant Breeding is mainly based on Genetics although it is also supported in other sciences such as Botany, Statistics, Physiology, and Plant Pathology. Moreover, Plant Breeding involves three main BIOTECHNOLOGICAL PLANT BREEDING

group of activities: i) searching for new sources of variation useful for the objectives and challenges faced by the breeder (and their exploitation); ii) the introgression of traits of interest in commercial materials from sources of variation; and iii) selection of the most suitable genotypes at two different levels: in screenings aimed to identify the new sources of variation mentioned in “i”, or in screenings of segregating progenies derived from ii within a breeding program. In this regard, biotechnology has developed invaluable new scientific methodologies and products useful in food and agriculture in the last 30 years. Thus, the modern biotechnology with innovative techniques and protocols such as in vitro culture, recombinant DNA techniques, plant transformation, DNA markers, genomics is very useful for breeders. All these biotechnological tools make the breeding process more efficient and allow reaching new frontiers unthinkable some years ago. Maybe the most representative of all is the ability to produce new varieties with genes transferred from other taxonomically distinct genera, families, orders or even kingdoms through plant transformation. Nevertheless, there are also many other helpful techniques and strategies, which have revolutionized Plant Breeding. This chapter aims to show how different biotechnological tools are used in the 21st century Biotechnological Plant Breeding.

2. Molecular Markers in Plant Breeding Molecular markers have many applications within the plant breeding process like the study of genetic diversity in plant populations, marker assisted selection of genotypes, the development of genetic maps, parental selection and cultivar fingerprinting. DNA markers identify specific regions of the genome. There are many types of molecular markers. One of the first molecular markers developed (by Alec Jeffreys) was the restriction fragment length polymorphism (RFLPs), which was based in the polymorphism derived from cutting the genome with restriction enzymes and the subsequent hybridization with a marked probe. However, the CHAPTER 4

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MARKER NAME

TECHNIQUES

RFLP, Random Fragment Length Polymorphism

DNA restriction Hybrization

RAPDs, Random Amplified Polymorphic DNA

PCR

AFLPs, Amplified Fragment Length Polymorphisms

DNA restriction PCR

ADVANTAGE Highly reproducible Codominant No seq. info needed No seq. info needed Cheap Highly polymorphic No seq. info needed Highly polymorphic Reproducible

DISADVANTAGE Time consuming Expensive Dominant Low reproducibility Dominant

SSR, Simple Sequence Repeats

PCR

Codominant Highly reproducible Polymorphic and evenly distributed in the genome

ISSR, Inter-Simple Sequence Repeats

PCR

Reproducible

STSs, Sequence-Tagged Sites

PCR

Useful for physical mapping Low polymorphism Highly specific

CAPs, Cleaved Amplified Polymorphic Sequence

PCR DNA restriction

Reproducible Specific

Need sequencing

SNPs, Single Nucleotide Polymorphism

Sequencing

Useful for genotyping platforms

Need sequencing

Need of seq. for primer development Dominant

Table 1. Main features of molecular markers frequently used in plant breeding.

RFLP analysis is slow, tedious (several steps required) and particularly, requires large amounts of DNA, which consequently limited its application. In this regard, Mullis developed in the 80s one of the most important advances in genetics: the Polymerase Chain Reaction technique (PCR). This technique enabled the amplification of a single (or a few) copies of a specific DNA sequence generating millions of new copies. The PCR provided basis for new molecular markers. Thus, the combined use of restriction enzymes, hybridization and PCR techniques has led to the development of many different molecular makers with different advantages and disadvantages (Table 1). Thus, depending on the primers used for the PCR, it was possible to amplify random sequences within the genome as in the RAPDs (random amplified polymorphic DNA) or for example, to detect differences in the length of genomic tandem repeats by microsatellite markers also called Simple Sequence Repeats (SSRs). Finally, the single nucleotide polymorphisms (SNPs) are the most informative and precise molecular markers since they are based in differences at the nucleotide level. Until recently, they have not been used mas40

sively as their detection requires knowing the DNA sequence. Nevertheless, because of the decrease in the prices of the sequencing process the use of these markers is increasing gradually particularly in genotyping platforms. This chapter does not aim to explain the molecular markers protocols, which are general to any biotechnological discipline but to show their main applications in plant breeding.

2.1 Marker Assisted Selection Plant Breeding is based on selecting genotypes carrying traits of interest to be the parents for the next generation. By doing this, breeders are able to increase the frequency of favorable alleles in each generation. Consequently, Plant Breeding involves comprehensive phenotyping activities on which breeders evaluate the plants for many traits as well as matings among plants (on many occasions done by hand pollination). Due to the need for large populations of plants, several trial fields and specialized staff to perform the crosses, those activities constitute the core of the expenses in a breeding program.



Sometimes it is possible to select those individuals with traits of interest at early stages before pollination and fruit set. For example, when breeding for resistance to seedling damping-off which kills or weakens seedlings of many species and can be caused by a range of fungi. In this case, it is possible to inoculate seedlings with the corresponding causal agent applying a selection pressure on the seedling population. Only resistant plants will survive and therefore, they will be selected as parents for the next generation, which simplifies the breeding process. However, many traits cannot be detected before pollination and fruit set. For example, in breeding programs aimed to increase the lycopene content of tomato or to increase yield in wheat. In such cases breeders are forced: i) to grown the plants till the end of the season; ii) to perform all the possible crosses; iii) to phenotype all the plants; iv) to select only the offspring of those parents with the best performance. This procedure increases dramatically the breeding cost in comparison with the first example. Therefore, any strategy that enables plant selection at the earliest stages of development saves much time, space and money in breeding programs. In this regard, when a specific molecular marker is linked to an allele of interest it is possible to select the genotypes bearing this allele at an early stage so avoiding or simplifiying the phenotyping process. This is the basis of the marker-assisted selection (MAS). Of course, the best molecular marker would be the allele responsible for the phenotype. Nowadays, there are many molecular markers which are being used daily in breeding programs. Of special interest are those markers linked with monogenic genes of resistance to viruses and other diseases (Figure 2).

Figure 2. Agarose gels with the results of a MAS screening of Capsicum annuum breeding lines for the presence of a dominant L4-linked marker (left) and a codominant Tsw-linked CAPs marker (right). L4 and Tsw genes provide, respectively, resistance to the Pepper Mild Mottle Virus (PMMoV) and Tomato Spotted Wilt Virus (TSWV) in peppers. Left picture: spots in the middle indicate individuals carrying, at least, one copy of the L4 gene (as dominant markers cannot distinguish between heterozygous L4/-- or homozygous L4/L4 genotypes), while no spot indicate individuals without the L4 gene (homozygous --/--). Right picture: bottom spots in the middle (marked Tsw) indicate the presence of the Tswmarker for resistance to TSWV, while top spots in the middle (marked --) indicate the Tsw-marker for susceptibility to TSWV. High signal only in the Tsw point or only in the – point indicates, respectively, homozygous plant for presence (Tsw/Tsw) or absence (--/--) of Tsw gene, while low-medium signals in both points indicates heterozygous (also resistant) plant (Tsw/--).

As mentioned above, MAS has been found to be very useful for monogenic traits. Unfortunately, many other traits of interest have a quantitative inheritance such as yield, tolerance to drought or salt stress or the content in sugars, vitamins or antioxidants, among others, show quantitative inheritance and are controlled by many loci, with a small additive effect on the trait or alternatively, several major genes combined with many other minor genetic factors. The loci controlling this type of trait are called quantitative trait loci (QTL). Finding QTL-related markers useful for breeding is not an easy task. First of all, it is necessary to have appropriate segregating progenies like F2 populations or preferably, recombinant inbred lines (RILs) or near-isogenic lines (NILs). Then,


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to obtain reproducible data and to assess effectively the value of a trait, it is necessary to consider the environment-dependence of the QTL expression (as a quantitative trait). In addition, a desirable QTL allele discovered in non-elite genetic material might not offer any improvement, because the allele may already be ubiquitous in current varieties. Finally, the effects of the positive allele may not be transferable to elite backgrounds due to unfavorable epistatic interactions. Despite all these difficulties many QTLs have been discovered in several crops and are being used through MAS for breeding (Table 2).

2.2 Other Uses of Molecular Markers

Species

Traits

Tomato

Ascorbic acid, phenolics, soluble solids

Peppers

Flavonoid content

2.2.1 Plant Genetic Diversity and Germplasm Management

Wheat

Protein content

Barley

Yield, yield stability

Corn

Tolerance to shading stress

Apple

Fruit acidity

Table 2. Some examples of QTLs developed for MAS in plant breeding.

BOX: Some advantages of MAS • Identification of those individuals carrying the (gene)

trait of interest at the seedling stage. Therefore, MAS allows accelerating the selection process or, at least, to decrease considerably the number of plants for subsequent evaluations of other traits, optimizing space in the field and the efforts of the breeders.

• MAS works at a DNA sequence-level. As a result,

this strategy enables breeders to detect the gene(s) involved in the trait of interest, regardless of environmental conditions. This prevents mistakes due to environmental effects that on ocassions distorts the phenotypic expression of the trait.

• MAS makes possible the selection of recessive genes or mutants (usually gene mutations are recessive against the wild variant of the gene).

• Gene pyramiding. This strategy consists of tracking

simultaneously by MAS two or more genes favourable for a specific trait. For example, this allows enhancing the response to a particular disease as the several loci providing different mechanisms of resistance can be piled up in one cultivar or breeding line.

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The possibility of producing saturated genetic maps or identifying individuals genetically diverse by means of molecular markers opens a wide range of applications related to plant breeding. The efficient management of plant germplasm, the prediction of heterotic progenies, the identification or legal protection of cultivars by means of DNA fingerprints or the development of genetic maps are also some other examples of the uses of molecular markers in plant breeding.

Any succesful breeding program requires the availability of genetic diversity at the beginning. Danish botanist Johansen, in his classical experiment demonstrated that it is not possible to breed within a pure line because of the lack of genetic diversity. Therefore, breeders are seeking for sources of diversity to improve current varieties (e.g. new sources of resistance to a new strain of a pathogen). To do this, they look for new sources of favorable alleles in the following order/levels: i) other cultivated varieties, ii) old and neglected cultivars or landraces, iii) wild related species. Once having detected a suitable source of variation, the usual procedure would be to perform sexual hybridization to introgress the trait of interest on the materials that need to be improved. If there are no interesting genes in the above-mentioned levels of diversity, then breeders must look at other species of the genera or even other taxons. In these cases, it would be necessary to use somatic hybridization or plant transformation to introgress the genes of interest. For example, many resistances to pests and diseases present in varieties of common pepper (C. annuum) have been introgressed from other varietal types like Serrano Criollo de Morelos (SCM-334) or from close relatives C. frutescens or C. chinense. Such a need for genetic variation, along with the genetic erosion produced by the generalized use of few varieties of each crop, leaded to an increasing



interest since the 70s in preserving plant genetic resources. Nowadays, there are many inter-governmental networks and institutions involved in plant genetic conservation including the European Cooperative for Plant Genetic Resources (ECPGR), Bioversity International, the Centre for Genetic Resources The Netherlands, and the Germplasm Resources Information Network (GRIN) of the United States Department of Agriculture. These institutions hold seeds or propagation material of many cultivated species in their genebanks. Genebanks are a kind of ex situ conservation places of plant diversity for plant breeders and future generations. In these genebanks it is necessary to characterize the plant material at both morphological and genetic levels. For this second task the use of molecular markers is essential. From a molecular point of view, characterization refers to the detection of DNA polymorphisms as a result of differences in random sequences or specific genes by using molecular marker techniques. In this regard, molecular markers can be useful to estimate many parameters related to genetic diversity and plant genetic populations such as: genetic diversity and genetic differentiation, inbreeding coefficients, effective number of alleles per locus, observed and expected heterozygosity, pair-wise comparison of random DNA polymorphisms to establish genetic similarity (S) or dissimilarity (1-S) between accessions. These parameters among others provide information about the range of diversity of populations or collections of crop species or wild relatives and are very helpful to define strategies for an efficient conservation, both in situ or ex situ, and exploitation of plant genetic resources. Core collections are an example of ex situ conservation strategy based on the diversity assessed by DNA markers. Thus, a core collection is a small group of accessions that encompass most of the genetic diversity present in a larger original collection. Comparing the DNA patterns of the different accessions of the original collection provides information to perform dendrograms and cluster analysis, showing phylogenetic relationships among accessions and as a result detecting redundancies and BIOTECHNOLOGICAL PLANT BREEDING

accessions genetically close, identifying different genetic lineages of accessions (clusters). As a result, a group of accessions can be selected as representatives of the whole collection. A core collection allows breeders to prioritize their efforts and evaluations on a smaller population of representative selections. If one of the accessions of the core collection (accession X) shows a trait of interest, then breeders can extend their trials to accessions out of the core collection but genetically close or related to accession X.

2.2.2 Hybrid Vigour Prediction Breeders tend to exploit the hybrid vigour, defined as the increase in size, vigor, fertility, and overall productivity of a hybrid plant over the parent values, which is the opposite phenomenon to inbreeding depression. The genetic distance among genotypes (GD) has been used on many occasions as a good predictor of heterosis or hybrid vigour. Thus, molecular markers are used to calculate GD among pairs of parent lines. Usually, GD is estimated on the basis of similarity indexes mentioned above as 1-S. High GD values can then be used by breeders as criteria to reduce the number of hybrids to be obtained and evaluated. This is of particular interest when huge collections of parent lines are available. Thus, breeders may plan only those hybrid crossings whose parents show high GD values, instead of obtaining hundreds or thousands of hybrids at random. Such hybrids, or a part of them, will be expected to show heterosis at a higher frequency than hybrids obtained randomly.

2.2.3 Cultivar Identification and Protection Conventional methods to identify cultivars and other plant materials are based on phenotypic characterization. Nevertheless, breeders can also obtain DNA polymorphism patterns to distinguish among different varieties or genotypes, which is also known as DNA fingerprinting. Generally, they are neutral markers. Thus, although they are not necessarily associated to traits of interest as in MAS they are useful to detect differences in DNA sequence between CHAPTER 4

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plant varieties. Genetic fingerprinting may be applied to any kind of commercial plant materials: F1 hybrids (or even their corresponding inbred parent lines), clone varieties (particularly fruit trees), cultivars of alogamous species, improved landraces or ecotypes. DNA fingerprinting is particularly useful for: i) complementary characterization of plant materials to protect or claim for proprietary rights; ii) to ensure the purity of propagating seed material; or iii) to discard genetic contamination in elite germplasm by uncontrolled cross pollination.

2.2.4 Genetic Maps Gene mapping or genome mapping lays in assigning different molecular markers to the regions of a genome (i.e. to state how these markers are distributed, ordered and separated in the chromosomes, covering the whole genome). Therefore, a genetic map is the graphic representation of a genome covered (or tagged) by a collection of DNA markers. Classical mapping is based on recombinant frequencies between each pair of markers, which are called centimorgans (cM). Thus, a distance of 1 cM between two markers means that they recombine at a 1% rate. Usually, map distances and the order in which markers are located on the genome are systematically determined by means of three-point testcrosses. Once x the distances (cM) between each pair of markers are estimated, the order can be established. For example, if marker A and marker B are separated by 0.75 cM and a third marker C shows map distances of 0.45 cM to marker A and 0.30 cM to marker B, the only possibility lies in the following order:

Alternatively, if marker A and marker B are separated by 0.75 cM and a third marker C shows map distances of 0.25 cM to marker A and 1.00 cM to marker B, the only possibility will be:

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It must be taken into account that cM is a distance unit based on recombinant frequencies and, therefore, does not fit exact physical distances. In fact, the frequency of crossing over may be different among chromosome regions. Consequently, genetic recombination between two points can be more frequent in some regions than in others. Moreover, the higher the number of markers mapped on the genome the more detailed the map. In this regard, a map can be based firstly in a relatively low number of markers and later scientists can gradually adding and mapping new markers, increasing the degree of saturation of the map. Additionally, by means of the latest molecular techniques (e.g. massive sequencing), these cM-based classic maps can be used to develop new maps, on which the distance between each two points is measured in nucleotids, also known as physical mapping. Finally, it is also possible to use information from the map of one species in the mapping process of other species less studied. This is called comparative mapping. In addition, the closer the species the more efficient the process, as differences between both species at the genome level (due to separate evolution) will be lower. In this way, a high density map of a model species may provide scientists and plant breeders with complementary and helpful information to develop the maps of other related species with agricultural interest. There are several examples of comparative mapping. Thus, the map of Arabidopsis thaliana, the most important model plant in genetics but with no agricultural use, has been used in the gene mapping of many crop species, particularly other Cruciferae from the genus Brassica like B. oleracea (cauliflower, broccoli) or B. juncea, among others. Furthermore, several Brassica species have been utilized to study the relationships between the different genomes encompassed in this genus. Comparative mapping has been also performed for the genomes of phylogenetically distant cereals like rice and wheat. Information from tomato, which previously has been developed using information from Arabidopsis, is being used in the maps of other fruit Solanaceae like Capsicum peppers, eggplants and even other neglected Solanum crop species.



BOX: Immediate applications of gene maps in plant breeding are: • To develop new markers for MAS. Thus, when

a trait of interest is detected in a new source of variation (e.g. a wild material showing resistance to a virus), a gene map may help to identify which marker(s) is (are) close to the DNA region responsible for this trait. In this regard, the most usual procedure lies in performing F1 crosses between the new source and the genotypes used to develop the map. A subsequent segregating progeny (F2: F1 selfing; BC1: backcrossings) can be then screened for the trait of interest, while in parallel these individuals are studied at a molecular level with the markers of the map. The objective is to determine which markers correlate at higher rates with the new trait (i.e. lower recombination frequency). Once identified as a useful marker, it can be directly used for MAS or, alternatively, used for assessing new markers still closer to the DNA region of interest.

• To search regions of the genome related to the

expression of traits that depend on a cascade of metabolic steps. This is very helpful in assessing the position of the codifying region for those enzymes which catalyze different steps of a biochemical pathway, in particular in quantitative traits like flavor components (e.g. sugars, acids, volatiles), or bioactive compounds (e.g. antioxidants, vitamins, carotenoids).

• To search candidate genes or allelic variants for a particular trait.

• To facilitate the development of near isogenic lines (NILs), recombinant inbred lines (RILs) and other introgression lines.

3. Plant Breeding in The Genomics Era The term genomics refers to a sub-discipline of genetics dealing with genetic mapping, sequencing and functional analysis of complete genomes, providing information not only about sequences, but also about the function and regulation of each sector of the genome at different development stages under different environmental conditions. Genomics can be divided into structural, functional and comparative genomics. BIOTECHNOLOGICAL PLANT BREEDING

- Structural genomics addresses physical characterization of the genomes. - Functional genomics characterizes the transcriptome (constituted by the complete set of transcripts produced by an organism), the proteome (comprising all the proteins encoded by a genome) and the metabolome (the complete set of metabolites of a cell, due to the function of the RNA and proteins) - Comparative genomics compare similarities among sequences present in different biological entities. By computer analysis of the sequences and using known genetic principles, it is possible: to make predictions about proteins encoded by those sequences, to establish genetic variations between different populations, to compare sequences from different species and understand evolutionary processes and to identify highly conserved sequences (motifs) in coding and non-coding regions related to functionally important regions. Comparative genomics has demonstrated that there is considerable synteny, that is preserved gene localization in equivalent positions, among related species. Genomics is very useful for plant breeding through the development of genomic physical maps and genetic maps, through the generation of molecular markers (specially SNPs) useful for high-thoughput genotyping, analyzing genetic diversity and determining the function of candidate genes. This discipline has been dramatically boosted by i) the so called next generation sequencing techniques which have reduced the price of sequencing; ii) multiplex genotyping platforms and iii) bioinformatics tools, which allow the processing of all the generated data. We refer to these tools as high-throughput genome techniques.

3.1 High-Throughput Genomic Techniques In the last thirty years Sanger sequencing was very useful to sequence several genomes and transcriptomes. However high-throughput DNA sequencing technologies, collectively known as next generation sequencing (NGS), Second Generation Sequencing or Massive Parallel Sequencing have lowered CHAPTER 4

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the price of sequencing more than one thousand times. NGS based on an in vitro amplification and a parallelized sequencing produces thousands of sequences at a time, avoiding the time consuming cloning and individual analysis of samples steps. These methods produce short and usually less accurate reads than the Sanger method, but this disadvantage is compensated for by the high number of samples analyzed. NGS produces a great amount of data which need a great computational power. Bioinformatic tools and programs are continually evolving and improving to keep pace with NGS technical advances. One of the main contributions of NGS to plant breeding is the wide discovery of molecular markers, such as SSR and SNPs, useful in high-throughput genotyping platforms, which permit the analysis of many markers and/or many individuals in one run. SNPs are highly used for their simplicity, uniform distribution along the genome and easy use in genotyping platforms. The primary source of SNP discovery is direct sequencing at high accuracy. This sequencing can be done on the genome or in the transcriptome. The transcriptome sequencing is based on sequencing the cDNA derived from mRNA of expressed genes in one specific tissue at a certain developmental stage or under certain stress. The reads usually do not cover the whole gene and they are called EST (Expressed sequence tag). Although transcriptomic sequences represent only some genes and do not cover un-transcribed regions of the genome, the EST approach is cheap and very useful in the SNP discovery (among other applications). High throughput sequencing and SNP mining using bioiformatic tools detect usually hundreds/tens of thousands SNPs. Genotyping assays usually require a previous process of selection of a set of SNPs that are appropriate for the assay objectives. Currently, there are a wide variety of SNP genotyping systems commercially available using different chemistries and detection systems. “Serial” systems allow testing of small to modest numbers of SNPs on many subjects in each reaction and are easy to customize. Others, called “parallel” methods, test up to a million SNPs on each subject at one time in 46

fixed panels. The cost per SNP for a serial method is much larger than for a parallel method, but the cost per subject is much less. High Throughput Genotyping techniques allow for deep and accurate genotyping of plant populations. But in plant breeding the most important point is the association of a genotype with a phenotype. Accurate phenotyping relies on evaluating many plant replicas during several years and under different conditions. In this sense, high throughput phenotyping systems are needed. Recently, new automated or semi-automated phenotyping platforms begin to be commercially available.

3.2 Some Plant Breeding Applications The possibility to assay many markers and individuals at the same time is a great opportunity in plant breeding for accelerating the generation of introgression lines such as RILs, NILs or CSSLs (chromosome segment substitution line). These lines are used as mapping populations to locate QTLs associated with complex traits. QTL detection based on the linkage analysis method is limited though. Association mapping, through genome-wide association (GWA) studies, uses the natural diversity to identify genetic loci associated with phenotypic trait variation in a collection of individuals. Natural populations have suffered many more recombination events than those that occurred during the development of the biparental mapping populations and therefore, provide better resolution. The ‘Breeding by Design’ strategy is an extension of the different levels of marker applications that are currently used in breeding. The process includes three steps: mapping loci involved in all agronomically relevant traits (using biparental populations, candidate genes or GWA), assessment of the allelic variation at those loci (in a broad population), and, finally, breeding by design (combining favourable alleles at all loci). Genotyping by sequencing (GBS) is the genotyping process referred to until now is mainly a twostep process involving marker discovery followed



by assay design and genotyping. In the sequencebased genotyping approaches the marker, discovery and genotyping are completed at the same time. This facilitates exploration of new germplasm sets or even new species without the upfront effort of discovering and characterizing polymorphisms. In addition, sequences obtained from GBS can be reanalyzed, uncovering further information (e.g., new polymorphisms, annotated genes) as bioinformatics techniques improve, reference genomes develop and the collection of sequence data increases. This strategy is especially useful in species with little previous information. Genomic selection (GS) is a new approach for improving quantitative traits in large plant breeding populations that uses wholegenome molecular markers (high density markers and highthroughput genotyping). Genomic selection predicts the breeding values of new lines in a population by analyzing their genotypic values. To calculate the genotypic values previously the marker effect has been calculated by integrating 1) phenotypic information from relevant breeding germplasm evaluated over a range of environmental conditions; (2) molecular marker scores; and (3) pedigree information using. Reverse genetics is an approach to discover phenotypes arising from known genetic sequences. It has been used widely to understand gene function. TILLING (Targeting Induced Local Lesions in Genomes) is a tool of reverse genetics able to identify all allelic variants of a DNA region present in an artificial mutant collection. TILLING consists in mutagenizing a population of plants, genotyping the population by searching for mutations in the target gene/s and then phenotyping plants bearing the different mutation to associate the mutation with a specific phenotype. TILLING is an opportunity to create de novo variation in plant genomes useful for breeding; it reduces the phenotyping to the selected genotypes and has been suggested as an alternative to plant transformation. EcoTILLING (ecotype TILLING) is a similar procedure used to identify allelic variants for target genes in natural collections.


4. In Vitro Culture Techniques In vitro culture is a biotechnological tool consisting of the cultivation of plant tissues, or even isolated protoplasts (plant cells without cell wall), in a nutrient medium under axenic (no other organisms present) conditions. The nutrient medium includes specific combinations of different mineral salts, vitamins, sugars, and usually hormones, antibiotics and other chemical compounds aimed at promoting tissue growth. In vitro culture is based on the ability of plant tissues to de-differentiate its cells and re-start a new development program. That is, the ability to regenerate a complete plant from isolated somatic cells. In vitro techniques have many applications in the Plant Breeding field. These applications can be divided into: i) those aimed at increasing the diversity or facilitating the introgression of genetic material into the species of interest and ii) those aimed at improving the breeding process. As stated previously in this chapter, breeders use the genetic diversity available to combine favorable alleles into a plant variety. Sometimes favorable alleles are found in already cultivated varieties of the same species ready to be used in breeding programs through conventional crosses. But other times, the favorable alleles are present in wild related species and wide crosses are necessary. In those cases incompatibility problems are usual. Along with other cultural techniques not referred to here, in vitro culture can help to overcome some of these problems with techniques such as in vitro pollination and embryo rescue. In addition, genetic diversity available in other plant species, not necessarily related to the target crop, can be exploited by somatic hybridization. Still the range of the genetic diversity available for plant breeders is not restricted to the plant kingdom but to the living organisms through plant genetic transformation. Despite all these tools, it is not rare that a plant breeder needs to generate de novo genetic diversity. In thise cases, induced mutagenesis or somaclonal variation is employed. CHAPTER 4

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The breeding process, from the identification of the alleles of interest to the release of a new plant variety, can take easily 5-10 years depending on the trait improved, the species used etc. In this sense, any technique that is able to shorten the process is of great interest in plant breeding. Large-scale screenings are usually performed not only to identify new phenotypes but also to select the best ones, in vitro selection offering an opportunity to evaluate thousands of genotypes in a very small area. Further, many breeding protocols use homozygous plants in one or several steps. Nevertheless, to obtain homozgous plants can be complicated in cross-pollinated species due to inbreeding depression. Even in self-pollinated species new homozygotes derived by hybridization are achieved after six to eight generations of selfing. Therefore, it is easy to understand how valuable a method is to obtain homozygous plants in just one generation. This is the case of double haploid production through androgenesis or gynogenesis. All these tools are going to be introduced briefly in the following paragraphs except for plant transformation, which deserves a separate section due to its relevance.

4.1 In Vitro Techniques Used to Increase Diversity 4.1.1 In Vitro Pollination and Embryo Rescue In vitro pollination can be used in the case of prezygotic incompatibility or pre-zygotic barriers, it is produced before the fertilization and formation of the zygote. Pre-zygotic barriers are a consequence of either the inability of pollen to germinate on the stigma or the inability of the pollen tube to reach the egg due to for example, a slow rate of growth or excessive style length. Within the name of in vitro pollination there are several techniques such as pollination of isolated ovules or placental pollination. In all of these techniques female organs are cultured in vitro and contacted with the pollen. It is interesting to note the difference between in vitro pollination and in vitro fertilization. The latter re48

fers exclusively to the controlled fusion of the gametes (egg cell and sperm cell) that is very difficult to achieve in crop species due to the anatomy of the ovary. In vitro fertilization and subsequent plant regeneration has been achieved in maize. Although in vitro pollination is mainly used for wide crosses it is also helpful in selfing auto-incompatible crops like many fruit trees. By contrast, post-zygotic barriers occur after the fertilization. In this regard, higher plants require double fertilization. Thus, the fusion of the gametes will produce the embryo, whereas the fusion of the secondary nucleus of the pollen with the polar nuclei will produce the endosperm. The endosperm is the tissue that nurses the embryo during its development. Post-zygotic barriers are due to problems in the chromosomes assortments during mitosis of the zygotic cells and/or problems in the development of the endosperm, provoking the abortion of the embryo. In those cases in which the endosperm does not evolve properly, but the embryo can divide properly, the embryo rescue technique allows isolating young immature embryos and growing them under in vitro conditions. It must be taken into account that the earlier the embryo abortion occurs the more difficult will be the technique and the lower the efficiency. The main application of this technique has been to regenerate potentially abortive embryos in many cases of interspecific crossings limited by postzygotic barriers. Consequently, embryo rescue has enabled breeders to introgress traits of interest between related species. Alternatively, the in vitro culture of immature embryos has also many other applications: i) shortening breeding cycles; ii) overcoming either seed dormancy or sterility; iii) obtaining valuable haploid materials; or iv) micropropagation among other strategies.

4.1.2 Somatic Hybridization This technique allows i) the genetic interchange between species unable to sexually hybridize; ii) the interchange of cytoplasmic material; iii) the production of polyploids and iv) the production of introgression lines. Somatic hybridization consists in



fusing somatic protoplasts (somatic cells without its cell wall) from different parents and to regenerate the fusion product (heterokaryons) into a complete plant. The first step in somatic hybridization protocol is to isolate intact protoplast. This is done by digesting the cell walls of mesophyll tissues (although other tissues can be used) by enzymatic preparations (cellulose, hemicelluloses) derived from fungus. The second step is protoplast fusion that can be done chemically or physically by applying an electric current (electrofusion). After the fusion there are several products: i) homokaryons, fused protoplasts from the same parental; ii) heterokaryons, fusied protoplasts from different parents; iii) multiple fusion products, more than two protoplasts have been fused and iv) unfused protoplasts. Therefore, the third step involves the cultivation of the products and selection of the hybrids. Some of the methods used to select hybrids include the use of selective media, genetic complementation of non-allelic mutants and mechanical isolation. The last step is to regenerate the hybrid plants. The result of a symmetric fusion protocol is an autopolyploid (when both parents used belong to the same species) or an alopolyploid (when each parent belongs to different species), because the hybrid plant has the full genome from each parent. However, in many cases, asymmetric fusions are produced. In this case only one part of the genome of one parent is inserted into the nucleus of the second parent. These hybrids are very useful to produce introgression lines. To produce asymmetric fusions, it is usual to employ irradiated nuclei in which the chromosomes are fragmented, or micronuclei which bear only few chromosomes. Another interesting case is the production of cybrids that are the combination of the nuclear genome of one parent and the cytoplasm of another. Cybrids are very useful for producing cytoplasmic sterile plants which are of great interest in breeding plants in which the control of pollination is difficult. Somatic hybridization has been used to produce new varieties of different species; it has been very useful especially with Citrus, Solanum and Brassica.


4.1.3 Somaclonal Variation On many occasions, during in vitro culture, genotypic variants appear spontaneously at high frequencies and is known as somaclonal variation. The amount of somaclonal variation that appears on in vitro culture depends on several factors like the tissue used, genotype, regulator levels in the growing media and tissue age. In micropropagation protocols where the objective is to achieve thousands of true-to-type clones, somaclonal variation is considered a great problem. However, it offers an opportunity for breeders to develop new genetic variation. It is an inexpensive and quick technique that can be used to improve sexual and vegetative propagation species with mutation rates relatively high when compared with rates of spontaneous mutations. Yet, the mutations are not directed and phenotypes of low interest for breeders can appear, as well as loss of morphogenic capacity in longterm cultures. Other disadvantages are the instability of some somaclones due to epigenic variation or the occurrence of aneuploidy, infertility and other disorders. This technique has been used sucessfully to breed new varieties of rice, tomato and banana among others.

4.1.4 Induced Mutation Mutations are the primary origin of genetic variability and therefore, induced mutagenesis can be considered a valuable tool for plant breeding. In the late 1920s, L.J. Stadler developed the basis of experimental mutagenesis in crop plants. During the 1950s, 1960s and 1970s, this technique boomed being relegated to a secondary position for breeders later, although it has been crucial for functional biology studies. Nowadays there is a renewed interest in this subject associated with advanced molecular analysis capabilities as in TILLING technique explained in section 3.2. Mutations are induced by either mutagenic chemicals such as nitrous acid, base analogs or alquilant agents e.g. ethyl methane sulfonate (EMS) or by physical mutagens such as ionizing radiation e.g. X-rays, gamma rays, neutrons, protons and alpha CHAPTER 4

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particles. The resulting mutations depend on the mutagenic agent and the dose. In general, ionizing radiation produces high levels of chromosome damage, originating single and double strand DNA breaks, and deleterious physiological effects leading to a high cell mortality and to high sterility of plants of the first generation.In contrast, chemical mutagens are more specific and of a precise effect. The steps to apply mutagenesis in plant breeding are: i) to apply the mutagenic agent to a plant tissue which is considered the first mutagenized generation (M1) (this tissue will show a mixture of mutated and non-mutated cells called a chimera); ii) to regenerate solid mutants and in the case of sexual reproduced plants; iii) to self-pollinate those mutants to recover the mutation in homozygosis (M2) and then select by the trait of interest. In many cases the selection can be made during the regeneration process using selective media.

nique, obtaining pure lines very quickly, in just one generation, instead of several generations needed in conventional breeding programmes. The production of haploids and double-haploids through genome duplication, is based on the regeneration of complete plants from gametes (which are haploids). When the plants are derived from microspores the process is called androgenesis, whereas when plants derive from oospores the process is called gynogenesis. The most popular haploid and double haploid production method, due to is simplicity and good results, is anther culture (Figure 3). Double haploids shorten breeding programs by obtaining 100% homozygous plants (i.e. inbred lines) in one generation. This method has been used successfully in many species. Additionally, double haploids are especially useful to develop mapping populations such as RILs and NILs.

4.2 In Vitro Techniques Used to Improve The Breeding Process 4.2.1 In Vitro Selection This consists of selecting the desired phenotypes from tissues grown in vitro. The great advantage of this type of selection is the possibility of screening a large number of individuals in a relatively small area. As an example, if we want to obtain individuals resistant/tolerant to salinity or any toxic agent (alluminium, boron, lead) or herbicide, cells or tissues are cultivated in a medium containing the agent. The main disadvantage of this technique is the possible differences among the in vitro responses and the plant in vivo response.

4.2.2 Double-Haploid Production Haploids are not economically useful per se as they are sterile (because of the lack of homologous pairs of chromosomes during meiosis). However, by treating these haploid individuals with certain physical or chemical agents (e.g. colchicine) it is possible to obtain diploids (actually double-haploids) that will be homozygous for all the loci (i.e. pure lines). This is the main interest of this tech50

Figure 3. In vitro culture of tobacco anthers to obtain double-haploids.

5. An Introduction to Transgenic Plants Modern plant biotechnology covers a wide range of techniques mostly applied to plant breeding. However, in terms of commercial activities and economic profits, at present ‘plant biotechnology’ almost exclusively means the exploitation of transgenic plants. Huge areas of cultivated land worldwide



are used to grow transgenic varieties of some of our major crops – known, in fact, as ‘biotech crops’ – due to the benefits they provide to the farmers as compared to conventional crops in terms of higher yields, more stable production and reduced costs. This chapter will focus first on a brief description of the methods commonly used to generate and analyse transgenic plants. The second part will refer to the major crops now growing commercially in our fields and the traits introduced into them giving also a very brief mention of minor commercial GM crops and other traits of interest.

5.1 What is a ‘Transgenic’ or ‘Genetically Modified’ Plant? The simplest definition of a transgenic plant would refer to a plant containing one or a few foreign genes, stably integrated into its genome together with the appropriate regulatory sequences (plant promoters and transcription termination signals) to allow expression of those genes which will confer a specific and pre-determined phenotype to the transformed plant. It is important to point out that genetic engineering techniques must be used in the process of genetic transformation; if the foreign genes – or even complete genomes – have been introduced by traditional breeding methods (i.e., by sexual crosses) or if genetic information of the original plant has been altered by spontaneous or induced mutations the plant variety is not considered as ‘transgenic’, even though it is obvious that it has been ‘genetically modified’.

5.2 ‘Classical’ or ‘Traditional’ Plant Breeding vs. Genetic Engineering of Crops The general goal of plant breeding is the introduction of a particular trait of interest into a specific variety or cultivar of a crop species. Classical methods rely on hybridization (sexual crosses) of the ‘receptor’ parental with another variety of the same crop species or with a wild relative, which will provide the required character; this will be followed by sevBIOTECHNOLOGICAL PLANT BREEDING

eral cycles of phenotypic selection and backcrosses of the individuals selected in each generation with the ‘receptor’ parental. In this way, the undesired characteristics of the donor are eliminated and the phenotype of the receptor, plus the additional trait, is recovered. It is also possible to cross two varieties or related species followed by several cycles of self-fertilisation from F1 hybrids with phenotypic selection of the individuals with the desired traits in each generation. In both cases, apart from the long time required to obtain the new variety, the main limitation of traditional breeding is that genes can only be transferred by natural (or forced) sexual crosses. On the other hand, generation of transgenic plants through genetic engineering involves the controlled transfer of one or a few genes, previously isolated and characterised, the expression of which confers the desired trait on the genetically modified plant. That is, plants are ‘designed’ with a specific, pre-determined purpose. Since the genes to be transferred can be isolated from any organism – bacteria, yeast, animals or other plants – the reproductive barriers that strongly limit traditional plant breeding are overcome. Classical and molecular approaches to plant breeding are not either opposite or alternative, but complementary. Plant varieties previously improved by classical breeding are used for genetic transformation. Once a ‘new’ transgenic plant is obtained and characterised in the laboratory and the greenhouse (a ‘prototype’), all further developments are made using traditional methods: field trials to check agronomic characteristics – such as growth, yield, etc. – transfer of the new trait to other varieties or largescale seed production and commercial propagation.

6. Generation of Transgenic Plants 6.1 Steps to Produce a Transgenic Plant Generation of GM plants usually involves three successive steps: CHAPTER 4

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• First, the foreign DNA is introduced into plant cells. Appropriated constructs, including the gene of interest and a selectable marker gene – conferring, for example, resistance to a specific antibiotic or herbicide – with the corresponding regulatory sequences (plant promoters and terminators) are prepared in vitro and transferred to either plant cells in culture or plant explants, using one of several available methods (see below). A procedure for in planta transformation has also been developed, but is used only for the model species Arabidopsis thaliana. • Second, the introduced DNA integrates into the plant genome, in one or more copies, at a single locus or at several loci. This process is not yet completely understood, but involves non-homologous recombination. Integration is essentially at random, although ‘open’ or ‘relaxed’ chromatin regions are preferred targets; therefore, integration is more frequent during DNA replication and in transcriptionally active regions of the plant genome. • Third, full plants are regenerated from the individual transformed cells, in in vitro cultures, generally by organogenesis, although other processes, such as somatic embryogenesis or androgenesis can also be used, depending on the starting material. Expression of the selection marker gene during regeneration allows elimination of non-transformed cells. This step is usually the bottle-neck of the whole process of transgenic plant generation. For many ‘recalcitrant’ species, genetic transformation has not yet been achieved or it is very difficult. In general, it is not for lack of efficient methods for DNA transfer to isolated cells or explants but because of the difficulties to regenerate whole plants in vitro. In cereals, for example, only embryogenic tissues (immature embryos, embryogenic calli) are competent for regeneration in tissue culture systems. The regenerated plants are analysed to confirm their ‘transgenic’ nature. Southern hybridisation or PCR methods are used to detect the physical presence of the transferred gene inserted into the plant genomic DNA. Transgene expression is assessed by 52

detecting the corresponding mRNA (by Northern blots or RT-PCR) and, at the protein level, by Western blots either with specific antibodies or by enzyme assays. Genetic and molecular analyses of the plant offspring (self-fertilizations / backcrosses) are also performed to check the segregation of the phenotype associated with the transgene and its stability. Finally, several independent homozygous lines are generally selected, carrying a single copy of the transgene, which should behave in a Mendelian dominant character.

6.2 Gene Transfer Methods 6.2.1 Agrobacterium Tumefaciens Mediated Transformation Agrobacterium tumefaciens is a Gram-negative soil bacterium, which infects plants – preponderantly dicotyledonous – at wound sites, inducing the formation of tumours (crown galls) and causing the socalled crown gall disease. Crown gall cells can grow in vitro in the absence of phytohormones, whereas normal plant cells require the presence of auxins and cytokinins to proliferate. Moreover, tumour cells produce unusual compounds, amino acid derivatives known as opines, which are not present in normal plant cells. Opines are used by virulent agrobacteria as a source of carbon, nitrogen, and energy but cannot be metabolised by plant cells or by other soil bacteria. In short, the induction of crown galls creates a niche favourable for growth of the oncogenic bacteria which colonise infected plants and ‘force’ them to produce nutrients that only agrobacteria can utilise. Induction of crown gall disease upon infection is due to transfer and expression in the plant cells of genetic information from the bacteria. The genes responsible for tumour formation and opine synthesis as well as those controlling the process of gene transfer to the plant cells, are included in a very large (ca. 200 kbp) bacterial plasmid, the Ti (for tumour-inducing) plasmid. The Ti plasmid contains two defined regions essential for genetic transformation of the plant cells by the bacteria.



· T-DNA (for ‘transferred DNA’): This is a region of ~ 13 kbp, flanked by short direct repeats, highly homologous sequences of 25-28 bp, known as the left and right T-DNA ‘borders’. A singlestranded copy of the T-DNA is transferred to the plant cell and integrated in the nuclear plant genome. This region contains genes encoding enzymes responsible for the synthesis of phytohormones, auxins and cytokinins. Expression of these genes in the plant cell, by increasing the levels of these growth regulators, blocks cell differentiation and induces cell division, leading to tumour formation. The T-DNA also encodes the enzymes responsible for opine biosynthesis. The genes included in the T-DNA are not expressed in Agrobacterium: their promoters and termination signals, although of bacterial origin, have evolved to be recognized by the plant RNA polymerase and functional only in the plant cells.

virG2 genes, recognises AS and activates transcription of the other vir genes.

· Vir (virulence) region: This region, of ~ 40 kbp, acts in trans and it is not itself transferred to the plant, but contains the genes encoding all proteins required for T-DNA transfer and integration into the plant genome: approximately 35 virulence genes, grouped in eight operons. These proteins are involved in recognition and endonucleolytic cleavage of the T-DNA borders, release of a single-stranded T-DNA through a strand-replacement mechanism, formation of a covalent complex with the T-strand (‘immature T-complex’) and its transfer to the plant cell by a mechanism similar to that of bacterial conjugation. Some vir proteins are transferred together with the immature T-complex to the plant cell where the ‘mature T-complex’ is assembled; they protect the T-DNA from plant nucleases and help to import it into the nucleus.

• Only the T-DNA ‘borders’ – but not the T-DNA itself – are recognised by the A. tumefaciens transfer machinery (i.e., the vir proteins). Any DNA fragment present between the right and left borders will be transferred to the plant cell.

Plant cells become susceptible to Agrobacterium infection when they are wounded; wounded cells produce and release phenolic compounds, such as acetosyringone (AS), which attract the bacteria to the wound sites (chemotaxis) and are specific inducers of vir gene expression. A two-component sensor/regulator system, encoded by the virA and


The Ti plasmid also includes a region, outside the T-DNA and the vir sequences, containing the genes necessary for opine catabolism and so the bacteria can profit from the ‘food’ produced by the transformed plant cells upon expression of the transferred T-DNA genes.

6.2.2 Agrobacterium Tumefaciens as a Vector for Gene Transfer to Plants In the mid 1980s, procedures for the controlled genetic transformation of plants by A. tumefaciens were developed, making possible, for the first time, the generation of transgenic plants. Two characteristics of Agrobacterium’s ‘natural’ gene transfer system have allowed its adaptation as a vector for the transfer of any foreign DNA to plant cells:

• The vir region products act in trans: it is not necessary for the vir genes and the T-DNA to be physically together in the same molecule (i.e., in the Ti plasmid). Based on these properties, so-called binary vectors have been developed for Agrobacterium-mediated genetic transformation of plants. Binary vectors are relatively small plasmids containing the construct to be transferred flanked by the T-DNA borders – and lacking all ‘natural’ T-DNA sequences: crown gall tumours will not be induced in the host plant. These constructs typically contain the foreign gene of interest together with the required regulatory sequences of plant origin (promoter, transcription termination signal) for its expression in the transgenic plant as well as a selection gene – conferring, for example, antibiotic (e.g. kanamycin or hygromycin) or herbicide (e.g., BASTA) resistance – also with the appropriate expression elements. In addition, outside the artificial T-DNA, the binary CHAPTER 4

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vector carries those elements essential for replication and selection in bacteria: an origin of replication for Escherichia coli, an origin of replication for A. tumefaciens (or one single origin of replication functional in both bacteria), and a bacterial selectable marker gene conferring resistance to an antibiotic; in this case, obviously, under control of a bacterial promoter. The binary vector is introduced into an Agrobacterium strain containing a ‘disarmed’ Ti plasmid, in which the whole T-DNA region has been removed but contains the virulence region: upon activation of vir gene expression all proteins necessary for gene transfer are synthesised in the bacteria. The DNA construct cloned in the binary vector in-between the T-DNA borders is then transferred to plant cells by the same mechanism used for transformation with the ‘natural’ T-DNA sequences.

6.2.3 Leaf-Disc Transformation The simplest procedure for Agrobacterium-mediated plant transformation is known as the ‘leaf-disc’ method. Leaf material is cut in small pieces, for example with a scalpel or a cork-borer, and cocultivated for ca. 30 min with the Agrobacterium strain containing the binary vector. During this period bacteria attach to the leaf pieces and vir gene expression is activated. Leaf fragments are then removed from the bacterial culture and incubated for one or two days in agar plates without any selective agent, to allow transfer of the T-DNA to the plant cells. Afterwards, the explants are washed with an antibiotic solution to kill Agrobacterium cells, and cultured in fresh solid medium supplemented with the appropriate antibiotic or herbicide for selection of transformed cells – depending on the specific selectable marker gene included in the T-DNA construct. After some weeks of culture, undifferentiated growth – callus tissue – will be detected on the leaf pieces. Phytohormones (one auxin and one cytokinin) are included in the agar medium, to stimulate regeneration by organogenesis; relatively high cytokinin to auxin ratios will promote formation of shoots, which can be rooted in the presence of high auxin to cytokinin ratios. In this way, presumably 54

transgenic plants are regenerated, although it will be necessary to confirm that the transgenes are stably integrated in the plant genome, as described above.

6.3 Particle Bombardment (Biolistics) Initially, the use of Agrobacterium as a vector for gene transfer to plants was limited by the range of its natural hosts, which did not include some of the economically most important crops, the cereals. Although at present there are protocols which allow the Agrobacterium-mediated transformation of species which are not infested in nature by the bacterium, these procedures are still difficult and with low efficiency. In any case, the search for an alternative to Agrobacterium prompted the development of different direct gene transfer methods, which rely on the transfer to plant cells of naked plasmids containing the appropriate constructs: the gen of interest and the selectable marker gene, with the corresponding plant promoters and transcription termination signals. Particle bombardment or ‘biolistics’ (or bioballistics) is the most successful and most effective direct gene transfer method and has been widely used particularly for transformation of cereal crops. Gold or tungsten microparticles (ca. 1 µm diameter) are coated with plasmid DNA, accelerated to high speed using different devices and shot into the target plant material – explants, cells cultures. The DNA is released within the cells, and integrates into the plant genome. The first particle bombardment system, developed in 1987 used an explosive charge to propel a plastic macroprojectile (‘bullet’) down a barrel until it was stopped by a disk with a small hole in the middle; the bullet carried a droplet of the DNA-coated tungsten microprojectiles, which were shot through the hole at high speed into a Petri dish with the plant sample placed below the barrel. The design of this first biolistic particle delivery system – popularly known as ‘DNA-gun’ or ‘gene-gun’ – was later improved, switching to non-explosive accelerating systems, and to gold microprojectiles, as tungsten



can be toxic to cells. A helium-driven particle bombardment apparatus, commercially produced by the BioRad company, has become the most widely used ‘DNA-gun’. Although the development of biolistic methods was the key for transformation of cereal crops, this technology has some disadvantages as compared to Agrobacterium-mediated transformation. Particle bombardment requires complex and expensive equipment and is less precise and less efficient. One major problem is that the vector DNA is often rearranged, which can lead to the loss of a functional transgene. In addition, particle bombardment frequently results in integration of multiple copies of the transgene in the plant’s chromosomal DNA at a single locus or at a few different loci. This high copy number of the integrated DNA complicates the genetic analysis of the GM plant and may lead to gene silencing phenomena. Therefore, Agrobacterium remains the method of choice for plant transformation, at least for those species that are hosts of the bacterium.

6.4 Other Methods for Direct Gene Transfer into Plants All commercially grown GM crops have been originally generated by one of the two transformation methods described above: Agrobacterium-mediated transformation (applied mostly to dicotyledonous species) or biolistics (for cereal crops). However, much effort has been invested in establishing alternative gene transfer procedures by either searching for simpler or more efficient plant transformation or just for intellectual property reasons. Here, only two of those techniques will be briefly described. • Direct gene transfer to protoplasts: Protoplasts are prepared from plant cells by enzymatic digestion of the cell wall, which represent a physical barrier for naked DNA transfer. Protoplasts can take up plasmid DNA through their plasma membranes by electroporation: protoplasts are incubated in an appropriated solution with the foreign DNA (the plasmid containing the construct to be transferred), in especial recipients BIOTECHNOLOGICAL PLANT BREEDING

with two opposite electrodes; a short, high-voltage electric pulse is then applied through the solution by discharging a capacitor. This physical treatment leads to formation of transient pores in the protoplast plasma membrane allowing plasmid DNA entry into the cell. • Chemical transformation is also possible, incubating the protoplasts in the presence of compounds such as polyethylene glycol (PEG) that also transiently modify the plasma membrane. In both cases, the protoplasts can regenerate the cell wall in culture in a few hours and from these cells, transgenic plants can be obtained as for any other gene transfer method. These procedures are historically important; they were developed in the early 1980s as a possible alternative to Agrobacterium and before biolistic methods were available. The model plant Nicotiana tabaccum was mostly used to optimise the electroporation and chemical transformation conditions and also for a systematic molecular and genetic analysis the GM plants initially obtained together with their offspring: integration of the foreign DNA in the tobacco genome, number of copies, segregation and stability of the transgene for several generations. • Microinjection: Plasmids containing the DNA constructs to be transferred can be injected into plant cells using micropipettes and micromanipulators under a microscope. This method works efficiently and allows introducing the DNA directly into the nucleus, increasing the rate of integration events. However, it requires very complex equipment and highly trained personnel and is very time-consuming as cells must be injected one-by-one. Other, less reproducible methods for gene transfer to plant cells, which will not be discussed further, include for example: electroporation of intact cells (not protoplasts), laser-mediated uptake of DNA, ultrasound and agitation (vortex) in the presence of silicon carbide fibers. Although regeneration of transgenic plants using some of these methods has been reported on rare occasions, they are mainly used for transient expression experiments. CHAPTER 4

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7. Biotech Crops Transgenic plants are extremely useful tools for the study of plant biology at the molecular level and have been used in basic research since the methods described above were developed in the mid 1980s. However, the first transgenic crops were not commercially grown until 1996. Since then, the area dedicated worldwide to cultivate genetically modified or genetically engineered (GE) crops – now more often called ‘biotech crops’ – has increased year by year at a very fast rate. In 2011, 16.7 million farmers in 29 countries – 10 industrial and 19 developing countries – grew biotech crops in 160 million hectares. This represents an impressive 94fold increase as compared to the relatively modest 1.7 million hectares cultivated in 1996, making biotech crops the fastest adopted crop technology in history. This land is now distributed about equally between developed and developing countries, but the growth rate for biotech crops was in 2011 twice as fast, and twice as large, in the latter as in the former. It is expected, therefore, that soon the cultivated area of biotech crops in developing countries will exceed that of industrial countries. It is also important to note that more that 90% of those farmers (15 million) were small, poor farmers in developing countries mostly in India and China, each owning on average less than 0.5 ha of arable land. In 2011, more than one million hectares were used to grow GM crops in ten countries, led by the USA and followed by Brazil, Argentina, India, Canada and China. Another nine countries had biotech crop land of between 105 and 106 ha and the remaining ten countries – including those EU countries which have not banned (legally or de facto) this biotechnology – grew biotech crops in less than 105 ha. All of them cultivated one or more of the aforementioned crop species, expressing the traits described below, independently of combined in the same crop. All these data clearly show the success of this technology. Biotech crops deliver substantial and sustainable socioeconomic benefits to farmers, big and 56

small, due to higher crop yields, more stable production and reduction in labour and energy costs. At present, these benefits are based almost exclusively, in the cultivation of only four major crops: soybean, maize, cotton and rapeseed (canola), and in the expression of only two major traits: herbicide tolerance and insect resistance.Therefore, there is still a big potential for increasing agricultural production by large-scale growth of additional biotech crops and commercialization of GM plants with new traits which are being developed at present in public and private laboratories all over the world.

7.1 Improvement of Agronomic Traits The biotech crops cultivated now in our fields belong to the so-called ‘first generation’ of GM plants and were designed to help farmers to obtain higher yields and more stable production, mostly by introducing two specific traits, herbicide tolerance (HT) and insect resistance (IR) as well as – to a much lesser extent – virus resistance (VR).

7.1.1 Herbicide Tolerance One of the factors that reduce crop productivity is the presence of weeds competing with the cultivated species for resources such as water and nutrients in the soil. The use of herbicides is, therefore, necessary to maintain the high crop yields characteristic of modern intensive agriculture. Most transgenic plants grown commercially are tolerant to the herbicide glyphosate. This compound inhibits specifically the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), blocking the synthesis of aromatic amino acids. Since EPSPS does not exist in animals – which do not synthesise aromatic amino acids and must include them in the diet – glyphosate has very low toxicity for animals including humans; moreover, it is easily inactivated by soil bacteria. Because of these characteristics, glyphosate has been used long before the first glyphosate-tolerant GM plants were produced by expressing an EPSP synthase of bacterial origin that is insensitive to the



herbicide. However, since glyphosate is not selective and affects all plants, it could only be applied as a pre-emergence herbicide to eliminate the weeds before sowing the crop. The availability of tolerant GM plants allows a more controlled and efficient use of glyphosate which is applied at lower doses during crop growing. Large-scale cultivation of glyphosate-tolerant biotech crops has contributed to a significant reduction in the worldwide use of ‘classical’, highly toxic and contaminating herbicides such as paraquat or synthetic auxins.

7.1.2 Insect Resistance The second common trait introduced in biotech crops is resistance against insect pests such as the European corn borer which attacks maize plants causing a substantial reduction of yields and, consequently, important economic losses for the farmer. This resistance phenotype is based in the expression in the GM plants of Bt proteins from the bacterium Bacillus thuringiensis. This proteins, also known as Cry proteins (from ‘crystal’, as they are found crystallised in the bacterial spores) are ‘biologic’ insecticides; commercial preparations of B. thuringiensis spores have been used for many years against insect pests and are some of the few insecticides recommended and accepted by ‘organic’ farmers. Different Bt proteins are toxic and very specific for different insects: some Lepidoptera, Coleoptera and Diptera species are killed when the ingested proteins form pores in the membranes of intestine epithelial cells. They do not have any effect on other organisms, including beneficial insects and higher animals which do not possess receptors for Bt proteins. Cultivation of Bt-expressing biotech crops has resulted in a reduction in the use of chemical insecticides which are highly toxic and contaminating and also allows a more efficient elimination of insects. In addition, Bt plants are more resistant to fungal infection which is favoured by insect attack. Therefore, GM insect resistant crops generally contain much lower levels of fungi-produced toxins than their conventionally grown (or ‘organic’) counterparts. BIOTECHNOLOGICAL PLANT BREEDING

7.1.3 Other Crops, Other Traits Apart from the four major biotech crops (soybean, maize, cotton and rapeseed), which together represent more than 99% of the land used worldwide to grow transgenic plants, there are other minor commercial GM crops cultivated in several countries including alfalfa, sugarbeet, squash, potato, sweet pepper, poplar, papaya and tomato. In some cases, the transgenic variety represents a very high percentage of the total production of the corresponding crop even higher than 90% for example alfalfa in USA. Regarding the traits expressed in biotech crops, herbicide tolerance and insect resistance represent more than 99% (in terms of cultivated area), either independently or combined; the trend in the last years have been to generate transgenic crops with ‘stacked’ traits expressing simultaneously two or more proteins conferring, for example, tolerance to glyphosate and one or more insect pests. Apart from these two characters a small percentage of transgenic crops have been engineered for virus resistance but they represent less than 1% of the global land area used to grow biotech crops – although they may be important at the local level; for example VR-resistant papaya has saved the production of this fruit in Hawaii devastated by infection with Papaya ringspot virus (PRSV).

7.2 Perspectives for The Near Future The arable land dedicated to grow GM crops worldwide will no doubt continue a steady increase, probably with additional countries, both industrial and developing, joining the club of ‘biotech’ nations. Apart from increasing the production of the present crops, transgenic varieties of other cultivated species already developed will be commercially grown. For example, China has started the large-scale production of its own Bt-rice. Years ago Monsanto developed and tested a HT-wheat that it has not yet commercialised. More recently, Brazil developed and approved a biotech bean resistant to the bean golden mosaic virus (BGMV) which causes the most devastating disease of bean CHAPTER 4

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in Latin America; it is expected that the GM bean will be in the market in the next few years after carefully conducted field trials have demonstrated their complete resistance to the virus. Biotech crops will significantly contribute to the increase in food production needed to feed a growing world population,which will reach over 9 x 109 people by 2050. However, the present commercial GM crops will not be sufficient, even if they are grown in larger areas. New traits are required to substantially increase crop productivity and the development of abiotic stress-resistant transgenic varieties represents the most promising approach to reach this goal especially for plants tolerant to soil salinity, drought and elevated temperatures. Until now, only a drought-tolerant maize variety, jointly developed by Monsanto and BASF, has been commercialised, but intensive research is in progress in many public and private laboratoriess to generate biotech crops resistant to environmental stress, the main cause of reduction of crop yields worldwide.

Review Questions and Answers Q1. What is the domestication syndrome? A1. Domestication made possible the evolution from wild traits (more competitive under nature conditions) to man-useful traits. Such modifications are called domestication syndrome. Q2. Provide some examples of plant traits modified during domestication. A2. Wild relatives usually show fruits with very high seed/flesh ratios (e.g. wild relatives of tomatoes, peppers, eggplants, cucumbers, melons), spines in the whole plant (e.g. wild eggplants), toxic, bitter or pungent compounds in the fruit or the whole plant (wild relatives of tomatoes, peppers, eggplants, watermelons), extremely sour fruits. By contrast, due to domestication, crop species are more productive and produce edible and larger and fleshier fruits in comparison to their wild relatives. Other traits developed during plant domestication are related to harvesting, particularly in species such as cereals or beans. In this regard, wild 58

relatives of wheat or corn (teosinte) show fragile ears/cobs when ripe, and spikes disarticulate upon ripening, which facilitates the dispersion of seeds by means of wind or animal hits, while the domesticated species keep the seeds in their rachis until harvesting. Similarly, many wild relatives of grain legumes (e.g. peas, chickpeas, lentils) are dehiscent to release their seeds, while domestication involved the development of partially dehiscent or even indehiscent genotypes. Q3. Explain the main group of activities in Plant Breeding. A3. Plant Breeding involves three main group of activities: i) searching for new sources of variation useful for the objectives and challenges faced by the breeder (and their exploitation), ii) the introgression of traits of interest in commercial materials from sources of variation, and iii) selection of the most suitable genotypes at two different levels: in screenings aimed to identify the new sources of variation mentioned in i, or in screenings of segregating progenies derived from ii within a breeding program. Q4. Explain the significance of PCR´s discovery for molecular markers. A4. The Polymerase Chain Reaction technique (PCR) developed by Mullis enabled to amplify a single (or a few) copies of a specific DNA sequence, providing millions of new copies. This technique, provided basis for new molecular markers. Q5. Mention some advantages provided by marker-assisted selection in plant breeding. A5. i) Identification of individuals carrying a particular gene/trait of interest at the seedling stage. which allows accelerating the selection process or decreasing the number of plants for subsequent evaluations of other traits, ii) MAS work at a DNA sequence-level. which enables breeders to detect the gene(s) involved in the trait of interest, regardless of environmental conditions, iii) MAS makes possible the selection of recessive genes or mutants (usually gene mutations are recessive against the wild variant of the gene), iv) gene pyramiding. This strategy



consists of tracking simultaneously by MAS two or more genes favourable for a specific trait. For example, this allows enhancing the response to a particular disease as several loci providing different mechanisms of resistance can be piled up in one cultivar or breeding line. Q6. How can molecular markers help breeders to predict hybrid heterosis? A6. The genetic distance among genotypes (GD) has been used on many occasions as a good predictor of heterosis or hybrid vigour. Thus, molecular markers are used to calculate GD among pairs of parent lines, which is estimated on the basis of similarity indexes (S) as 1-S. High GD values can then be used by breeders as criteria to reduce the number of hybrids to be obtained and evaluated. Such hybrids, or a part of them, will be expected to show heterosis at a higher frequency than hybrids obtained randomly. Q7. What does genetic fingerprint mean? Explain it uses in plant breeding. A7. DNA polymorphisms patterns can be used to distinguish varieties or genotypes, which is also known as DNA fingerprinting. Generally, they are neutral markers. Thus, although they are not necessarily associated to traits of interest as in MAS, they are useful to detect differences in DNA sequence between plant varieties. DNA fingerprinting is particularly useful for: i) complementary characterization of plant materials to protect or claim for proprietary rights, ii) to ensure the purity of propagating seed material, or iii) to discard genetic contamination in elite germplasm by uncontrolled cross pollination. Q8. Are recombinant frequencies and physical distances equivalent in gene mapping? Please explain. A8. No. It must be taken into account that cM is a distance unit based on recombinant frequencies and therefore, they do not fit exactly physical distances. In fact, the frequency of crossing over may be different among chromosome regions. Consequently, genetic recombination between two points can be BIOTECHNOLOGICAL PLANT BREEDING

more frequent in some regions than in others. Q9. How can genetic maps assist breeders? A9. Gene maps may help breeders: i) to develop new markers for MAS. Thus, when a trait of interest is detected into a new source of variation (e.g. a wild material showing resistance to a virus), a gene map may help to identify which marker(s) is(are) close to the DNA region responsible for this trait. Once identified a useful marker, it can be directly used for MAS or, alternatively, used for assessing new markers still closer to the DNA region of interest; ii) to search regions of the genome related to the expression of traits which depend on a cascade of metabolic steps, iii) to search candidate genes or allelic variants for a particular trait, iv) to facilitate the development of near isogenic lines (NILs), recombinant inbred lines (RILs) and other introgression lines. Q10. Define in vitro rescue of immature embryos and mention some applications. A10. Embryo rescue involves the excision and isolation of immature embryos from developing seeds and their subsequent cultivation under in vitro conditions to obtain the plantlets. This is of particular interest to regenerate potentially abortive embryos in many cases of interspecific crossings limited by post-zygotic barriers. In those cases on which the endosperm does not evolve properly, but the embryo can divide, this strategy allows isolating and regenerating young immature embryos before abortion takes place. Moreover, the in vitro culture of immature embryos has also other applications: i) shortening breeding cycles, ii) overcoming seed dormancy or sterility, iii) obtaining valuable haploid materials, or iv) micropropagation. Q11. Explain the somatic hybridization and the exploitation of somaclonal variation techniques and their application in plant breeding. A11. Somatic hybridization consists in fusing two protoplasts (somatic cells without cell wall) from two different parent lines (heterokaryon) and to regenerate this fusion product into a complete plant. Protoplast fusion can be done using chemiCHAPTER 4

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cal agents or physically by electrofusion. After that, heterokaryons are selected from a mixture of fusion products (heterokaryons, homokaryons, unfused protoplast, and even fusion products of ≥3 protoplast) and then, cultivated in vitro to regenerate the (somatic) hybrid plants. This technique allows i) the genetic exchange between species unable to hybridize sexually, ii) the exchange of cytoplasmic material, iii) the production of polyploids and iv) the development of introgression lines. Finally, another application is the production of cybrids, which are the combination of the nuclear genome from one parent cell line and the cytoplasm of another. These materials are very useful to produced cytoplasmic sterile plants which are of great interest in breeding plants in which control pollination is difficult. Somatic hybridization has been used to produce new varieties of different species, especially in Citrus, Solanum and Brassica. Somaclonal variation involves the spontaneous appearance of genotypic variants under in vitro culture conditions at relatively high frequencies. The frequency of this phenomenon depends on several factors like tissue type and age, genotype, levels of growth regulators in the culture media. Although it is considered a problem in common micropropagation protocols (the objective is to achieve thousands of true-to-type clones), somaclonal variation may provide breeders with new genetic variation useful for their breeding programs in both sexually and vegetatively propagated species. Unfortunately, mutations are not directed and phenotypes of low or nil interest for breeders can appear, as well as the loss of morphogenic capacity on long term cultures. Moreover, some somaclones may show instability due to epigenic variation or the occurrence of aneuploidy, infertility and other disorders. This technique has been used sucessfully to breed new varieties of rice, tomato and banana, among others. Q12. Explain the advantages of double haploid production in plants. A12. Although haploids are not economically useful per se as they are sterile, they can be used to obtain double-haploids by treating these hap60

loid individuals with certain physical or chemical agents (provoking genome duplication). In practice, these double-haploids are 100% homozygous, which means that breeders can achieve highly inbred lines very fast, in just one generation, instead of the several generations needed in conventional breeding programmes. Thus, the production of haploids and subsequent double-haploids is firstly based on the regeneration of complete plants from (haploid) gametes followed by the duplication of their (haploid) chromosome set. The most popular haploid and double haploid production method, due to is simplicity and good results, is the culture of anthers. This method has been used successfully in many species. Additionally, double haploids are especially useful to develop mapping populations such as RILs and NILs. Q13. What are binary vectors and their role? A13. Binary vectors have been developed for Agrobacterium-mediated genetic transformation of plants. They are relatively small plasmids containing the construct to be transferred, flanked by the T-DNA borders and lacking all ‘natural’ T-DNA sequences: crown gall tumours will not be then induced in the host plant. These constructs typically contain the foreign gene of interest together with the required regulatory sequences of plant origin (promoter, transcription termination signal) for its expression in the transgenic plant, as well as a selection gene – conferring, for example, antibiotic (e.g., kanamycin or hygromycin) or herbicide (e.g., BASTA) resistance – also with the appropriate expression elements. In addition, and outside the artificial T-DNA, the binary vector carries those elements essential for replication and selection in bacteria: an origin of replication for Escherichia coli, an origin of replication for Agrobacterium tumefaciens (or one single origin of replication functional in both bacteria), and a bacterial selectable marker gene, conferring resistance to an antibiotic. In the latter case, a bacterial promoter is required. Q14. Explain briefly the main steps in plant transformation. A14. Generation of GM plants usually involves



three successive steps. Firstly, the foreign DNA is introduced into plant cells. Suitable constructs, including the gene of interest and a selectable marker gene, with the corresponding regulatory sequences (promoters and terminators) are prepared in vitro and transferred to plant cells in culture, or to plant explants, using a range of methods (Agrobaterium-mediated transformation, biolistics, microinjection, protoplast transformation by electroporation or chemichal agents). Secondly, the introduced DNA integrates into the plant genome, in one or more copies, at a single locus or at several loci. This process is not yet completely understood, but involves non-homologous recombination. Integration occurs essentially at random, although ‘open’ or ‘relaxed’ chromatin regions are preferred and, therefore, integration is more frequent during DNA replication and in transcriptionally active regions of the plant genome. Thirdly, full plants are regenerated from the individual transformed cells, using in vitro culture, generally by organogenesis, although other processes, such as somatic embryogenesis or androgenesis can also be used, depending on the starting material. Expression of the selection marker gene during regeneration allows the elimination of non-transformed cells. This step is usually the bottle neck of the whole process of transgenic plant generation. Q15. Explain briefly the methods for gene transfer in plant transformation. Q15. The most common plant transformation method is the Agrobacterium-mediated plant transformation. Thus, a binary vector is introduced into an Agrobacterium strain containing a ‘disarmed’ Ti plasmid, in which the whole T-DNA region has been removed but contains the virulence region (vir): upon activation of vir gene expression, all proteins necessary for gene transfer are synthesised in the bacteria. The DNA construct cloned in the binary vector in-between the T-DNA borders is then transferred to plant cells by the same mechanism used for transformation with the ‘natural’ T-DNA sequences. The simplest procedure for Agrobacterium-mediated plant transformation is known as the ‘leaf-disc’ method, on which leaf maBIOTECHNOLOGICAL PLANT BREEDING

terial is cut in small pieces and co-cultivated with the Agrobacterium strain containing the binary vector. During this period, bacteria attach to the leaf pieces and vir gene expression is activated. Finally, leaf fragments are then removed from the bacterial culture and incubated for one or two days in agar plates, without any selective agent, to allow transfer of the T-DNA to the plant cells. Although there are protocols which allow the Agrobacteriummediated transformation of many species which are not infested in nature by this bacterium, these procedures are still difficult. As an alternative, direct gene transfer methods have been developed, which rely on the transfer to plant cells of naked plasmids containing the appropriate constructs: the gen of interest and the selectable marker gene, with the corresponding plant promoters and transcription termination signals. As direct methods we have: i) particle bombardment or ‘biolistics’ (or bioballistics), ii) direct gene transfer to protoplasts and iii) microinjection. Biolistics is the most successful and effective direct gene transfer method. Gold or tungsten microparticles (ca. 1 µm diameter) are coated with plasmid DNA, accelerated to high speed using different devices, and shot into the target plant material. The DNA is released within the cells, and can get integrated into the plant genome. In the direct gene transfer to protoplasts, they can take up plasmid DNA through their plasma membranes. Such process can be stimulated physically by electroporation which leads to formation of transient pores in the protoplast plasma membrane, allowing the entrance of the DNA plasmid into the cell or chemically by using agents such as polyethylene glycol (PEG), that also modify transiently the plasma membrane. Finally, in microinjection, plasmids containing the DNA constructs to be transferred can be injected into plant cells using micropipettes and micromanipulators under a microscope. Q16. Describe the main applications of transgenic plants in agriculture. A16. The main examples of transgenic plants in agriculture are: i) herbicide tolerance and ii) insect resistance. In this regard, the use of herbicides is necessary to maintain the high crop yields characCHAPTER 4

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teristic of modern intensive agriculture as weeds compete with the cultivated species for resources such as water and nutrients in the soil. Thus, many transgenic plants grown commercially are tolerant to the herbicide glyphosate. GM plants were produced by expressing an enzyme of bacterial origin that is insensitive to the herbicide. However, since glyphosate is not selective and affects all plants, it could only be applied as a pre-emergence herbicide to eliminate the weeds before sowing the crop. Availability of tolerant GM plants allows a more controlled and efficient use of glyphosate, which is applied at lower doses during crop growing. Insect resistance follows herbicide resistance in terms of importance in biotech crops. Thus, GM maize resistant to the European corn borer has been developed. This insect can cause remarkable yield losses. GM resistant cultivars are based in the expression of Bt proteins (also known as Cry proteins) from the bacterium Bacillus thuringiensis. Bt proteins are toxic, and very specific for different insects (e.g. Lepidoptera, Coleoptera, Diptera), which are killed when the ingested proteins form pores in the membranes of intestine epithelial cells. Although these proteins have been utilized in ‘biologic’ insecticides (commercial preparations of B. thuringiensis spores) for many years, cultivation of Bt-expressing biotech crops has resulted in a reduction in the use of chemical insecticides, which are highly toxic and contaminating, and also allows a more efficient elimination of insects.

Further Readings 1. Abbo S, Pinhasi van-Oss R, Gopher A, Saranga Y, Ofner I, Peleg Z. Plant domestication versus crop evolution: a conceptual frame work for cereals and grain legumes. Trends in Plant Science 2014; in pres. 2. Amrhein N, Schab J, Steinrücken HC. The mode of action of the herbicide Glyphosate. Naturwissenschaften 1980;67:356-357. 3. Antanaviciute L, Fernández-Fernández F, Jansen 62

J, et al,. Development of a dense SNP-based linkage map of an apple rootstock progeny using the Malus Infinium whole genome genotyping array. BMC Genomics 2012;13:203. 4. Bateman A, Quackenbush J. Bioinformatics for next generation sequencing. Bioinformatics 2009;25:429. 5. Beard JB, Cookingham PO. WJ. Beal: pioneer applied botanical scientist and research society builder. Agronomy Journal 2008;100:4-10. 6. Borlaug NE, Dowswell CR. Feeding a world of ten billion people: a 21st century challenge. Edited by Tuberosa R, Phillips RL, Gale M, In the wake of the double helix: from the green revolution to the gene revolution. Proceedings of the international congress Bologna: Avenue Media 2005;3-24. 7. Bosland PW, Votava E. Peppers: vegetable and spice capsicums. New York: CABI Publishing 2000. 8. Bravo A, Gill SS, Soberón M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 2007;49:423-35. 9. CIMMYT. Genomic selection and prediction in plant breeding 2013. 10. Collins NC, Tardieu F, Tuberosa R. Quantitative Trait Loci and Crop Performance under Abiotic Stress: Where Do We Stand? Plant Physiology 2008;147:469-86. 11. Comai L, Young K, Till BJ, et al,. Efficient discovery of DNA polymorphisms in natural populations by EcoTILLING. Plant J 2004;37:778-786. 12. Diamond J. Guns, Germs and Steel. New York: WW Norton & Co 1997. 13. Edenberg HJ, Liu Y. Laboratory methods for high-throughput genotyping. Edited by Al-Chalabi A, Almasy L, Genetics of complex human diseases: a laboratory manual. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press 2009;183-93. 14. Falconer DS, Mackay TFC. Introduction to quantitative genetics. Essex: Pearson Education Ltd. 1996.



15. Fiorani F, Schurr U. Future Scenarios for Plant Phenotyping. Annual Review of Plant Biology 2013;64:267-91. 16. Forster BP, Heberle-Bors E, Kasha KJ, Touraev A.The resurgence of haploids in higher plants. Trends in Plant Science 2007;12:368-75. 17. Harlan JR. Crops & Man (2nd edition). Madison: American Society of Agronomy and Crop Science Society of America;1992. 18. Heffner EL, Sorrels MR, Jannink JL. Genomic selection for crop improvement. Crop Science 2009;49:1-12. 19. Hellens R, Mullineaux P, Klee H. A guide to Agrobacterium binary Ti vectors. Trends in Plant Science 2000;5:446- 51. 20. Herrera-Estrella L, Depicker A, Van Montagu M, Schell J. Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector. Nature 1983;303:209-13. 21. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoot RA. A Binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 1983;303:179-80. 22. Hooykaas PJJ, Schilperoot RA. The Ti-plasmid of Agrobacterium tumefaciens: a natural genetic engineer. Trends in Biochemical Science 1985;10:307-9. 23. Horsch RB, Fry J, Hoffmann N, Neidermeyer J, Rogers SG, Fraley RT. Leaf disc transformation. Edited by Gelvin SB, Schilperoort RA, Dordrecht: Kluwer Academy Publishers 1988. 24. James C. Global status of commercialized Biotech/GM crops: 2011. ISAAA Brief No 43. Ithaca: ISAAA 2011. 25. Johnson AT, Veilleux RE. Somatic Hybridization and Applications in Plant Breeding. Edited by Jannick, Plant Breeding Reviews, vol. 20. New York: John Wiley & Sons, Inc 2010;167-225. 26. Kaczmarek M, Koczyk G, Ziolkowski PA, et al,. Comparative analysis of the Brassica oleracea genetic map and the Arabidopsis thaliana genome. Genome 2009;52:620-33.


27. Kalloo G, Bergh BO. Genetic Improvement of Vegetables. Oxford: Pergamon Press 1993. 28. Kanta K. Intra-ovarian pollination in Papaver rhoeas L. Nature 1960;188:683-84. 29. Klein TM, Wolf ED, Wu R, Sanford JC. High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 1987;327:70-3. 30. Klug W, Cummings MR. Concepts of Genetics. Upper Saddle River (NJ): Prentice Hall 1997. 31. Kraakman ATW, Niks RE, Van den Berg PM, et al,. Linkage disequilibrium mapping of yield and yield stability in modern spring barley cultivars. Genetics 2004;168:435-46. 32. Larkin P, Scowcroft W. Somaclonal variation - a novel source of variability from cells cultures for plant improvement. Theoretical and Applied Genetics 1981;60:197-214. 33. Macek T, Pavlikova D, Mackova M. Phytoremediation of metals and inorganic pollutants. Edited by Singh A, Ward OP, Applied bioremediation and phytoremediation. Berlin Heidelberg: Springer-Verlag; 2004;135-157. 34. McCallum CM, Comai L, Greene EA, Henikoff S. Targeted screening for induced mutations. Nature Biotechnology 2000;18:455-57. 35. Pacurar DI, Thordal-Christensen H, Pacurar M, Pamfil D, Botez C, Bellini C. Agrobacterium tumefaciens: From crown gall tumors to genetic transformation. Physiological and Molecular Plant Pathology 2011;76:76-81. 36. Panjabi P, Jagannath A, Bisht NC, et al,. Comparative mapping of Brassica juncea and Arabidopsis thaliana using Intron Polymorphism (IP) markers: homoeologous relationships, diversification and evolution of the A, B and C Brassica genomes. BMC Genomics 2008;9:113. 37. Park M, Jo S, Kwon JK, et al,. Comparative analysis of pepper and tomato reveals euchromatin expansion of pepper genome caused by differential accumulation of Ty3/Gypsy-like elements. BMC Genomics 2011;12:85. 38. Paszkowski J, Shillito RD, Saul M, et al,. Direct gene transfer to plants. EMBO Journal 1984;3:2717-22. CHAPTER 4

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39. Peleman JD, Van der Voort JR. Breeding by design. Trends in Plant Science 2003;8:330-334.

49. Sharma DR, Kaur R, Kumar K. Embryo rescue in plants a review. Euphytica 1996;89:325-37.

40. Pérez-de-Castro AM, Vilanova S, Cañizares J, Pascual L, et al,. Application of Genomic Tools in Plant Breeding. Current Genomics 2012;13:179– 95.

50. Sorrells ME, La Rota M, Bermudez-Kandianis CE, et al,. Comparative DNA sequence analysis of wheat and rice genomes. Genome Research 2003;13:1818-27.

41. Pistorius R. Scientists, Plants and Politics: A History of the Plant Genetic Resources Movement. Rome: International Plant Genetic Resources Institute 1997.

51. van Hintum ThJL, Brown AHD, Spillane C, Hodgkin T. Core Collections of Plant Genetic Resources. Rome: International Plant Genetic Resources Institute 2000.

42. Poland JA, Rife TW. Genotyping-by-Sequencing for Plant Breeding and Genetics. The Plant Genome 2012;5:92-102.

52. Weising K, Nybom H, Woll K, Kahl G. DNA fingerprinting in plants: Principles, methods and applications (2nd edition). Boca Raton: CRC Press 2005.

43. Potrykus I. Gene transfer to plants: Assessment of published approaches and results. Annual Review Physiology and Molecular Biology of Plants 1991;42:205-25. 44. Russell PJ, Genetics (5th edition). Menlo Park (CA): Benjamin/Cummings Publishing Company 1998. 45. Russo EB. Taming THC: potencial cannabis synergy and phytocannabinoid-terpenoid entourage effects. British Journal of Pharmacology 2011;163:1344-64. 46. Sacco A, Di Matteo A, Lombardi N, et al,. Quantitative loci pyramiding for fruit quality traits in tomato. Molecular Breeding 2013;31:217-22. 47. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain terminating inhibitors. Proceedings of the National Academy of Sciences 1977;74:5463–7.

53. Xu Y. Molecular Plant Breeding. London: CABI Pub¬lishing 2010. 54. Yuan L, Tang J, Wang X, Li C. QTL analysis of shading sensitive related traits in maize under two shading treatments. PLoS ONE 2012;7:e38696. 55. Zambryski P, Tempe J, Schell J. Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants. Cell 1989;56:193-201. 56. Zhang Q, Ma B, Li H, et al,. Identification, characterization and utilization of genome-wide simple sequence repeats to identify a QTL for acidity in apple. BMC Genomics 2012;13:537. 57. Zohary D, Hopf M, Weiss E. Domestication of Plants in the Old World. Oxford: Oxford University Press 2012.

48. Senda M, Takeda J, Abe S, Nakamura T. Induction of cell fusion of plant protoplasts by electrical stimulation. Plant and Cell Physiology 1979;20:1441-3.

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PRODUCTION OF THERAPEUTIC RECOMBINANT PROTEINS IN TRANSGENIC ANIMALS

CHAPTER 5 PRODUCTION OF THERAPEUTIC RECOMBINANT PROTEINS IN TRANSGENIC ANIMALS Haydar Bağış

CONTENTS Summary ...............................................................................................................................

67

68

1. Production of Transgenic Mice Expressing Human Interferon Gamma ........................

1.1 Construction of The Hybrid Human Interferon Gamma Genes ................................. 68

1.2 Generation of Transgenic Mice ..................................................................................

69

1.3 Identification of Transgenic Mice ...............................................................................

69

1.3.1 PCR Analysis of The Integrated Transgene ........................................................

69

1.4 Milk Collection for hIFN-g Analysis .........................................................................

70

1.5 Concluding Remarks .................................................................................................

70

Review Questions and Answers ..............................................................................................

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CHAPTER 5 / H. BAGIS


Summary

T

ransgenic animals are animals which have been genetically transformed by splicing and inserting foreign animal or human genes into their chromosomes. The production of the first transgenic mice with the aid of microinjection techniques was published in 1980. Since then hundreds of transgenic mouse lines have been created. Due to the similarity in physiology and gene function between humans & rodents, transgenic mice can mimic aspects of human disease. For example excellent mouse models have facilitated the study of the pathophysiology of Type 2 diabetes and much progress has been made in the development of mouse models of human Hepatitis Virus B. A compelling example of the power of transgenic technology in dissecting human disease pathogenesis is the work on Alzheimer’s disease. A transgenic animal has a piece of foreign DNA stably integrated into its genome. This foreign DNA usually consists of a construct containing a specific promoter region, a gene coding for the protein of interest, and other regulatory elements to protect or enhance gene expression. Transgenic animals are extensively used to study in vivo gene function as well as to model human diseases. The most commonly used techniques for producing transgenic mice involves the pronuclear injection of foreign DNA into fertilized oocytes. Most eggs do not survive or do not have the transgene, but between 1% and 30% of the eggs injected can produce a live transgenic animal. Two techniques, pronuclear microinjection and nuclear transfer (cloning) have been the predominant techniques used to produce transgenic animals. However, nuclear transfer using pre-selected transgenic donor cells is rapidly superceding pronuclear microinjection as the method of choice. The protein coded by the transgene is known as a recombinant protein as it results from the recombination of the desired gene with the controlling elements. Transgenic production of domestic animals has been moving steadily from the research laboratory to commercial use. Many human proteins such as human gamma interferon, hormones, Antitrombin III, Protein C

and Factor VIII and other pharmaceuticals have been successfully expressed so far exploring the transgenic animal system. In our previous studies, it was proven that the Mouse Whey acid protein (WAP) promoter induced proper levels of foreign proteins including human gamma interferon with the highest level up to a few milligrams per milliliter of milk even though no detectable amount of protein (less than nanogram per milliliter) was expressed in some transgenic lines. Here, we review the main strategies for introducing recombinant protein production into the transgenic mouse, as well as in other mammalians. Proteins are widely used in research, medicine and industry, but the extraction of proteins from their natural sources can be difficult and expensive. Also, the use of pharmaceutical proteins from natural sources can pose risks. For example, many people have contracted diseases from contaminated blood products or hormones. Transgenic animals are used for production of recombinant proteins for scientific, pharmaceutical, and agricultural purposes. Pharmaceutical proteins are products with much higher profit margins than those found in traditional agricultural products. Transgenesis in mice is the most often used approach to generating models of human disease. The ability of transgenic animals to produce biologically active recombinant proteins in an efficient and economic manner was demonstrated a long time ago and has attracted substantial attention and investments. February 6, 2009 – GTC Biotherapeutics and Ovation Pharmaceuticals, Inc. announced today that the US Food and Drug Administration (FDA) approved ATryn® (Antithrombin [Recombinant]) for the prevention of peri-operative and peri-partum thromboembolic events in hereditary antithrombin deficient patients. ATryn®, GTC’s recombinant human antithrombin, has been approved for use in the United States and Europe. Gene ‘pharming’ entails the production of recombinant biologically active human proteins in the mammary glands of transgenic animals (Table 1). The benefits of transgenic protein production include safety from human viral contamination, low-cost, high volume


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production, correct post-translational modifications, and applicability to a wide range of complex proteins and peptides. All of the recombinant proteins produced and marketed to date are produced in bacterial, yeast, and mammalian cell culture systems. Currently, human pharmaceutical proteins are either isolated from human blood or produced as recombinant proteins in fermentation systems. The first method involves the risk of microbial contamination. The second method, production of recombinant therapeutic proteins through mammalian cell culture and bacterial fermentation systems, is very expensive. Production of these proteins in transgenic dairy animals provides significant advantages in areas of health risk and production costs. Transgenic animals are costly to produce and they have high value. The cost of making one transgenic animal ranges from $21.000 to $350.000 and only a small portion of the attempts succeed in producing a transgenic animal. A Wisconsin firm that clones transgenic calves for human pharmaceutical production estimated that one transgenic animal can produce, in its lifetime, $220 to $310 million worth of pharmaceuticals. A recent Financial Times article reported that a herd of 610 transgenic cows could supply the worldwide demand of some pharmaceuticals, for example, human serum albumin used in the treatment of burns and traumatic injuries. A number of “new” technologies, including Chinese hamster ovary (CHO), insect cell culture and baby hamster kidney (BHK), transgenic plants, and milk from transgenic livestock, have products in development. The ability of transgenic animals to produce biologically active recombinant proteins in an efficient and economic manner has been demonstrated a long time ago and has attracted substantial attention and investments. Besides economic considerations, the other advantages of the transgenic animal bioreactors are: 1) easy purification of the recombinant protein; 2) biological activity of the protein; 3) possibility for production of glycosylated proteins; 4) increased stability of the proteins in the milk. Several investigators are the pioneers in expressing recombinants 68


in the milk of transgenic mice under the control of mammary gland-specific promoters. For example; Human interferon-gamma (hIFN-g) is a key cytokine endowed with multiple biological activities such as antiviral, antibacterial, antiparasitic, antiproliferative, and immunomodulatory activity. We demonstrated that hIFN-gamma was efficiently expressed in the mammary gland of transgenic mice under the control of the mWAP gene promoter and exhibits biological properties without any adverse. Drug

Disease/Target

Animal

Alpha-lactalbumin

Anti-infection

Cow

Alpha-1 Antitrypsin (AAT)

Deficiency leads to emphysema

Sheep

CFTR

Cystic Fibrosis

Sheep, Mouse

Human Protein C

Thrombosis

Pig, Sheep

Tissue Plasminogen Activator (TPA)

Thrombosis

Mouse, Goat

Human Antithrombin III

Thrombosis

Goats

Factor VIII

Hemophilia

Cow

Glutamic Acid Decarboxylase

Type 1 Diabetes

Goat

Human protein C

Thrombosis

Goat

Collagen I-II

Rheumatoid arthritis

Cow

Table 1. List of therapeutic proteins produced in transgenic animals milk.

1. Production of Transgenic Mice Expressing Human Interferon Gamma 1.1 Construction of the Hybrid Human Interferon Gamma Genes To construct transgenic mice, the human Human Interferon Gamma open reading frame was cloned downstream of the murine whey acidic gene promoter. Schematic diagram of the transgene construct is presented in Figure 1. Briefly, the pTZ57R vector, containing hIFN-g open reading frame was


used as template for the amplification of hIFN-g open reading frame by PCR. Subsequently, the hIFN-g amplicon was digested by KpnI and BclI restriction enzymes and cloned into KpnI and BclI-digested pWAP-T vector. The resulting vector has been designated as pWAP-hIFN-g. The latter was digested by SalI and EcoRI restriction enzymes and the 2.383 bp linear DNA was purified from an agarose gel by NucleoSpin Extract II and reconstituted in the microinjection buffer. The pure DNA was quantified and diluted to 2–4 ng/ml in 10 mM Tris-HCl and 0.25 mM EDTA buffer at pH 7.4 and stored at -20oC until further use.

followed by an i.p. injection of 7,5 IU human chorionic gonadotropin (hCG; Pregnyl, Organon) 48 hr later, then placed individually with stud males (Hogan et al., 1994; Bagis et al., 1997). Embryos were collected at 20 h post-hCG. And pronuclear stage embryos were selected under an inverted DIC microscope at 200X magnification for the presence of the second polar body and two pronuclei (Figure 2). Approximately 2 pl of DNA solution (2-4 ng/ml) was microinjected into the male pronuclei (Figure 3). Following microinjection the embryos were transferred into the right oviducts of CD1 females (10 weeks old) with 0.5-day pseudo pregnancy and allowed to develop to term.

Figure 2. Embryos Collection from Oviduct in Mouse.

Figure 3. Pronuclear Injection into One-Cell Stage Mouse Embryos.

Figure 1. Gene Construct.

1.2 Generation of Transgenic Mice All animal care and use procedures were in accordance with the International Guide for the Care and Use of Laboratory Animals and were approved by Animal Care and Use Ethics Committee. As embryo donors, 4 to 5 weeks old hybrid (C57BL/6J x BALB/c) CB6F1 mice were used. The animals were kept in an animal house under a 14:10 hr light/ dark cycle (lights on at 05.00 hr), at 21 ± 0.5oC and 50-60% humidity conditions. Females were superovulated by intra-peritoneal (i.p.) injections of 10 IU pregnant mare serum gonadotropin (PMSG; Sigma; G-4877) at 10.00 hr,

1.3 Identification of Transgenic Mice 1.3.1 PCR Analysis of The Integrated Transgene Total genomic DNA was extracted from tail tissue according to Hogan et al. (1994). Polymerase chain reaction (PCR) was performed on a Biometra Personal Thermocycler (Tampa, FL). PCR was carried out in 20 µl total reaction volumes, each containing 20 pmol of each primer, 1x PCR master mix (MBI Fermentas, Hannover) and 100 ng of template genomic DNA. The reaction mixture was heated to 95oC for 5 min, followed by 30 cycles each consisting of 60 sec denaturation at 95oC, 60 sec annealing at 60 oC, 90 sec of primer


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extension at 72 oC and then a final 10 min extension at 72 oC. Specific primers used to amplify a 495 bp fragment of the target sequence were 5’-CCAGGAGAAGTCACCCTCAGATG-3’ (sense) and 5’-ACTTGTATATTTCATGGTGGCG-3’ (antisense) (Figure 4). As an internal control, mouse b-globulin primers were used to amplify the 494 bp target sequence.

Figure 5. Milk Collection from Transgenic Mouse Mammary Glands (from picture Haydar Bagis Bilateral project, TUBITAK).

1.5 Concluding Remarks • The promise of biopharming, that is the actual commercial production of pharmaceuticals and other bioproducts, is nearing fulfilment. Improvements in molecular and reproductive techniques and strong economic incentives have continued to drive the implementation of transgenic technology to domestic animals. • The world market is growing for human pharmaceutical products. Figure 4. PCR.

1.4 Milk Collection for hIFN-g Analysis At day 8-19 of lactation, the mothers were separated from their pups for 3 h. They were anaesthetized using ketamine and xylazine combination (diluted with 4 ml sterile saline to get 15 mg/ml ketamine and 1 mg/ml xylazine). After 15 min they were injected i.p. with 5 IU of oxytocin (Sigma). Milk was collected from both lactating transgenic and wildtype mice at the 8th -19th day after parturition. Milk samples were harvested every other day by a special mouse breast pump developed by The Scientific and Technological Research Council of Turkey (TUBITAK) and Marmara Research Center (MRC), Gebze) (Figure 5). Generally, approximately 70-350 μl of milk was collected from the mouse in a single milking. Milk samples were diluted 1:2 with Ca+/Mg+ - free 1.5x Dulbeccos’s and defeated by repeated centrifugation (14.000 x g) for 30 min. Milk samples were stored frozen at -800C until use. 70


• Producing transgenic animals is still relatively expensive, however, costs are trending down and transgenic animals have certain advantages over traditional laboratory methods for producing human proteins. • The two major animal systems to produce pharmaceutical proteins in milk and egg white have recently been technically improved and their use as an essential source. • The different mammals which participate in this industrial activity are rabbits, pigs, sheep, goats and cows. • Rabbits are sufficient to produce several kilos of proteins per year. This species is particularly flexible allowing a rapid generation of founders and scaling up. • For large scale recombinant protein production transgenic cows and goats are needed. • The number of companies involved in the production of recombinant pharmaceutical proteins is expected to increase. This will result from the improvement of the different systems and from the fact that the oldest patents are becoming obsolete in the coming years. Competition may thus become very intense (Houdebine et al 1994).


• Other recombinant proteins expressed in the milk of transgenic animals are currently in development, including human albumin, human growth hormone, C1-esterase inhibitor, alpha1-antitrypsin, as well as monoclonal antibodies. It is likely that in the coming years several regulatory filings in various jurisdictions will be submitted with some of these products, realizing the promise of transgenic technology in offering a safe cost-efficient alternative for the production of complex recombinant proteins (GTC Biotherapeutics. Press Release. 2006 June 2. Available at www.gtc-bio.com)

Review Questions and Answers

Q5. Transgenic mice have been used to produce which of the following proteins that is used against cancer cell line? a. Casein b. Plasminogen activator (tPA) c. Amyloid precursor proteins d. Gamma İnterferon A5. (d) Q6. Animal pharming can be defined as? a. Growing animals for farming b. Programming animals to produce novel products c. Generating transgenic animals for farming d. None of the above

Q1. What are transgenic animals?

A6. (b)

A1. Animals harboring foreign DNA in their genome.

Q7. DNA microinjection into eggs has been used to produce which of the following transgenic animals?

Q2. Which of the following is a genetic vector for transgenic animal production? a. Plasmid b. Phage c. Cosmid d. All of these A2. (a) Q3. Which protein has been produced generating a transgenic sheep that is used for replacement therapy for indivuduals at risk from emphysema? a. Plasminogen activator (tPA) b. Alpha anti trypsin (AAT) c. Casein d. Amyloid precursor proteins A3. (b) Q4. Where abouts is DNA injected into mice? a. Pronuclei b. Cytoplasm c. Both (a) and (b) d. None of these A4. (a)

a. Mice b. Pig c. Chicken d. All of the these A7. (d) Q8. When is DNA microinjected into the fertilized egg? a. After the fusion of male and female nuclei b. Before the fusion of male and female nuclei c. At the time of fusion of male and female nuclei d. Any time, it can be infected. A8. (b)

Further Readings 1. LeRoith D, Gavrilova O. Mouse models created to study the pathophysiology of Type 2 diabetes. International Journal of Biochemistry & Cell Biology. 2006;38:904-12. 2. Quitschke WW, Steinhauff N, Rooney J. The effect of cyclodextrin-solubilized curcuminoids on amyloid plaques in Alzheimer transgenic mice: brain uptake and metabolism after intravenous and subcutaneous injection. Alzheimer’s Research & Therapy 2013;28;5:16.


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3. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proceedings of the National Academy of Sciences 1980;77:7380-7384. 4. Clark AJ, Bessos H, Bishop JO, Brown P, Harris S, Lathe R, McClenaghan M, Prowse C, Simons J, Whitelaw CBA, Wilmut I. Expression of human anti-hemophilic factor IX in the milk of transgenic sheep. Biotechnology 1989;7:487-492. 5. Drohan WN, Zhang DW, Paleyanda RK, et al,. Inefficient processing of human protein C in the mousse mammary gland. Transgenic Research 1994;3:355- 364 6. Ziomek C, Kutzko J, Sherman L, Gavin W, Hendry C, Hayes M, Cole ES. Viral and prion safety of recombinant human antithrombin. Annals of Hematology 2003;82:S95. 7. Houdebine LM. Production of pharmaceutical proteins from transgenic animals. Journal of Chemical Technology and Biotechnology;34:269287. 8. Houdebine, M. Transgenic animal bioreactors. Transgenic Research 2000;9:305-320. 9. Devinoy E, Thpot D, Stinnakre MG, et al,. High levels of production of human growth hormone in the milk of transgenic mice: the upstream region of the rabbit whey acidic protein (WAP) gene targets transgene expression to the mammary gland. Transgenic Research 1994;3:79-89. 10. Bagis H, Aktoprakligil D, Gunes C, Kankavi O, Akkoc T, Cetinkaya G, Taskin AC, Arslan K, Arat S, Sekmen S, Turgut G, Tas A, Dundar M, Vania L. Tsoncheva, Ivan G. Ivanov. “Expression of Biologically Active Mouse Whey Acidic Protein/ Human Interferon Gamma Hybrid Gene in the Milk of Transgenic Mice. Biochemical Genetics 2011;49:251-7. 11. Bagis H, Akkoc T, Tas A, Aktopraklıgil D. Cryogenic Effect of Antifreeze Protein on Transgenic Mouse Ovaries and Production of Live Offspring by Orthotopic Transplantation of Cryopreserved Mouse Ovaries. Molecular Reproduction and Development 2008;75:608-613. 12. Bagis H, Tas A, Kankavi O. Determination of the Expression of Fish Antifreeze Protein (AFP) in several Tissues and Serum of Transgenic Mice in F7 72


generation at the Room Temperatura. The Journal of Experimental Zoology Part A 2008;309:25561. 13. Bagis H, Aktoprakligil D, Odaman H, Yurdusev MN, Gazi Turgut, Sekmen S, Arat S, Cetin S. Stable transmission and transcription of Newfoundland ocean pout type III fish antifreeze protein (AFP) gene in transgenic mice and hypothermic storage of mouse gamets with AFP. Molecular Reproduction and Development 2006;73:14041411. 14. Bagis H, Arat S, Mercan Odaman H, Aktopraklıgil D, Caner M, Turanlı Tahir E, Baysal K, Turgut G, Sekmen S, Çırakoğlu B. Stable transmission and expression of the hepatitis B virus genome in hybrid transgenic mouse until F10 generation. The Journal of Experimental Zoology Part A 2006;305:420. 15. Bağış H, Papuççuoğlu S. Studies on The production of Transgenic Mice. Turkish Journal of Veterinary & Animal Sciences 1997;21: 287-292. 16. Bağış H, Odaman H, Sağırkaya H, Dinyéss A. Production of Transgenic Mice from Vitrified Pronuclear Stage Embryos. Molecular Reproduction and Development 2002;61:3. 17. Wall RJ, Pursel VG, Shamay A, McKnight RA, Pittius CW, Hennighausen L. High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine. Proceedings of the National Academy of Sciences 1991;88:1696-1700. 18. Stinnakre M-G, Devinoy E, Thepot D, Chene N, Bayat-Samardi M, Grabowski H and Houdebine L-M. Quantitative collection of milk and active recombinant proteins from the mammary glands of transgenic mice. Animal Biotechnology 1992;3:245-255 19. Rudolph NS. Biopharmaceutical production in transgenic livestock. Trends in Biotechnology 1999;17:367-74. 20. Gordon K, Lee E, Vitale JA, Smith AE, Westphal H, Hennighausen L. Production of human tissue plasminogen activator in transgenic mouse milk. Bio/Technology 1987;5:1183-1187. 21. Yann Echelard, Carol A Ziomek, Harry M Meade Production of Recombinant Therapeutic Proteins in the Milk of Transgenic Animals. BioPharm International 2006; 19:8.

RECENT ADVANCES IN ANIMAL BIOTECHNOLOGY

CHAPTER 6 RECENT ADVANCES IN ANIMAL BIOTECHNOLOGY Edo D’Agaro


1. Selective Breeding of Farm Animals ..............................................................................

75

1.1 BLUP Procedure (Sire model) .....................................................................................

76

1.2 Single Trait Animal Model ..........................................................................................

77

2. Molecular Methods ......................................................................................................... 78

2.1 Genetic Markers ............................................................................................................ 78

2.2 High-Throughput Marker Genotyping .......................................................................... 79

2.3 DNA Chips .................................................................................................................. 79

3. Identifying Markers for Specific Traits ........................................................................... 79

3.1 Quantitative Trait Loci Mapping in Livestock .............................................................. 80

3.2 Genetic Mapping and Linkage Analysis ......................................................................... 80

3.3 Identification and Analysis of a QTL ............................................................................. 81

4. Genomic Selection .......................................................................................................... 83



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74

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Summary

T

raditionally, improvement of livestock has mainly focused on the selective breeding of individuals with superior phenotypes. This simple approach was, in the past, extremely successful in increasing the quantity of agricultural output. However, new information, now, available on the organisation and functioning of the genome could be used in breeding programmes to improve a range of traits. Many production traits are under the control of several genetic loci, each of which contributes to the genetic variation and hence are called quantitative trait loci (QTL). Genetic markers linked to QTLs and single point polymorphisim (SNPs) within the trait genes, can be used to choose superior animals by means of genomic selection procedures.

1. Selective Breeding of Farm Animals So far, selection of farm animals has been extremely successful in increasing the quantity and quality of products and reducing production costs. Nowadays, in order to respond to the new public demands and to develop a sustainable industry, it is necessary to fully exploit the new technologies available for the selection of genetically superior animals. The major challenge that faces animal geneticists is to identify markers for genes that control the genetic variation in the target traits. Two types of marker can be considered. First, markers those are sufficiently close to the gene on the chromosome such that, in most cases, alleles of the marker and gene are inherited together. This type of marker is called a linked marker. At the population level, alleles of linked markers cannot be used to predict the phenotype until the association between the marker and gene is known (called ‘phase’). To determine the phase, inheritance of the marker and gene has to be studied in a family. However, information on phase is only valid within that family and may change in subsequent generations through recombination. The second type of marker is a

functional polymorphism of the gene that controls the trait. These markers are called direct markers. Once the functional polymorphism is known, it is possible to predict the effect of particular alleles in all animals of the population, without having to determine the phase in advance. Therefore, direct markers are more useful than linked markers for predicting the phenotypic variation of target traits within a population. A further complication is that the mechanisms of genetic control differ between traits. The genetic variation of some traits is directly controlled by a single gene (monogenic traits), which may have a limited number of alleles. In the simplest situation, a gene will have two alleles: one allele is associated with one phenotype and the other allele with a different phenotype (e.g. black versus brown coat colour in cattle: the brown coat colour occurs as a result of a mutation). However, traits that are important in livestock production are generally more complex and have a very large range of variation in the observed phenotype. Growth rate and milk yield are examples of two traits that exhibit a continuous phenotypic variation. Such traits are called quantitative traits. The variation in quantitative traits is controlled by several genetic loci, each of which is responsible for a small amount of the overall variation. The following steps should be considered in planning a genetic program in farm animal population: • identify clear selection objectives for all the heritable and economically important traits. Objectives must be clearly defined, keeping in mind that the project goals are not static but they can be changed following the acquisition of new techniques. The main target of a genetic improvement program is to achieve a greater production output. • apply an appropriate selection criteria; • use effective techniques for genetic evaluation (e.g. BLUP animal model); • effective control of inbreeding. The following techniques can be used for the genetic evaluation of broodstock using information from relatives:


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• selection index: combines information from different sources in order to get the highest genetic progress; • one single trait index: combines information from different classes of relatives; • multiple trait index: combines information from different traits.

1.1 BLUP Procedure (Sire Model) The phenotypic expression of a quantitative trait, within an animal population, is the result of the action of numerous genetic and environmental factors. For example, we assume that milk production of a cow is the result of sum of the genetic effects inherited from the father, mother, grandmother, etc. and several environmental factors such as the lactation number, year, age, season of calving, farm in which production takes place and other as the health status, food, etc. Each of these genetic and environmental effects can be positive or negative, large or small and interact each other. The BLUP is a modern statistical method that allows to evaluate simultaneously all these different genetic and environmental effects. This technique allows disentangling genetic from management and feeding effects giving accurate predictions of the breeding values for all the animals in the population. BLUP method (Best Linear Unbiased Prediction) is a mixed model which allows estimating of fixed effects (BLUE estimate) and predictions of breeding values (BLUP) of animals. Some advantages of the BLUP procedure, compared to the traditional methods, can be summarized as follows: • variance of estimation errors is a minimum (best estimation); • estimation of random effects is correct (unbiased estimation); • correlation between estimated and true expected breeding value (EBV) is maximised; • uses the same weights as a selection index but fixed effects are more properly corrected; • is a flexible method.

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The BLUP method has the following disadvantages: • heritability coefficients have be to known, • several models can be used. The sire model was the first mixed model to be used in selection for schemes of dairy cattle (progeny EBV = 0.5 of sire EBV). In Example 1 a simple milk production system using three farms and 20 cows is described. Example 1 The average milk production of 20 cows reared in 3 different farms (A, B and C) and daughters of 3 bulls (1, 2, 3) was the same 90,000 (kg). Bull Farms

A B C Average

4

1

2

-

9,000

2

3 1 3 9,000

3

- 2 5 9,000

Average 9,230 9,000 8,800 Table 1. Milk production (kg).

Phenotypic records (Yijk), effects of farms (α) and bulls (β) of the example 1 (Table 1) are expressed by means of the following linear model: Yijk = μ + αi + βj + εijk

(1)

equation 1 can be rewritten as follows: Yijk = μ + α1 + α2 + α3 + β1 + β2 + β3 + εijk

(2)

Cow 1, reared in farm 1 and daughter of the bull 1 is: Y111 = 1 1 0 0 1 0 0 + ε 111 In a similar way, for each cow in the population, an equation can be calculated and a matrix (called incidence matrix) constructed. All equations can be easily solved using the following mixed linear model: y = Xb + Zu + e

(3)


where: y = n x 1 vector of observations where n = number of records; b = p x 1 vector of fixed effects where p =number of levels for the fixed effects; u = q x 1 vector of random animal additive genetic effects where q = number of levels for the random effects; e = n x 1 vector of residual effects; X = incidence matrix n x p ; Z = incidence matrix n x q. The variance-covariance of y : V (y) = ZGZ’ + R

(4)

equation for each animal in the population. The main difference with the BLUP model is that the additive genetic relationship matrix (A) (G = A σ2u ) considers all the relationships between the evaluated animals. A matrix accounts for covariances between random additive effects of related animals and effects of selection over generations. The simpler model is a single animal model: one trait, fixed effects and additive genetic effects. In the example 2 consider the following data of eight calves (Table 2) (after Mrode, 2005) (five with phenotypic records and three without records linked though the pedigree): Example 2 Calf

Sex

R = dispersion matrix for the error term

1 S4

male

The solutions of equations are:

2 S5

where:

Dam

Sire

Pre-weaning gain (kg)

-

S1

4.5

female

S2

S3

2.9

3 S6

female

S2

S1

3.9

4 S7

male

S5

S5

3.5

5 S8

male

S3

S3

5.0

G = dispersion matrix for random effects

a 1 = 9,1707 a 2 = 9,000 a 3 = 8,766 b1 = -0,60

b 2 = -0,06 b 3 = 0,66

Solutions of the mixed model are BLUP estimates for sires and their sum is equal to 0. According to the statistical analysis of fixed effects, milk yield in farm A was better than in farm B, which reflects the overall average (9,000 kg), while farm C shows a negative effect on milk production. Notice that, if we consider only the milk yield of daughters, the production level would have been the same for the three groups (9,000). Six daughters of bull 1 were reared in farm A with a positive effect on milk yield. This is an advantage for bull 1 and therefore the genetic value is negative. On the other hand, bull 3 who had 5 out of 7 daughters that were produced in farm 3C (negative value) has a positive value.

Table 2. Example of a single trait animal model.

The BLUP animal model is as follows: yij = + si +ai + eij (5) where: yij = pre-weaning gain (kg)

μ= overall mean si = sex effect

aj = additive genetic effect eij = random error The variance-covariance of y is:

V (y) = ZAZ’ σ2a + I σ2e (6)

1.2 Single Trait Animal Model

where:

The BLUP animal model method is, among the BLUP procedures, the most advanced technique for the evaluation of all animals in the population. Conceptually, the animal model is an evolution of previous mixed model allowing to construct an

A = numerator relationship matrix I = identity matrix

Assuming σ2a = 20 kg2 and σ2ae = 40 kg2 and after several matrix computations (R software) we can obtain the following solutions:


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S1 = 0.0984 S2 = -0.0187 S3 = -0.0410 S4 =-0.0086 S5 =-0.1857 S6 = 0.1768 S7 = -0.2494 S8 = 0.1826 Multi trait models are an extension of the single trait case.

2. Molecular Methods 2.1 Genetic Markers Genetic markers can take a number of forms and in the simplest definition are: an observable genetically controlled variation that follows a Mendelian pattern of inheritance. An example of such a marker is the coat colour, which was used in early selection programs for establishing various breeds of livestock. More recently, protein polymorphisms, particularly blood groups, have been used to verify pedigrees in several species including man, horses, cattle, and dogs. Such protein markers are generally impractical for use as genomic markers as they are relatively infrequent and, in some cases, the protein is expressed at low levels, making detection difficult, or is only found in specific tissues. The earliest form of deoxyribonucleic acid (DNA) marker to construct the first true genomic maps was the restriction fragment length polymorphism (RFLP). Restriction fragment length polymorphism analysis uses bacterial restriction enzymes to bind and cut DNA molecules at highly specific recognition sequences that are typically four to six base pairs (bp) long. There are a large number of restriction enzymes, each of which has a different specific recognition site. In theory, variations at restriction enzyme recognition sites in the genome could be identified by digesting genomic DNA with a restriction enzyme and observing the pattern of fragments produced by gel electrophoresis. However, due to the large number of recognition sites within the genome, too many fragments are produced for simultaneous examination. Therefore, the fragments produced by a genomic digest are separated by gel electrophoresis and transferred onto a solid matrix. Polymorphisms within a particular gene are then revealed by hybridisation with a radioactively

78


labelled gene-specific probe. This approach allows studying each locus separately and is a powerful way to examine variations at a particular point in a given gene. The RFLP technique has been used to screen for carriers of genetic defects, e.g. bovine leukocyte adhesion deficiency (BLAD) in Holstein cattle. On occasion, it is desirable to screen several loci simultaneously, e.g. to produce a ‘genetic fingerprint” to identify specific individuals. Micro and mini-satellite are repeated sequences (five to 20 copies of 2-4 and 20-50 bp tandem repeats, respectively) which may occur at 10 to 100 different sites in the genome. Variations in the number of repeats, present at a particular locus, give rise to a large number of alleles. These variable number tandem repeat (VNTR) markers have been used to identify relationships between individuals in wild populations, to verify pedigrees in many species, and in genetic mapping studies. Insertions or deletions of DNA sequences (indels) and single changes of the nucleotide sequence (SNPs) may occur throughout the animal genome. Single nucleotide polymorphisms (SNPs) are much more frequent than indels and occur at high frequency in both non-coding regions and coding regions of the genome. Current estimates from genome sequencing projects indicate that SNPs occur every 200 bp on average. Single nucleotide polymorphisms, within coding regions, may have no effect on the protein coded by the gene (silent polymorphisms) or may result in a change in a single amino acid in the protein sequence. The latter are most likely to be the functional polymorphisms that are responsible for the phenotypic variation of important traits. However, in some cases, the functional polymorphism responsible for variations in a trait may occur in intergenic regions (DNA sequences located between genes), e.g. insulin-like growth factor 2 (IGF2). An advantage of using single nucleotide polymorphism markers is that they can be detected by automatic methods. It is now possible to rapidly genotype hundreds to thousands of individuals for several thousand SNP markers within a few hours.


2.2 High-Throughput Marker Genotyping Species-specific genome sequences and large expressed sequence tag (EST) collections aid in extensive data mining for genetic markers. Genome sequences provide direct access to large numbers of microsatellite markers and by database sequence mining or re-sequencing to single nucleotide polymorphisms (SNPs). This marker resource can be used to map and fine map QTL, and identify quantitative trait genes and causative quantitative trait nucleotides. Microsatellites provide an efficient medium-resolution mapping tool for initial QTL mapping, but often lack resolution for fine mapping when investigating breeds with limited effective population size, instead of experimental crosses. Furthermore, microsatellites appear to be much more common in mammals than in birds. The SNPs occur at a frequency of approximately 1/200 base pairs of nucleotide sequence in pigs, cattle and chickens and represent a vast marker resource. Considering the costs of high-throughput genotyping, it should be noted that successfully applying DNA pooling strategies for QTL mapping in farm animals can significantly reduce the number of individuate that need to be analysed. High-throughput microsatellite genotyping, performed in multiplex polymerase chain reactions (PCR) with capillary electrophoresis instruments, is cost effective, reliable and straightforward. Various concepts and techniques are available for high-throughput SNP genotyping. All SNP genotyping technologies have two components, as follows: • a method for discriminating between alternative alleles; • a method for reporting the presence of alleles in a given DNA sample. The general methods for allele discrimination are hybridisation/annealing, primer extension and enzymatic cleavage. However, methods for reporting the presence of alleles in a given sample are much more varied. Most signal detection platforms follow the fate of a label in real time or at the assay

end point. Mass spectrometry (MS) is unique in that it can be employed to detect the product of the discrimination assay directly.

2.3 DNA Chips In this approach, PCR-amplified DNA which contains the polymorphisms of interest is hybridised to an oligonucleotide microarray with thousands of different oligonucleotides of known sequence. These oligonucleotides are gridded onto a solid substrate such as a glass microscope slide or silicon wafer. Genotyping in most commercially available devices is achieved by allele-specific hybridisation or allele-specific primer extension. Allele identification results from the emission of signals from specific positions on the chip, which allows the sequence around the polymorphism to be deduced. An example is the oligonucleotide chip technology offered by Affymetrix and Illumina. Genomic DNA is digested with a restriction enzyme, ligated to adapters and amplified by PCR. The amplified DNA is then fragmented, labelled and hybridised to the array. Each allele is represented and interrogated by overlapping oligonucleotides and after washing allele-specific hybridisation (i.e. fluorescence) intensities are recorded and used for genotype calling.

3. Identifying Markers for Specific Traits Identifying genes that control particular traits can be approached in a number of ways. For simple monogenic traits it may be possible to postulate which gene(s) control the observed variation through studying the physiology of the trait and identifying the biochemical pathways that are involved. This information can be coupled with patterns of expression in various tissues, which can then be used to facilitate cloning of the gene involved. In some cases, knowledge of the gene that controls a similar phenotype in another species may suggest a potential analogous candidate gene that could be considered in the species of in-


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terest. The candidate genes are then studied to determine if the polymorphisms within the gene can account for the observed variation in the trait. This approach clearly requires good prior knowledge of the trait and the underlying physiology or relevant information regarding the trait from other species. For more complex traits, several genes are likely to contribute to the observed variation. Even with a good knowledge of the physiology of such a trait, other genes may be involved that are not obviously part of the biochemical pathways known to contribute to the variation in the trait. Therefore, for complex traits it may be better to make no prior assumptions regarding the physiology of the trait or the possible candidate genes that control the trait. Instead, a genetic mapping approach can be used. Marker assisted selection (MAS) is very useful in the following cases: • heritability is low; • traits are sex limited; • traits are not measurable for selection (e.g. longevity, fertility); • traits are difficult to measure (e.g. disease resistance, feed efficiency, carcass traits).

3.1 QTL Mapping in Livestock Animal production traits (e.g. milk production, pre-weaning gain) are under the control of a large number of loci (polygenes) whose effects are very small (infinitesimal). However, it is unlikely that all genes coding for milk production have an infinitesimal effect. A more realistic hypothesis is that two types of genes are present in the genome: many genes that have an infinitesimal effect, which represent the majority and, others showing a greater effect (major genes). The latter are called also QTL, i.e. genes that have a great effect on a quantitative trait. Identification of QTLs is essential in order to obtain a significant genetic improvement in the animal breeding schemes. These new insights are important in terms of better understanding of the genetic structure of quantitative traits as, in particular, in for improving the efficiency of selection 80


schemes, especially in the case of low heritability traits or when selecting traits that are sex limited. Genome scans for QTLs have been realized in the most important farm livestock species.

3.2 Genetic Mapping and Linkage Analysis Gene mapping techniques can be used to assign a gene to a precise chromosomal position. The advent of new DNA techniques has made available a set of powerful tools to determine the location of genes. There are several types of genetic maps, each characterized by its resolution: • cytogenetic map – genes are localized within chromosomal regions by means of banding patterns. BAC based clones are used to generate a detailed cytogenetic map. Cytogenetic maps have not a high resolution; • genetic map – the observed recombinations between two loci are used as a measure of the distance that separates them. The unit of measurement is a centimorgan (cM). The cM is defined as the distance between two genes for which 1 out of one hundred is recombinant; • physical map – genes and markers are sorted along a chromosome assigning the real distance (expressed in base pairs, bp). This map is obtained by dividing the genome into fragments and then reassembling them to form contigs (clones partially overlapping) covering the entire region of interest; • linkage maps – these maps indicate the order of genes on chromosomes and their relative distance. The most important use of linkage maps is to identify associations between makers and QTLs; • radiation hybrid maps – these maps are obtained by x-rays fragmentation of a hybrid cell line and fusion of the chromosome fragments to a recipient rodent cell. Radiation hybrid maps can be used to localize loci distances and to construct integrated linkage and physical maps;


• nucleotide sequence – this is the map with the highest resolution, indicating the exact nucleotide sequence between two loci. Over the years several methods have been developed for gene mapping: • somatic cell hybrids – cell cultures of different organisms are treated with Sendai virus or polyethylene glycol. Sendai virus has the ability to simultaneously attack two different cells and, having small size, the cells merge into a single cell. With the subsequent union of nuclei, a hybrid cell is produced. By these means, specific cellular functions or protein products are revealed allowing the localization of specific genes on chromosomes; • in situ hybridization on chromosomes - due to its high resolution, the fluorescence in situ hybridization (FISH) technique is often used. This method allows to construct a specific probe for a given gene and to study its hybridization. The localization of the probe reveals the chromosomal region of the gene; • linkage mapping- linkage analysis of genes and markers is used to study their segregation over generations. Genetic distance maps are used to analyze relationships between markers and genes (QTLs). A unit of distance map (centimorgan (cM)) depends on the number of recombinations which occurs between two genes loci. During meiosis, homologous chromosomes pairs exchange segments in a process called crossing over. Gametes, after meiosis can be parental or recombinant. The smaller is the distance between two genes, the smaller the chance of recombination. The term recombination fraction (Ө) indicates the proportion of recombinant gametes. Recombinant gametes have a frequency Ө and non recombinant, 1 - Ө. Maximum Ө is equal to 0.5 (two homologous chromosomes). Recombination frequency is used to measure the genetic linkage and create linkage maps. Two loci are said to be in linkage if their recombination frequency is less than 50%. The linkage analysis requires a number of markers, distributed along all the

chromosomes, which allows to verify the possible segregation of a gene of interest with any other point of the genome. However, recombination frequencies are not additive and are inappropriate as physical distance measures. A number of mapping formulas can be used to predict the number of crossing-over from the observed recombination frequencies.

3.3 Identification and Analysis of a QTL A marker in order to be useful for QTL mapping should have the following features: • it can be detected easily and at low cost (for example with a simple PCR); • it must be evenly distributed throughout the genome; • it must be highly polymorphic, to ensure a good chance that an animal chosen at random in the population is heterozygous. Figure 1 shows an example of a marker (alleles, M1 and M2) and a QTL (alleles, Q1 and Q2).

Figure 1. Example of QTL mapping (reproduced from Gjedrem, 2005).

If two genes are very close together on a chromosome, the possibility that a recombination (crossing over) occurs between them is rather low. In this case, loci are said to be associated or in linkage. The consequence of linkage is that, during meiosis, the transmission of alleles of two associated genes is not independent. The association between the marker and gene of interest varies from family to


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family and, within the same family, it may change over generations (due to the occurrence of crossing over). Figure 2 shows an example of a marker (alleles, M1 and M2) and a QTL (Q1 and Q2) in the first generation.

Figure 2. Example of QTL mapping and segregation analysis (reproduced from Gjedrem, 2005).

The maximum distance between genetic markers and gene of interest should not exceed 10 cM. In the first instance linkage maps were based predominantly on anonymous, microsatellite markers, but more recently, SNPs and expressed sequence tags (ESTs) have been added. These genetic maps have been used to select markers that are distributed across whole genomes. These markers are then used in QTL mapping studies to study the inheritance of specific regions of chromosomes. At the beginning, microsatellite markers were used in these studies because they usually have several alleles and hence the parental origin of a particular marker can easily be determined. However, in recent years, the development of new technologies has allowed use SNPs as markers. Although presenting a much lower average heterozygosity (biallelic polymorphisms) have a denser and less uniform distribution compared to the microsatellite markers. In a family, once observed the segregation of a marker and a QTL, the probability that this segregation is due to a true relationship between the loci and not obtained by chance (two loci may be inherited together in some individuals, even if they are not associated) is calculated. Results are reported as logarithms (base 10) of the odds (LOD) scores calculated as the ratio between probability that the loci analyzed are associated and the probability that the loci are not associated. 82


LOD = log 10 (P (θ)/P(θ=0.5)

(7)

A LOD score greater than or equal to 3 means that there is an association between loci while values less than -2 are indicative of a lack of association between loci. In a population, the common origin of parts of chromosomes from one or a few ancestors (founders) characterizes a phenomenon known as linkage disequilibrium. If two alleles of two loci tend to be inherited together, with a frequency greater than expected, such alleles are said to be in linkage disequilibrium. A specific combination of alleles on a chromosome is called haplotype. The presence of this condition is closely dependent on the evolutionary history of each population and is more observable in the case of geographically isolated or selected populations. Different formulas (D, D’, r2) were proposed to measure the linkage disequilibrium (LD). D index can be expressed in terms of the rate of recombination θ: D = 0 indicates a stage of linkage equilibrium and θ=0.5; positive values of the D index indicates the presence of linkage equilibrium and thus θ is less than 0.5. In practice, identification of QTLs is achieved using a combination of genetic mapping, to localise the QTL region on a chromosome, and candidate gene or positional cloning approaches, to identify the gene within the QTL region. The analysis of QTLs is based on a statistical method that uses two types of information: phenotypic records (measurement of the trait of interest) and genotypic data by means of molecular markers. Several statistical methods are used to make the QTL analysis. The most popular techniques are the following: • Haley-Knott regression; • maximum likelihood estimation. In QTL analysis, several types of markers have been used: SNPs, microsatellites and restriction enzymes fragment length polymorphism (RFLP). In a population, animals could be sorted, theoretically, on the basis of the marker genotype and compared for differences in the productive trait under study. In practice, most experimental designs used for QTL studies, exploit the pedigree structure of livestock


populations. In this case, the aim is to find linkage disequilibrium between QTLs and genetic markers. A high power is obtained using the back cross or F2 design. However, in dairy cattle due to the within breed selection, these designs can not realistically be used. In this situation, the following experimental schemes (using paternal half sib) such as “Selective DNA Genotyping” and “Daughter Design”are used. In the first experimental design, animals from two divergent lines (high and low tails of the phenotype distribution) are genotyped. The use of specific statistical techniques, such as the interval mapping (analysis of multiple markers) is used to calculate the probability that a marker is associated with a QTL that influences the trait. Results of this analysis are presented in graphical form, where the x axis shows the results of the statistical test and the y axis the chromosome map, in units of recombination (cM). The second scheme is the Daughter Design. In this case, a heterozygous bull at a locus marker M (M1 M2) is used. The bull daughters are sorted on the basis on marker allele M that they have inherited from her father. If the averages of two groups (M1 and M2), adjusted for the main environmental factors that influence livestock production, are statistically significant. We can conclude that the marker M is associated to a QTL affecting the trait, e.g. the milk production. Fernando and Grossman (1989) showed how to use the information on a single marker linked to a QTL using the standard BLUP procedure. The notation can be easily extended to any number of QTLs linked to markers. The mix model is: y = Xb + Zu +ZQq + e where: y = vector of phenotypic records; b = vector of fixed effects; u = vector of random effects; q = vector of random QTL effects; e = vector of residual effects; X = incidence matrix n x p; Z = matrix n x u; Q = matrix n x q.

(8)

Putting λ = Ve/Vu and α = Ve/Vq (Ve = environmental variance; Vq = variance of QTL) we can estimate b and u solving conventional mixed model equations. Bayesian estimation is similar to BLUP estimation, with the exception that the variation of allelic effects is assumed to be different for every gene and is estimated using a prior distribution. In practice, several QTLs have been identified in different livestock species and countries. However, most of these QTLs were mapped with moderate to large confidence intervals, limiting their use in marker assisted selection programs. In order to transfer the QTL information to selection programs, the identification of quantitative trait nucleotides (QTNs) is needed.

4. Genomic Selection The complete sequencing of the bovine genome is a milestone in molecular biology applied to livestock. This is the result of a five year work of a large group of international researchers. The Hereford bull “Domino” and his daughter “Dominet” were the donors of biological materials used by researchers and, thus, the first animals to be completely sequenced. The latest version of the genome sequence is currently labelled 4.2 and is available free on the website http://www. ensembl.org. In general, for animal and plant breeding, the most important practical application generated by the high throughput sequencing efficiency is without doubt the feasibility of applying genomic selection schemes in breeding. Recent molecular techniques allow to typing (by means of a single high-throughput SNP genotyping assay) up to 750000 bovine SNPs spread across the whole genome. This map density is enough to find LD between markers and QTLs without a specific knowledge of the genetic population structure. Although, the theoretical principles of genomic selection were proposed in a pioneering work published in 2001 by Meuwissen et al., the practical application of this idea had to wait about 10 years. In fact, in practice, it was


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possible to apply that theory, only then new and affordable molecular technologies were made available to genotype animals. Briefly, a genomic selection scheme requires a “training” population made up of animals subjected to progeny tests and known breeding values. These animals are genotyped using a beadchip and with a very large number (50.000750.000) of molecular markers (SNPs). Using this information, the whole genome can be divided into “small” segments identified by the SNPs markers (Figure 3) and then estimate the effect of each segment on the trait of interest. SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP7 ... SNPn ------------------------------------------------------------I1 I2 I3 I4 I5 I6 Figure 3 – Segmentation of the genome in thousands of intervals (lower than 1 cM) by contiguous SNPs markers

Have established the value of each segment of the genome between two markers, the “DGV” (direct genomic index), is calculated as the algebraic sum of each of them. This information may be used to estimate the genetic value of young bulls without knowledge of the progeny testing results. In fact, these animals are genotyped for the same set of markers used in the training phase. The direct genomic and pedigree index are used to calculate the “G-EBV” that is the genomic index. The mix model is: y = Xb + Zg + e

(9)

where: y = vector of phenotypic records; b = vector of fixed effects; g = vector of random SNPs effects; e = vector of residual effects; X = incidence matrix of fixed effects; Z = incidence matrix (n individuals x m makers). This genomic model is equivalent to a conventional animal model in which the A matrix (additive relationship) is replaced by the G matrix (calculated from marker data).This idea has created a new 84


paradigm for the genetic improvement of livestock. Several advantages can be envisaged from the use of this method: • reduction of the generation interval that leads to an increase of the genetic progress and economic output; • effective selection of low heritability traits; • better estimate of relationships between animals due to the calculation of genomic kinship (genetic relationships); • better control of the level of inbreeding due to a broader animal base population.

Review Questions and Answers Q1. How is the animal model method used to evaluate animals in a population? A1. The animal model method is an evolution of previous mixed model allowing construction of an equation for each animal in the population. Q2. Outline the main features of a QTL, QTN and SNP A2. QTL (Quantitative Trait Loci) is a gene with a great effect on a quantitative trait; QTN: Quantitative Trait Nucleotide; SNP: single change of the nucleotide sequence Q3. Describe how a QTL can be identified on a chromosome A3. Identification of QTLs is achieved using a combination of genetic mapping, to localise the QTL region on a chromosome and candidate gene or positional cloning approaches. Q4. What are the main features of the linkage disequilibrium A4. Different formulas (D, D’, r2) can be used to measure the linkage disequilibrium (LD). For instance, D index can be expressed in terms of the rate of recombination θ: D = 0 indicates a stage of linkage equilibrium and θ=0.5; positive values of the D index indicate the presence of linkage equilibrium and thus θ is less than 0.5.


Q5. How is the genomic selection method used to evaluate animals in a population? A5. The actual genomic map density in bovines is sufficient to find LD between markers and QTLs without a specific knowledge of the genetic population structure.

Further Readings 1. Falconer D.S. Introduction to quantitative genetics. Longman, Harlow 1989. 2. Gjedrem T. Selection and breeding programs in aquaculture. Spinger, Dordrecht 2005.

3. Haley CS, Knott SA. A simple regression method for mapping quantitative trait loci in line crosses using flanking markers. Heredity 1992;69:315324. 4. Meuwissen THE, Hayes BJ, Goddard ME. Prediction of total genetic value using genome-wide dense marker maps. Genetics 2001;157:18191829. 5. Mrode RA. Linear models for the prediction of animal breeding values. CAB International, Wallingford 1996.


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REMOVAL OF HEAVY METALS FROM AQUEOUS SOLUTIONS THROUGH BIOSORPTION

CHAPTER 7 REMOVAL OF HEAVY METALS FROM AQUEOUS SOLUTIONS THROUGH BIOSORPTION H.Lalhruaitluanga, M.N.V. Prasad


1. Toxic Metallic Elements .................................................................................................. 89

2. Achievements over the Past Decade ................................................................................ 91

3. Biosorption .................................................................................................................... 91

3.1 Sorption ........................................................................................................................ 91

4. Activated Charcoal/Carbon ............................................................................................ 92

5. Mechanism of Heavy Metals Removal ............................................................................ 93

6. Factors Affecting Biosorption ......................................................................................... 94

6.1 Influence of pH ........................................................................................................... 94

6.2 Influence of Temperature ............................................................................................. 95

6.3 Effect of Initial Metal Concentration ........................................................................... 95

6.4 Effect of Adsorbent Dosage .......................................................................................... 95

7. Biosorption Equilibrium Models and Isotherms-Assessment of Sorption Performance .. 96

7.1 Langmuir Isotherm ....................................................................................................... 96

7.2 Freundlich Isotherm ..................................................................................................... 96

7.3 Dubinin-Radushkevich Isotherm ................................................................................. 97

7.4 Adsorption Kinetics ..................................................................................................... 97

7.5 Pseudo-First-Order Kinetic Equation ........................................................................... 97

7.6 Pseudo-Second-Order Kinetic Equation ....................................................................... 97



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88

CHAPTER 7 / H. Lalhruaitluanga, M.N.V. Prasad


Summary

W

ater is one of the most important natural resources on which life sustain. Therefore water must be clean and pure to be available for the health of human beings and other biota. Unfortunately considerably high level of pollution is globally common for water resources. Rapid industrialization and extraction of huge amount of natural resources have resulted in contamination and pollution of water resources. Large amounts of toxic waste have been dispersed in thousands of sites spread across the globe resulting in varying degrees of contamination and pollution. Thus every one of us are being exposed to contamination from past and present industrial practices and release to water resources even in the most remote regions. The risk to human and environmental health is rising and there is evidence that a cocktail of pollutants is a contributor to the global epidemic of cancers, lung and other degenerative diseases. These pollutants belong to two main classes: inorganic and organic. The challenge is to develop innovative and cost-effective solutions to decontaminate polluted environments, to make them safe for human habitation and consumption, and to protect the function of the ecosystems which support life. Much progress has been made as is highlighted in developed countries like UK, USA, Canada, Australia, Japan and European countries. Constituents of concern in groundwater include volatile organic compounds (VOCs such as chlorinated solvents.) and inorganics (metals). Heavy metals are directly or indirectly discharged into the environment by industries such as metal plating facilities, mining, fertilizer, tanneries, batteries, paper and pesticides etc. besides anthropogenic and geogenic sources.

1. Toxic Metallic Elements Toxic metallic elements do enter the aquatic ecosystems via industrial effluents such as mining, refining ores, fertilizers, tanneries, batteries, paper and pesticides etc. and poses a serious threat to environment. The major toxic metal ions hazardous to REMOVAL OF HEAVY METALS FROM AQUEOUS SOLUTIONS THROUGH BIOSORPTION

human as well as other forms of life are Fe, Se, Cr, V, Cu, Co, Ni, Cd, Hg, As, Pb, Zn etc. According to the World Health Organisation, cadmium, chromium, copper, lead, mercury and nickel are the most toxic metals. These heavy metals are of health concern due to their toxicity, bio-accumulation and persistence. Heavy metal pollution is a major problem leading to the public health hazards and environmental degradation. Heavy metals are non-mutable. Therefore, heavy metal removal from wastewater is a priority area of research for the protection of the environment and human health. Metals are essential mineral for all humans, animals, plants and organisms. However, it has been proven that large amounts of many heavy metals cannot process and dispose by human body. As a result they are deposited in various internal organs. Large deposits may cause adverse reactions and serious damage to the body. Industrial sources of various metals are shown in Table 1. The development of low-cost adsorbents and their tech-economic feasibility for wastewater and water treatment are currently extensively investigated. Traditional methods used for the removal of heavy metals from the environment are in general expensive and potentially risk due to the possibility of the generation of hazardous by-products. To remove heavy metals from the wastewater and water, physical and chemical methods have been proposed and applied, but in general, these methods are commercially impractical, either because of high operating costs or the difficulty in treating the solid wastes generated. The use of conventional methods for removal of metals ions from aqueous solutions include chemical precipitation, ion exchanger, chemical oxidation/reduction, reverse osmosis, electro dialysis, evaporative recovery and ultra filtration, solvent extraction, membrane processes, evaporation and coagulation etc. Nevertheless these techniques have disadvantages including incomplete metal removal, high consumption of reagent and energy, low selectivity, high capital and operational cost and generation of secondary wastes that are difficult to be disposed off and ineffective, when metal ions present in wastewater is at a low conCHAPTER 7

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Figure 2. Metal removal from ground water (leached into ground water from metalliferous surface water) following permeable reactive barrier.

Chromium (Cr)

Corrosion inhibitor, dyeing and tanning industries, plating operations, alloys antiseptics, defoliants and photographic emulsions. Mining, industrial coolants, chromium salts manufacturing, leather tanning

Lead (Pb)

Battery industry, fuel additives, paints, herbicides manufacturing of ammunition, caulking compounds, solders, pigments, and insecticides, Smelting operations, coal- based thermal power plants, lead acid batteries, paints, E-waste

Mercury (Hg)

Electrical apparatus manufacture, electrolytic production of Cl and caustic soda, paints, pharmaceuticals, plastics, paper products, batteries, pesticides and burning of coal and oil Chlor-alkali plants, thermal power plants, fluorescent lamps, hospital waste (damaged thermometers, barometers, sphygmomanometers), electrical appliances etc.

Arsenic (As)

Production of pesticides, veterinary pharmaceuticals and wood preservatives,Geogenic/natural processes, smelting operations, thermal power plants, fuel burning

Copper (Cu)

Textile mills, cosmetic manufacturing and hardboard production sludge, Mining, electroplating, smelting operations

Vanadium (Va)

Spent catalyst, sulphuric acid plant

Nickel (Ni)

Production of stainless steel, alloys, storage batteries, spark plugs, magnets and machinery, Smelting operations, thermal power plants, battery industry

Cadmium (Cd)

Cd-Ni battery production, pigments for plastics and enamels, fumicides, and electroplating and metal coatings

Molybdenum (Mo)

Spent catalyst

Zinc (ZN)

Figure 1. Mechanisms of metal ion interaction in soil-liquid phase.

Metal Industrial source as contaminant

Brass and bronze alloys production, galvanized metal production, pesticides and ink. Zinc smelting, waste batteries, e-waste, paint sludge, incinerations & fuel combustion Smelting, electroplating

Cobalt Tin (Co) (Sn)

centration (1-100 mg/L). An alternative method like adsorption gained important credibility due to high removal of heavy metals to very low concentration. The advantages of adsorption over conventional treatment methods include: high efficiency in detoxifying very dilute effluents, minimization of chemical and biological sludge, no additional nutrient requirement, these advantages have served as the primary incentives for developing full-scale adsorption processes to clean up heavy-metal pollution (Figures 1 and 2).

Soft drink, beer and beverage Can production Steel and alloy production, paint and varnish drying agent and pigment and glass manufacturing

Table 1. Industrial sources of metals.

90



2. Achievements Over The Past Decade Although many biological materials bind heavy metals, only those with sufficiently high metalbinding capacity and selectivity for heavy metal are suitable for use in a full-scale adsorption process. The first major challenge for the adsorption field was to select the most promising types of adsorbent from an extremely large pool of readily available and inexpensive biomaterials. A large number of adsorbents have been tested for their metal-binding capacity under various conditions. Although several proprietary adsorption processes (such as AlgaSORBTM and AMT-BioclaimTM) were developed and commercialized in the early 1990s, a lack of understanding of the mechanism underlying the metal-sorption process has hindered adequate assessment of process performance and limitations. The next real challenge for the field of adsorption was to identify the mechanism of metal uptake by dead adsorbent. Several possible mechanisms for metal adsorption have been scrutinized in Figure 3. A. Chemical precipitation 1. Hydroxide precipitation 2. Sulfide precipitation 3. Chemical precipitation combined with other methods 4. Heavy metal chelating precipitation B. Adsorption 1. Activated carbon adsorbents 2. Carbon nanotubes adsorbents 3. Low-cost adsorbents 4. Bipadsorbents C. Membrane filtration 1. Ultrafiltration 2. Reverse osmosis 3. Nanofiltration 4. Elecrodialysis

(1) biomass of higher plants and animals such as bark, (2) lignin, shrimp, krill, squid, crab shell, etc.Microbial biomass includingaggal: bacteria, fungi and yeast.

D. Ion exchange E. Coagulation and flocculation F. Flotation G. Electrochemical treatment

Figure 3. Important strategies for removal of heavy metals in wastewater. REMOVAL OF HEAVY METALS FROM AQUEOUS SOLUTIONS THROUGH BIOSORPTION

3. Biosorption Biosorption is the removal of substances (compounds, metal ions, organic etc) by inactive, nonliving, as well as excreted and derived products materials (materials of biological origin) due to high attractive forces present between the two. Biosorption consists of several mechanisms, mainly adsorption, ion exchange, chelating and diffusion through cell walls and membrances, which differ depending on the species used, the origin and processing of the biomass and solution chemistry. Adsorption is one of the processes, which besides being widely used for treatment of low soluble contaminants in water. The term adsorption refers to a process wherein a substance is concentrated at a solid surface from its liquid surroundings. It is now customary to differentiate between two types of adsorption. If the attraction between the solid surface and the adsorbed molecules is physical in nature, the adsorption is referred to as physical adsorption (physiosorption). Generally, in physical adsorption the attractive forces between adsorbed molecules and the solid surface are van der Waals forces and they being weak in nature result in reversible adsorption. On the other hand if the attraction forces are due to chemical bonding, the adsorption process is called chemisorption. In view of the higher strength of the bonding in chemisorption, it is difficult to remove chemisorbed species from the solid surface. Ion exchange is basically a reversible chemical process wherein an ion from solution is exchanged for a similarly charged ion attached to an immobile solid particle. Ion exchange shares various common features along with adsorption, in application in batch and fixed-bed processes and they can be grouped together as‘‘sorption processes’’for a unified treatment to have high water quality. Ion exchange has been fruitfully used too for the removal of colours.

3.1 Sorption The most important chemical removal process in aqueous solution is sorption, which results in short-term retention of long-term immobilization of several classes of contaminants. Sorption is transfer of ions from water to the adsorbent i.e. from CHAPTER 7

91


solution phase to the solid phase. Sorption actually describes a group of processes, which includes adsorption and precipitation reactions. Heavy metals are adsorbed on solid particles by either cation exchange or chemisorption. Cation exchange involves the physical attachment of cations (positively charged ions) to the surfaces of adsorbent by electrostatic attraction. The heavy metals once adsorbed on to the adsorbent will remain as metal atoms, unlike organic pollutants, which will ultimately decompose. Their speciation may change with time as the conditions change. Many constituents of wastewater and run off exist as cations, including most of the trace metals such as Cu, Zn, Pb, Ni and Cd. Chemisorption represents a stronger and more permanent form of bonding than cation exchange. The adsorption capacity by cation exchange or non-specific adsorption depends upon the physicochemical environment of the medium, the properties of the metals concerned and the concentration and properties of other metals and soluble ligands present. The adsorption of metals varies with the fluctuation of pH in the out flow water. The precipitated hydroxides also act as absorption sites for phyto-toxic metals present in the water. Living as well as dead (metabolically inactive) biological materials have been sought to remove metal ions. It was found that various functional groups present on their cell wall offer certain forces of attractions for the metal ions and provide a high efficiency for their removal. The mechanisms of uptake by living materials (bioaccumulation) and removal by dead ones (sorption) are entirely different. Use of dead materials has several advantages because there is no need of growing, no growth media is required and these materials are available as wastes or by-products. Biomass from seaweeds, some higher plants, all of these has been effectively and successfully utilized in metal removal studies. Many adsorbents are used in this field for the removal of heavy metals, which included adsorbent from bacteria, fungi, algae, agricultural wastes, aquatic weeds, carbon/charcoal from different sources, etc. 92


4. Activated Charcoal/Carbon Commercial activated carbon is a preferred adsorbent for the removal of micro-pollutants from the aqueous phase; however, its widespread use is restricted due to high associated costs. To decrease treatment costs, attempts have been made to find alternative activated carbon precursors. High surface areas can be obtained using either physical or chemical activation; however, combined treatment might enhance the surface properties of the adsorbent, therefore increasing its adsorption capacity. Activated carbon (AC) is known as very effective adsorbents due to their highly developed porosity, large surface area, variable characteristics of surface chemistry and high degree of surface reactivity (Figure 4). Generally, the physical activation requires high temperature and longer activation time as compared to chemical activation, however, in chemical activation the activated charcoal need a thorough washing due to the use of chemical agents. The product formed by either of the methods is known as activated carbon/charcoal and normally has a very porous structure with a large surface. The removal of heavy metals by activated carbon is economically favorable and technically easy. Therefore, activated carbons are widely used to treat waters contaminated with heavy metals. The applicability of activated charcoal for water treatment has been reported in various literatures. A compilation of some studies on different sources of activated carbons for adsorption of heavy metals is presented in Table 2.

Figure 4. Production of Bamboo charcoal in traditionally built charcoal kilns. Pictorial representation of the formation of micropores in the carbon upon activation with KOH.


5. Mechanism of Heavy Metals Removal Adsorption applications have derived their usefulness though the metal binding capacities. Adsorption is made possible by the ability of adsorbent materials to accumulate heavy metals from waste-

water through physico-chemical uptake pathways. Metal adsorption is the removal of metal ions by inactive, non-living biomass due to highly attractive forces present between the adsorbent and adsorbate. Due to the interaction of several factors on specific adsorbents, it is almost impossible to propose a general mechanism. Metal ions are attracted and bound to the biomass by a complex process

Activated carbon/charcoal

Heavy metal

Reference

Year

Potato peel

Cu(II)

Moreno-Piraján et al

2011

Palm shell

Cu(II)

Issabayeva et al

2010

Tunisian olive-waste cakes

Cu(II)

Baccar et al

2009

Pecan shell

Cu(II)

Klasson et al

2009

Hazelnut husks

Cu(II) and Pb(II)

Imamoglu and Tekir

2008

Walnut shell

Cu(II)

Kim et al

2001

Cola edulis shell

Pb(II)

Eba et al

2011

Enteromorpha prolifera

Pb(II)

Li et al

2010

Carica papaya seed

Pb(II) and Cu(II)

Omeiza et al

2010

Pumpkin seed shell

Pb(II)

Okoye et al

2010

Polygonum orientale

Pb(II)

Wang et al

2010

Euphorbia rigida

Pb(II)

Gerçel et al

2007

Coconut shell

Pb(II)

Goel et al

2005

Nut shell

Cd(II)

Tajar et al

2009

Olive stone

Cd(II)

Kula et al

2008

Ceiba pentandra hulls

Cd(II)

Rao et al

2006

Corn-stalks

Cd(II)

Youssef et al

2004

Coconut coirpith

Cd(II)

Kadirvelu et al

2003

Walnut shell

Hg(II)

Zabihi et al

2010

Organic sewage sludge

Hg(II)

Zhang et al

2005

Sago waste

Hg(II)

Kadirvelu et al

2004

Bagasse pith

Hg(II)

Krishnan et al

2003

Syzygium jambolanum

Cr(VI)

Muthukumaran et al

2011

Tamarind wood

Cr(VI)

Acharya et al

2009

Olive bagasse

Cr(VI)

Demiral et al

2008

Coconut activated carbon

Cr(VI)

Liu et al

2007

Hevea Brasilinesis sawdust

Cr(VI)

Karthikeyan et al

2005

Lotus stalks

Ni(II)

Huang et al

2011

Melocanna baccifera

Ni(II) and Zn(II)

Lalhruaitluanga et al

2011

Apricot

Ni(II)

Erdogan et al

2005

Almond husk

Ni(II)

Hasar et al

2003

Coconut shell

Zn(II)

Yanagisawa et al

2010

Olive pulp

Zn(II)

Galiatsatou et al

2002

Table 2.Different sources of activated charcoal/carbon used in heavy metals adsorption REMOVAL OF HEAVY METALS FROM AQUEOUS SOLUTIONS THROUGH BIOSORPTION

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that comprises of a number of mechanisms like adsorption on the surface and pores, chemisorption by ion-exchange, surface precipitation, complexation and chelation, entrapment in capillaries, spaces of polysaccharide network, physical adsorption, etc. The actual mechanism of metal adsorption is still not fully understood. One of the important mechanisms of heavy metals removal is ion exchange. It is reversible chemical reaction where an ion within a solution is replaced by a similarly charged ion attached onto an immobile solid particle. Ion exchange was later ruled out as the most plausible mechanism by data for the adsorption energy value, E, obtained from the D-R isotherm model. Due to the complexity of the biomaterials used, it is quite reasonable that at least some of these mechanisms are acting simultaneously to varying degree depending on the biosorbent and the solution environment. The Pb(II) removal mechanism by Rhodotorula glutinis involved direct biosorptive interaction with the biomass through ion exchange and precipitation by phosphate released from the biomass. The interaction between Pb(II) ions and the functional groups on the surface of the fungus Aspergillus parasiticus’s cell wall is also believed to occur by a combination of ion exchange and complexation processes. The interaction between zinc and Saccharomyces cerevisiaewas analysed by a combination of SEM-EDX and XAFS methods. The existence of both covalent and ionic bonds between Zn(II) and the available functional groups on a yeast cell surface was verified by displacement of H+ ,K+, Mg2+, Ca2+ and Na+ ions during zinc uptake. The release of K+, Mg2+, Ca2+, and Na+ from the biosorbent during heavy metal ion attachment was taken as evidence that ion exchange was the dominant mechanism. Metal sequestration during biosorption adheres to complex mechanisms that mainly include ionic interactions, formation of complexes between metal cations and ligands contained within the cell wall biopolymer structure and precipitation on the cell wall matrix. In their study, Cd(II) ions were attached to chemical groups containing oxygen, carbon, nitrogen and sulfur. In addition, the binding process did not al94


ter the magnesium and calcium concentration in solution, which indicates that ion exchange might not be the main mechanism for Cd(II) biosorption on the brown marine macro-algae Sargassum vulgaris. Another frequently encountered metal binding mechanism is chelation. Chelation can be properly defined as a firm binding of a metal ion with an organic molecule (ligand) to form a ring structure. As mentioned before, various functional groups including carboxylate, hydroxyl, sulfate, phosphate amides, and amino groups can be considered responsible for metal sorption. Among these groups, the amino group is the most effective for removing heavy metals, since it does not merely chelate cationic metal ions but also adsorbs anionic metal species through electrostatic interaction or hydrogen bonding. The presence of various functional groups and their complexation with heavy metals during adsorption process has been reported by different researchers using spectroscopic techniques. The structural changes were also studied using spectroscopic techniques like FT-IR, XPS, Raman microscopy, EDS, XRD, EPR, etc. Each one can reveal certain information and thus can contribute to explain the mechanism of adsorption.

6. Factors Affecting Biosorption The investigation of the efficacy of the metal sorption by the adsorbent is essential for the industrial application. The efficiency is strongly influenced by the physico-chemical characteristics of the solutions, e.g., pH, metal concentration, time, etc. A large portion of biosorption studies has been devoted to investigating this relationship.

6.1 Influence of pH pH is one of the most crucial factor in heavy metals biosorption process. It significantly influences the solution chemistry of the metals and the dissociation site on the surface of adsorbent, e.g., hydrolysis, complexation by organic and/or inorganic ligands, redox reactions, and precipitation, as well as the speciation and sorption availability for heavy metals. In a particular pH range, most metal sorp-


tion is enhanced with pH, increasing to a certain value followed by a reduction on further pH increase. In principle, the dependence of metal uptake on pH can be associated with both the surface functional groups on the adsorbent as well as the metal chemistry of the solution. Adsorbents in general are considered to contain various functional groups like hydroxyl, carboxyl, sulphydryl etc. With the change in pH of the solution, the behaviour of each of these functional groups also changes. In highly acidic pHs, these are protonated and act as positively charged species. Deprotonation of these functional groups occur on increasing pH and these behave as negatively charges moieties. It starts attracting the positively charged metal ions and there is a competition between hydrogen ions and positively charged metal ions. The “winner” can be estimated through the amount of metal adsorbed at a certain pH value. As the pH is increased from highly acidic to slightly acidic region, the positive character of adsorbent is converted to negative one. The solution chemistry is also influenced by pH. In acidic pHs, metal ions are generally positively charged and are attracted by negatively charged on the adsorbent. When the pH is increased, the amount of OH- ions is increased in the solution. Metal ions react with these OH- ions and are precipitated as metal hydroxide at some pH value. In general, metal ions are precipitated out in alkaline pH range and do not contribute towards the adsorption. This indicated that the effect of pH has upper limit to be studied. For example, cadmium is present as free Cd+ species along the whole acid pH range. Above pH 7.5, it starts to precipitate as Cd(OH)2 and thus no more available for adsorption.

6.2 Influence of Temperature Depending on the structure and surface functional groups of a biosorbent, temperature has an impact on the adsorption capacity, to a certain extent. It is well known that a temperature change alters the adsorption equilibrium in a specific way determined by the exothermic or endothermic nature of a process. Quite a number of biosorption studies have REMOVAL OF HEAVY METALS FROM AQUEOUS SOLUTIONS THROUGH BIOSORPTION

been performed concerning the effect of temperature on isotherms, metal uptake and also with respect to biosorption thermodynamics parameters. The uptake of metal ions increased with higher temperature. A more enhanced level of uptake in parallel with a temperature rise resembles the nature of a chemisorption mechanism (endothermic process). In contrast, the opposite behaviour for the temperature effect on adsorption capacity was obtained baker’s yeast that higher temperature leads to a lower adsorption capacity.

6.3 Effect of Initial Metal Concentration The initial heavy metal concentration can alter the metal removal efficiency through a combination of factors, i.e., the availability of specific surface functional groups and the ability of surface functional groups to bind metal ions (especially at high concentrations). The initial concentration acts as a driving force to overcome mass transfer resistance for metal ion transport between the solution and the surface of the biomass. At a fixed biosorbent dose, pH and temperature, the equilibrium sorption capacity improved with higher initial ion concentration. The ion removal was highly concentration dependent. The increase in the biosorbent’s loading capacity as a function of metal ion concentration was believed to be due to a high driving force for mass transfer. For most cases with low initial ion concentration, the sorption capacity or metal uptake was enhanced with higher initial ion concentrations. However, the opposite phenomenon occurred when starting with high initial ion concentration. This opposite tendency was caused by saturation of the available active sites on surface functional groups, thus preventing further metal ion uptake.

6.4 Effect of Adsorbent Dosage Adsorbent dosage is an important parameter because it determines the capacity of an adsorbent for a given initial concentration of the adsorbate. By increasing the adsorbent dose, the adsorption efficiency increases even though the amount adsorbed per unit mass decreases. In principle, with more CHAPTER 7

95


adsorbent present, the available adsorption sites or functional groups also increase and the amount of adsorbed heavy metal ions is also increased, which brought about improved adsorption efficiency. In some cases, with increased in adsorbent dose, the adsorption capacity was lesser at a higher adsorbent dose. This is due to overlapping and aggregation of adsorption sites resulting in the decrease of the surface area available to metal ions was observed. Many factors can affect biosorption. The type and nature of the biomass or derived product can be very important. Physical and chemical treatments such as boiling, drying, autoclaving and mechanical disruption will all affect binding properties while chemical treatments such as alkali treatment often improve biosorption capacity, especially evident in some fungal systems.

7. Biosorption Equilibrium Models and Isotherms– Assessment of Sorption Performance Examination and preliminary testing of solid-liquid sorption system are based on two types of investigations: a) equilibrium batch sorption tests; b) dynamic continuous-flow sorption studies. Batch equilibrium sorption studies can provide useful information on relative adsorbent efficiencies and important physico-chemical factors that affect adsorption.The equilibrium of the adsorption process is often described by fitting the experimental points with models usually used for the representation of isotherm adsorption equilibrium. Flow and other continuous system are more complex, but many column studies use breakthrough curves to assess sorbent efficiency that occur when column contents become saturated with the sorbate. A variety of models have been used to describe adsorption isotherm. The widely accepted linear adsorption equilibrium isotherm models for a single solute system are: 96


7.1 Langmuir Isotherm The Langmuir adsorption isotherm describes the surface as homogeneous assuming that all binding sites possess an equal affinity for the adsorbate, adsorption is limited to formation of a monolayer. There is no interaction between adsorbed species and the number of adsorbed species does not exceed the total number of surface sites. The adsorption isotherm equation is given as: qe = qmaxbCe/1+bCe (non-linear form) Ce/qe = 1/qmaxb + Ce/qmax (linear form)

(1) (2)

where qe= equilibrium value of sorbate uptake by the sorbent Ce= concentration of sorbate in solution (mg/L) at equilibrium qmax (mg/g) = maximum adsorption capacity corresponding to complete monolayer coverage b (L/mg) = affinity parameter related to the bonding energy of the sorbate species to the surface

7.2 Freundlich Isotherm The Freundlich isotherm defines adsorption to heterogeneous surfaces i.e. surfaces possessing adsorption sites of varying affinities. A monolayer formation accompanied by interaction between adsorbed molecules. qe = Kf Ce1/n (non-linear) (3) logqe = log Kf + (1/n) logCe (linear) (4) where Ce (mg/L) = equilibrium concentration of the sorbate qe(mg/g) = amounts of metal ions adsorbed per specified amount of adsorbent at equilibrium Kf (mg/g) = adsorption capacity n= intensity of adsorption


7.3 Dubinin-Radushkevich Isotherm

7.5 Pseudo-First-Order Kinetic Equation

In order to explain the nature of sorption processes as physical, chemical or ion exchange sorption, Dubinin-Radushkevich (D-R) isotherm model was used Inqe = InXm – βε2 (5) where qe (mol/g) = metal ion concentration on the adsorbent at equilibrium

The Pseudo-first-order equation or the so-called Lagergren model for solid/liquid systems of adsorption stated that the rate is proportional to the number of unoccupied sites. It is expressed as:

Xm (mol/g) = maximum adsorption capacity

β= activity coefficient related to the mean free energy of adsorption ε =Polanyi potential.

The Polanyi potential (ε) can be expressed as, ε = RT In (1 + 1/Ce) (6) where R (J/mol) = universal gas constant T = absolute temperature in Kelvin Ce (mol/L) = metal ion concentration in the solution at equilibrium. The activity coefficient β was further used to calculate the adsorption mean free energy ‘E’ (kJ/mol). E = 1/√-2β (7) E is the free energy for the transfer of one mole of metal ions from the infinity in the solution to the surface of the adsorbent.

7.4 Adsorption Kinetics With the scope of the literature review, two kinetic models, namely pseudo-first- order and pseudosecond-order equations have been widely used to describe adsorption data. In most adsorption kinetic studies, both pseudo- first- order and pseudo-second-order kinetic equations have been commonly employed in parallel, and one is often claimed to be better than another according to marginal difference in correlation coefficient.

log(qe– qt) = logqe – k1 t/2.303 (8) where qe(mg/g) = amounts of cadmium adsorbed on the adsorbent at equilibrium qt(mg/g) = amounts of cadmium adsorbed on the adsorbent at any time ‘t’ k1 (/min) = rate constant of pseudo-first-order adsorption The slopes and intercepts of plot of log (qe– qt) versus t were used to calculate the first-order rate constant k1 and equilibrium adsorption capacity qe.

7.6 Pseudo-Second-Order Kinetic Equation The Pseudo-second-order assumes that the rate of sorption is proportional to the square of the number of unoccupied sites. It is expressed as: t/ qt= 1/k2qe2+ t/qe (9) where k2(g/mg/min) = equilibrium rate constant of pseudo-second-order adsorption. The slopes and intercepts of plots t/qtvs t is used to calculate the pseudo-second-order rate constants k2 and qe. The shape of graph and comparison of experimental and calculated qe values can help deciding which kinetic model is followed by adsorption system. Another important factor that determined a decision is the correlation coefficient of determination R2. The kinetic models showing higher R2 value is followed by adsorption system. Water Quality Problems: The major constraints for the availability of safe drinking water are:


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• excess salinity due to geological conditions. • contamination of surface sources mainly by bioorganisms. • contamination of surface groundwater with toxic metals. Table 3 show various methods for treating Ground Water and Vadose-Zone

Figure 6. Populus sp. growing in southern India for possible application in bioremediation and biorefinery industry.

Figure 5. Plant with high hydraulic function are useful in removing volatile contaminants from ground water.

Potential Corrective Measure Overview

Limitations

In Situ Air Sparging Vapor Extraction

Clean air injected underground. Volatile constituents volatized and captured at surface.

Its constituents are brought to surface and may not be completely captured. Also this method does not treat inorganics.

Subterranean Physical Barriers

Build a wall to prevent groundwater flow to the creek (Figure 2).

Drawback is that no treatment provided and may need to be utilized with other corrective measure. Also very difficult to design and maintain a perfect barrier.

In Situ Enhanced Bioremediation

Hydrogen release compounds are injected. Indigenous microorganisms utilized to break down organics.

Drawbacks are repeat treatment required, difficult to effectively distribute injected materials, does not treat inorganics.

Pump & Treat

Drawback are constituents brought to surface Groundwater pumped to surface for for treatment, systems require extensive treatment by various methods. maintenance.

Monitored Natural Attenuation

Monitoring of reduction in plume due to naturally occurring physical, chemical, and biological processes (Figures 5-6).

Data base to be established to different ecological regions.

Table 3. Methods of treating Ground Water and Vadose-Zone water [Vadose Zone- The zone between land surface and the water table within which the moisture content is less than saturation (except in the capillary fringe) and pressure is less than atmospheric. Soil pore space also typically contains air or other gases. The capillary fringe is included in the vadose zone].

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Review Questions and Answers Q1. What are the environmental sources of metals as pollutants? A1. Toxic metallic elements enter to the ecosystems via industrial effluents such as mining, refining ores, fertilizers, tanneries, batteries, paper and pesticides etc. Q2. Explain biosorption? A2. Biosorption is the removal of substances (compounds, metal ions, organic etc.) by inactive, non-living, as well as excreted and derived products materials (materials of biological origin) due to high attractive forces present between the two. Biosorption consists of several mechanisms, mainly adsorption, ion exchange, chelation, which differs depending on the species used, the origin and processing of the biomass and solution chemistry. Adsorption is one of the processes, which besides being widely used for treatment of low soluble contaminants in water. The term adsorption refers to a process wherein a substance is concentrated at a solid surface from its liquid surroundings. It is now customary to differentiate between two types of adsorption. If the attraction between the solid surface and the adsorbed molecules is physical in nature, the adsorption is referred to as physical adsorption (physiosorption). Generally, in physical adsorption the attractive forces between adsorbed molecules and the solid surface are van der Waals forces and they being weak in nature result in reversible adsorption. On the other hand if the attraction forces are due to chemical bonding, the adsorption process is called chemisorption. In view of the higher strength of the bonding in chemisorption, it is difficult to remove chemisorbed species from the solid surface. Ion exchange is basically a reversible chemical process wherein an ion from solution is exchanged for a similarly charged ion attached to an immobile solid particle. Chelation can be properly defined as a firm binding of a metal ion with an organic molecule (ligand) to form a ring structure. Q3. What is Sorption? A3. Sorption is transfer of ions from water to the REMOVAL OF HEAVY METALS FROM AQUEOUS SOLUTIONS THROUGH BIOSORPTION

adsorbent i.e. from solution phase to the solid phase. Sorption actually describes a group of processes, which includes adsorption and precipitation reactions. Heavy metals are adsorbed on solid particles by either cation exchange or chemisorption. Cation exchange involves the physical attachment of cations (positively charged ions) to the surfaces of adsorbent by electrostatic attraction. The heavy metals once adsorbed onto the adsorbent will remain as metal atoms, unlike organic pollutants, which will ultimately decompose. Their speciation may change with time as the conditions change. Many constituents of wastewater and run off exist as cations, including most of the trace metals such as Cu, Zn, Pb, Ni and Cd. Chemisorption represents a stronger and more permanent form of bonding than cation exchange. The adsorption capacity by cation exchange or non-specific adsorption depends upon the physico-chemical environment of the medium, the properties of the metals concerned and the concentration and properties of other metals and soluble ligands present. The adsorption of metals varies with the fluctuation of pH in the out flow water. The precipitated hydroxides also act as absorption sites for phyto-toxic metals present in the water. Q4. What are the benefits of activated charcoal/ carbon? A4. Commercial activated carbon is a preferred adsorbent for the removal of micro-pollutants from the aqueous phase; however, its widespread use is restricted due to high associated costs. To decrease treatment costs, attempts have been made to find alternative activated carbon precursors. High surface areas can be obtained using either physical or chemical activation; however, combined treatment might enhance the surface properties of the adsorbent, therefore increasing its adsorption capacity. Activated carbon (AC) is known as very effective adsorbents due to their highly developed porosity, large surface area, variable characteristics of surface chemistry and high degree of surface reactivity. Generally, the physical activation requires high temperature and longer activation time as compared to chemical activation, however, in chemiCHAPTER 7

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cal activation the activated charcoal need a thorough washing due to the use of chemical agents. The product formed by either of the methods is known as activated carbon/charcoal and normally has a very porous structure with a large surface. The removal of heavy metals by activated carbon is economically favorable and technically easy. Therefore, activated carbons are widely used to treat waters contaminated with heavy metals. Q5. Briefly explain the mechanism of heavy metals removal? A5. Adsorption is made possible by the ability of adsorbent materials to accumulate heavy metals from wastewater through physico-chemical uptake pathways. Metal adsorption is the removal of metal ions by inactive, non-living biomass due to highly attractive forces present between the adsorbent and adsorbate. Due to the interaction of several factors on specific adsorbents, it is almost impossible to propose a general mechanism. Metal ions are attracted and bound to the biomass by a complex process that comprises of a number of mechanisms like adsorption on the surface and pores, chemisorption by ion-exchange, surface precipitation, complexation and chelation, entrapment in capillaries, spaces of polysaccharide network, physical adsorption, etc. The actual mechanism of metal adsorption is still not fully understood. One of the important mechanisms of heavy metals removal is ion exchange. It is reversible chemical reaction where an ion within a solution is replaced by a similarly charged ion attached onto an immobile solid particle. Due to the complexity of the biomaterials used, it is quite reasonable that at least some of these mechanisms are acting simultaneously to varying degree depending on the biosorbent and the solution environment. The Pb(II) removal mechanism by Rhodotorula glutinis involved direct biosorptive interaction with the biomass through ion exchange and precipitation by phosphate released from the biomass. The interaction between Pb(II) ions and the functional groups on the surface of the fungus Aspergillus parasiticus’s cell wall is also believed to occur by a combination of ion exchange and complexation processes. The inter100


action between zinc and Saccharomyces cerevisiaewas analysed by a combination of SEM-EDX and XAFS methods. The existence of both covalent and ionic bonds between Zn(II) and the available functional groups on a yeast cell surface was verified by displacement of H+, K+, Mg2+, Ca2+ and Na+ ions during zinc uptake. The release of K+, Mg2+, Ca2+, and Na+ from the biosorbent during heavy metal ion attachment was taken as evidence that ion exchange was the dominant mechanism. Metal sequestration during biosorption adheres to complex mechanisms that mainly include ionic interactions, formation of complexes between metal cations and ligands contained within the cell wall biopolymer structure and precipitation on the cell wall matrix. In their study, Cd(II) ions were attached to chemical groups containing oxygen, carbon, nitrogen and sulfur. In addition, the binding process did not alter the magnesium and calcium concentration in solution, which indicates that ion exchange might not be the main mechanism for Cd(II) biosorption on the brown marine macro-algae Sargassum vulgaris. Another frequently encountered metal binding mechanism is chelation. Chelation can be properly defined as a firm binding of a metal ion with an organic molecule (ligand) to form a ring structure. Various functional groups including carboxylate, hydroxyl, sulfate, phosphate amides, and amino groups can be considered responsible for metal sorption. Among these groups, the amino group is the most effective for removing heavy metals, since it does not merely chelate cationic metal ions but also adsorbs anionic metal species through electrostatic interaction or hydrogen bonding. The presence of various functional groups and their complexation with heavy metals during adsorption process has been reported by different researchers using spectroscopic techniques. The structural changes were also studied using spectroscopic techniques like FT-IR, XPS, Raman microscopy, EDS, XRD, EPR, etc. Each one can reveal certain information and thus can contribute to explain the mechanism of adsorption. Q6. Briefly explain the various factors affecting biosorption of heavy metals?


A6. The efficiency of the metal sorption by the adsorbent is strongly influenced by the physicochemical characteristics of the solutions, e.g., pH, temperature, metal concentration, and adsorbent dosage. a) Influence of pH pH is one of the most crucial factor in heavy metals biosorption process. It significantly influences the solution chemistry of the metals and the dissociation site on the surface of adsorbent, e.g., hydrolysis, complexation by organic and/or inorganic ligands, redox reactions, and precipitation, as well as the speciation and sorption availability for heavy metals. In a particular pH range, most metal sorption is enhanced with pH, increasing to a certain value followed by a reduction on further pH increase.

driving force to overcome mass transfer resistance for metal ion transport between the solution and the surface of the biomass. At a fixed biosorbent dose, pH and temperature, the equilibrium sorption capacity improved with higher initial ion concentration. The ion removal was highly concentration dependent. The increase in the biosorbent’s loading capacity as a function of metal ion concentration was believed to be due to a high driving force for mass transfer. For most cases with low initial ion concentration, the sorption capacity or metal uptake was enhanced with higher initial ion concentrations. However, the opposite phenomenon occurred when starting with high initial ion concentration. This opposite tendency was caused by saturation of the available active sites on surface functional groups, thus preventing further metal ion uptake.

b) Influence of temperature Depending on the structure and surface functional groups of a biosorbent, temperature has an impact on the adsorption capacity, to a certain extent. It is well known that a temperature change alters the adsorption equilibrium in a specific way determined by the exothermic or endothermic nature of a process. Quite a number of biosorption studies have been performed concerning the effect of temperature on isotherms, metal uptake and also with respect to biosorption thermodynamics parameters. The uptake of metal ions increased with higher temperature. A more enhanced level of uptake in parallel with a temperature rise resembles the nature of a chemisorption mechanism (endothermic process). In contrast, the opposite behaviour for the temperature effect on adsorption capacity was obtained baker’s yeast that higher temperature leads to a lower adsorption capacity. c) Effect of initial metal concentration The initial heavy metal concentration can alter the metal removal efficiency through a combination of factors, i.e., the availability of specific surface functional groups and the ability of surface functional groups to bind metal ions (especially at high concentrations). The initial concentration acts as a REMOVAL OF HEAVY METALS FROM AQUEOUS SOLUTIONS THROUGH BIOSORPTION

d) Effect of adsorbent dosage Adsorbent dosage is an important parameter because it determines the capacity of an adsorbent for a given initial concentration of the adsorbate. By increasing the adsorbent dose, the adsorption efficiency increases even though the amount adsorbed per unit mass decreases. In principle, with more adsorbent present, the available adsorption sites or functional groups also increase and the amount of adsorbed heavy metal ions is also increased, which brought about improved adsorption efficiency. In some cases, with increased in adsorbent dose, the adsorption capacity was lesser at a higher adsorbent dose. This is due to overlapping and aggregation of adsorption sites resulting in the decrease of the surface area available to metal ions was observed. Many factors can affect biosorption. The type and nature of the biomass or derived product can be very important. Physical and chemical treatments such as boiling, drying, autoclaving and mechanical disruption will all affect binding properties while chemical treatments such as alkali treatment often improve biosorption capacity, especially evident in some fungal systems. CHAPTER 7

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Further Readings 1. Gadd GM. Biosorption:critical review of scientific rationale, environmental importance and significance for pollution treatment. Journal of Chemical Technology and Biotechnology 2009;84:13– 28 2. Fu F, Wang Q. Removal of heavy metal ions from wastewaters. A review Journal of Environmental Management 2011;92:407-418.

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3. Gavrilescu M. Removal of heavy metals from the environment by biosorption. Engineering in Life Sciences 2004;4:219-232. 4. Volesky B. Biosorption of Heavy metals. CRC Press Boca Raton FL 2000. 5. Volesky B, Holan ZR. Biosorption of heavy metals. Biotechnology Progress1995;11:235–250.

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CHAPTER 8 INDUSTRIAL ENGINEERING Edisher Kvesitadze, Teo Urushadze, Tinatin Sadunishvili, Giorgi Kvesitadze CONTENTS Summary ............................................................................................................................... 105

1. Production of Enzymes by Microorganisms ................................................................. 106

1.1 Enzymes ..................................................................................................................... 106

1.2 Strains of Microorganisms Producing Enzymes ........................................................... 108

1.3 Fermentation Process .................................................................................................. 111

1.4 Extremozymes ............................................................................................................ 113

1.4.1 Enzymes From Extremophiles ............................................................................ 114

1.4.2 Immobilized Enzymes ........................................................................................ 116

1.4.3 Genetic and Protein Engineering of Enzymes ..................................................... 116

2. Ecological Potential of Plants ....................................................................................... 117

2.1 Higher Plants Organic Contaminants Detoxifiers ....................................................... 117

2.2 Organic Contaminants Transformation in Plant Cells ................................................. 119

2.3 Enzymes ..................................................................................................................... 121

2.4 Contaminants Action on Plant Ultrastructure ............................................................. 124

2.5 General Considerations ............................................................................................... 125

3. Bacterial Viruses Against Crop Pathogens .................................................................... 127

3.1 Bacterial Diseases of Plants ......................................................................................... 3.2 Plant Disease Control ................................................................................................. 3.3 Bacteriophages as Biological Control Agents .............................................................. 3.4 Challenges in Using Phages for Disease Control ......................................................... 3.5 Strategies for Optimization in Using Phages for Disease Control ................................ 3.6 Phages as Part of integrated Disease Management Strategies ....................................... 3.7 Other Application of Bacteriophages ........................................................................... 3.8 Engineered Bacteriophages .......................................................................................... 3.9 Bacteriophages as Potential Bioterrorism Agents and Anti-Terrorism Tools ...............

127 128 129 132 133 135 136 136 136

Review Questions and Answers .............................................................................................. 137 Further Readings ................................................................................................................... 139 INDUSTURIAL ENGINEERING

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CHAPTER 8 / E. Kvesitadze, T. Urushadze, T. Sadunishvili, G. Kvesitadze


Summary

T

his review aims to present short, successive description of all procedures of such complicated and important process as initial selection and creation of industrially important microbial strains, including their genetic manipulations, for large scale production of enzymes from microorganisms. The whole spectrum of operations for the production of industrial enzymes, including selection of strains, their purification from natural samples, inoculums preparation, determination of their belonging to corresponding taxonomic group, genera and family; mode of cultivation and composition of nutrient media for effective growth under Solid State Fermentation or Submerged cultivation technology, including selectively chosen organic and inorganic sources of C, N, P, etc.; detection of inducible character of intra- and extracellular enzymes production; scaling up cultivation process from flasks level to industrial volume is discussed. Special section is devoted to microorganisms growing under extremes of conditions – extremophiles, and quite often producing stable enzymes – extremozymes, with increased resistance against extreme conditions of reaction mixture they catalyze, such as: high and low temperature and pH, high salt concentrations, low water activity, and high hydrostatic pressure. Separately, there are discussed immobilized enzymes, as the most appropriate ways for their effective, long-term repeated application, in practice. Perspectives of genetic and protein engineering, manipulations with the strains enzyme producers, for the creation of stable forms of enzymes and cloning of their genes in nontoxic and fast growing organisms are considered as a future of industrial enzymes production. As a result of increased military activities, production of chemicals, unpredictable growth of industry and transport, urbanization, the permanent increase of contamination of all biological sources by chemical compounds of toxic nature are observed. Naturally formed emission of poisonous gases, the washing of toxic elements out of ore during INDUSTURIAL ENGINEERING

floods or earthquakes, formation by microorganisms toxic compounds etc. are a very little as compared with human anthropogenic contribution in environments contamination. According to some data around 600 millions of tons of chemicals of are annually produced in the world. By different ways large amounts of these hazardous compounds or their incomplete metabolic transformations, still having high toxicity, are accumulated in biosphere significantly affecting the ecological balance. The great majority of chemically synthesized stable compounds do not undergo intracellular enzymatic transformations (plant protection and pest control agents, solvents and emulsifiers, etc.), are especially dangerous for all kinds of organisms. Disposal of municipal sewage and wastes accumulated by industry should be also considered a priority for human settlements, as serious contamination source. Uncontrolled discharge of all kinds of wastes always creates functioning biological source of contamination. The elimination of contaminants from the environment by microorganisms of different taxonomic groups is a well- established genetically determined property, which has already been widely discussed. The decrease in land availability for agriculture, climate change and environmental contamination are main problems, the world is facing to feed the growing human population. Among the threats to food production are plant diseases. Environmentally friendly advanced biological technologies, based on application of naturally existing and engineered microbial organisms are attractive and even cost-effective options to solve the above listed challenges. The use of bacterial viruses or bacteriophages for bacterial diseases control is a fast expanding area of plant protection. Advantages and challenges of their application for treatment and prophylaxis, including as part of integrated disease management strategies are described in the chapter. Non-therapeutic applications of phages, potential of these bacterial viruses to be used as bioterrorism agents as well as anti-terrorism tools are discussed. CHAPTER 8

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1. Production of Enzymes by Microorganisms 1.1 Enzymes After many centuries of intuitive use of enzymes, finally, in the 19th century, absolutely unique biological catalysts were named as enzymes. Evidently, the given name “biological catalysts” does not express the essence of the nature of enzymes because in chemistry, catalysts are known as factors accelerating the reaction rate, when enzymes themselves perform all transformation potential and just in some cases require supporting small molecular weight organics or metal ions. After discovering of enzymes functionally important unique three-dimensional structure, catalytically important active sites, the number side functional groups, penetration of molecular biology and organic chemistry in creation of active and more stable enzymes significantly broadened their application. Today in spite of expensive cost of enzymes, enzymes industry is a very rapidly emerging field and represents really successful branch of whole bio industry. Among the industrial sources of enzymes, microorganisms seem to be the best, as the fastestgrowing organisms producing enzymes; containing a broad range of genetic information of enzymes production; organisms to be genetically engineered; having no limits on production capacity; the existence of “extremophilic” forms of microorganisms growing under extreme conditions and quite often synthesizing enzymes with increased stability against critical conditions, etc. This chapter presents a short description of technically complicated and industrially important process of initial selection of microbial strains from natural substrates representing the whole taxonomic gamut of microorganisms - producers of enzymes, both gram-negative and gram-positive; further possibility of their genetic manipulations, determination of nutrient medium for selected strains and most effective type of fermentation, determination of fermentation conditions and in case of submerged 106

fermentation scaling up the cultivation volume (up to industrial level) of selected strains in bioreactors (fermentors) above 10 m3. Below the whole spectrum of operations for the production of industrially important enzymes is discussed including the selection of strains, their purification from natural samples, inoculums preparation, determination of their belonging to corresponding taxonomic group, genera and family; selection of cultivation mode and composition of nutrient media (selectively chosen organic and inorganic sources of C, N, P, metal ions, specific organic additives, etc.) for effective growth under solid-state fermentation (SSF) or submerged fermentation technology (SMF); scaling up cultivation process from flasks to industrialscale bioreactors (fermentors). Special section is devoted to extremophiles - microorganisms growing under extreme conditions and quite often producing more stable enzymes than their mesophilic analogues – extremozymes, with increased stability at extreme reaction conditions such as: high and low temperature and pH, high salt concentrations, low water activity, and high hydrostatic pressure. Some enzymes from extremophiles - microorganisms inhabiting under extreme conditions show stability at extremes of temperature, ionic concentrations (pH), high salt concentrations and high hydrostatic pressure. Aqueous and nonaqueous media allow the modification of reaction equilibrium, creating conditions for synthesizing novel compounds. Used in combination with such media, extremozymes reveal great potential as shown by their unique properties in aqueous media. This review also introduces organic media biocatalysis before addressing the state of the art of recent fundamental and applied aspects of extremozyme biocatalysis. The aim is to encourage further exploitation of this technology, drawing on the limited works published in this field and important methods developed using mesophilic enzymes. Enzymes from three classes of extremophile will be considered: psychrophiles, halophiles, and thermophiles. Low temperature processes using psychrophilic (cold-active) enzymes may enhance yields of heatsensitive products and reduce energy consumption. Halophilic enzymes require KCl/NaCl from 1 M to



saturation, i.e. low water activity media, a feature in common with organic solvent systems. Thermophilic enzymes can be active and stable at up to 130°C and are highly resistant to proteases, detergents, and chaotropic agents. These features may afford resistance to the effects of organic solvents. Enhancing extremozyme performance via chemical modifications, complexation, immobilization, and protein engineering is also discussed. Denaturation involves conformational changes that tend to unfold the protein and cause dissociation and depending on denaturation level can be reversible. Disruption of the active site reduces the catalytic activity. When followed by aggregation (due to the entropically unfavorable exposure of hydrophobic residues), denaturation becomes irreversible. Stabilization also reduces enzyme activity because of rigidification of the enzyme, in a reversible manner by restricting the conformational changes required for catalysis. Inhibition also can occur when solvent molecules bind to the active site. The perspectives and current application of immobilized enzymes, as the most appropriate way for effective, long-term repeated application are analyzed. Some data concerning the choice of carriers, coupling methods of enzymes, types of bioreactors for long-term application of enzymes immobilized forms are presented in the review. Perspectives of genetic and protein engineering, manipulations with the strains of enzyme producers and enzyme molecules for the creation of stable forms of enzymes and cloning of their genes in nontoxic, fastgrowing organisms are considered as future methods of improving industrial enzymes. Finally, enzymes are suggested as highly promising candidates for research and wide spectrum of applications in different nanotechnologies. Enzymes are complicated protein molecules catalyzing above 95-97% of all chemical transformations in living organisms. With the advancement of biochemistry, microbiology, physiology (animals, plants, microorganisms), molecular biology and related scientific disciplines it has become clear that no life form can exist and propagate without enzymes. Although all enzymes are synthesized inside cells and originally they are INDUSTURIAL ENGINEERING

typical intracellular molecules (biopolymers), some of them can be isolated from cells and act in vitro conditions. Due to these unique features, the application of enzymes, especially of microbial origin, has become quite successful in a number of industrial technologies. The catalytic function of enzymes is realized by their complex three-dimensional structures and folding configuration of the entire molecule. In spite of molecular liability, enzymes act at a temperature range from +2° C to above 100° C and at a pH range of 2-13. The processes catalyzed by enzymes are carried out mostly in normal physiological conditions, without high pressures, elevated temperatures and do not require the presence of strong chemicals as catalysts. All these results in fewer by-products, formation of sole high purity products, are energy efficient and environmentally friendly technologies. As with catalysts, all known enzymes are non-toxic, non-pathogenic, biodegradable polymers. In spite of restrictions of enzyme sources, their extraction from plants tissues is comparatively rarely used for enzymes isolation; production of enzymes from microorganisms has no limitation in quantity and is the base for the production of industrially important enzymes. The use of enzymes in different branches of industry, science, medicine and agriculture contributes to the solution of vitally important problems in food processing, agriculture, industry, medicine, environment, etc. The production of enzyme preparations holds one of the leading positions in modern biotechnology and refers to those spheres of industry where production volume grows and the areas of usage are increasing in spite of the world economy downturn. So, nowadays, when the anthropogenic pollutants are annually increasing, it is highly important to create energy saving, ecologically friendly technologies. For these purposes, enzymes are the best candidates. Currently, enzymes are in the category of low volume, high value products. The main obstacle to their wide usage is high cost and low stability. Increasing attention is being paid to enzymes for the development of various technologies for industrial and non-industrial use. Enzymes are CHAPTER 8

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extensively used for various research purposes, in everyday detergents and other household items.

1.2 Strains of Microorganisms Producing Enzymes The latest achievements in molecular biology, genetic and protein engineering, allow creating qualitatively new generations of microbial enzymes. Enzymes synthesized by genetically modified microorganisms acquire improved properties and give an opportunity to extend potential for their industrial usage. However, the strains mostly selected from unusual, naturally occurring environment produce enzymes which are quite often more stable than enzymes from regular strains (mesophiles) and represent good opportunities for production of qualitatively new enzymes. Stability of the commercial preparations of widely used polysaccharides, oligosaccharides, proteins and lignin-degrading enzymes, etc. most often do not correspond to meet critical industrial requirements. A new era in enzyme applications is connected with the production of biofuel on a large scale and industrial realization of enzymatic hydrolysis of wooden cellulose, the main renewable recourse of our planet, on average accumulating cellulose in amount of 140-160 billion of tons annually. All industrial technologies based on the use of enzymes from microorganisms include the isolation of wild strains from nature, determination of their belonging to particular taxonomic group of microorganisms, genera, family, etc. Isolation of microorganisms is possible by enrichment techniques, or single cell isolation by capillary method. The realization of such methodologies requires an inordinate amount of time, large group of skilled labor force and expenses, since only few species of a particular genus are isolated from one sample. Once, the wild strain is isolated and synthesized enzyme corresponds to technology requirements, the further procedure of development of aerobic strains is carried out according to the following steps: purification of the strains from undesirable microorganisms; determination of their belonging to a particu108

lar taxonomic group, genus and family; selection of cultivation mode and composition of nutrient media for effective growth under solid-state fermentation or submerged fermentation, including selectively chosen organic and inorganic sources of C, N, P, metal ions, specific organic additives, etc. Scaling up a technology requires cultivation of microorganisms in special bioreactors, upstream processing, of different volume and constriction, greatly determining the success of enzyme production technology. The subsequent steps, downstream processing, consist in isolation and purification of enzymes. Most often, genetic modification of the enzyme producer strains is carried out in order to increase the effectiveness of enzyme production. Lately, microbial diversity and belonging of the enzyme producing strains should be described at a molecular level by 16S rRNA or FAME (fatty acid methyl ester). On the basis of microbiologists’ experience, isolation of microorganisms by current procedure recovers no more than 1% of microorganisms inhabiting in different ecological niches. The determination of inducible character of microbial enzyme synthesis expressed in synthesis de novo or significant increase of activities of the number of enzymes acting on different substrates, mainly biopolymers, as a response on existence of their substrates or similar to substrates compounds even as a minor constituent part of nutrient media is one of the steps of microbial enzyme synthesis. Some of inducible enzymes after penetration of microbial cell carry out the transformations of nutrient media polymeric compounds by decreasing their molecular weight to the size allowing penetrating into microbial cell wall for further intracellular transformations. Regarding other ideas, there is no need to isolate microorganisms from the nature by well-known classical technique since interesting fragment of DNA and particular genes can be isolated directly from the nature. Quite often currently used eco-physiological technique of microbial strains isolation includes high or low temperatures procedure and extreme pH, different ionic strength and substrate concentra-



tion, etc., and allows isolating more strains from samples than the classical isolating procedure. But even in this case, the amount of isolated microorganisms would expose just several percent of the actual diversity of microorganisms in natural samples. So, there is no warrantee that any single technique would allow to isolate all or almost all varieties of microorganisms from extremely diverse natural samples. There are some techniques for the isolation of microorganisms depending on the type of sample, its chemical composition, ecological parameters, information on possible dissemination of microorganisms of different taxonomic groups, etc. The combination of isolation techniques would be the best for the real evaluation of microbial diversity but even in this case there is no warrantee to cover all existing forms of microorganisms. It should be underlined that the revelation of actinomycetes strains, bacteria and fungi in natural samples, such as: soil, plant material, water samples, rocks, etc., requires specific technique for each taxonomic group and many details (isolation media, diluents, temperature, pH, etc.). High synthesizing potential of microorganisms, their diversity and uniqueness is a base for the formation of the great amount of organic compounds, further development of natural products chemistry. Most of strains used for enzyme production have been modified by well known, so-called classical methods of selection. Mutagenesis by UV rays and chemicals having mutagenic effect are often used to get more useful variants, with increased potential of searching enzyme synthesizes. Cells of the strain producer also should be subjected to recombination procedure and that tested for the characteristics determining their industrial application. Mutation makes changes in protein/enzyme structure and correspondingly in the genome of strain. The success of active strain receiving depends on the method of selection. Usually for finding one mutant strain with genetically stable novel properties, 10.000-100.000 colonies should be analyzed. In general mutation and selection are directed primarily toward higher overall productivity than mutation of a specific function; loss of regulatory function is rather probable. INDUSTURIAL ENGINEERING

Purification procedures of isolated from nature microorganisms the most often of different taxonomic groups have much specificity and first of all require the use of specific nutrient media (source of C, N, salts, and specific organic additives such as: agar, vitamins, amino acids, yeast extract, antibiotics, etc.) differing/specific for each taxonomic group of microorganisms. The successful isolation of diverse actinomycete genera and their species greatly depends on the sample (freshness, place of isolation, content of water, temperature, etc.). While growing of actinomycetes isolates on typical nutrient medium, it is important to identify the difference in growth forms of actinomycete colonies and isolate them. The conformation of previously identified strains by a “trained eye” requires conformation by molecular techniques as 16S rRNA- based molecular techniques or by chemical method such as FAME. Purification of bacteria is quite a complicated procedure. The purification of any 683 genera described in Bergey’s “Manual Systematic Bacteriology” requires specific techniques and different nutrient media for the isolation and purification of bacterial strains. Once colonies are developed they should be sorted according to the types of grown organism. The individual colonies are transferred to special plates and several times reincubated until mono colony growth is observed. All steps in growth and development of colonies should be controlled by the use of a microscope. Strains isolation procedure from natural samples is the first step for fungi isolates. While repeatedly growing (for purification reasons) on Petri dishes, initial observation should be made by dissecting microscope. For microscopic fungi can take up to 10 days, basidial fungi to emerge from wood samples require 4–6 weeks, for yeasts time lasts up to 24 hours. A distinct needle is used to make single spore isolations excision. The propagules can be removed from isolation plate and inoculated in growth promoting for identification of fruiting body formation. Microorganisms of all taxonomic groups are exceptionally rich, diverse and easily accessible sources for the great amount of metabolites they synthesize. To discover desirable metabolites, first of all a CHAPTER 8

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reliable and sensitive method for screening microorganisms is required to identify producers. These methods are used for screening antihypercholesterolemic, antihypertensive and antiviral activities. For the detection of enzymes formed by fermented microbial monoculture, methods of directly assaying their activities are used. The activity and corresponding productivity of strains can be expressed in the following ways: - Total enzyme activity related to total DNA of cultivated strain. That would indicate the specific activity expressed on the unit of DNA. The comparison of different strains and several nutrient media by this index would explain the real productivity of strain; - The enzyme activity relating to the amount of total intracellular protein. For some cases (solid-state fermentation) this methodology seems to be quite useful, indicating the effective digestion of C/N sources from nutrient media; - The enzyme activity could be measured by relation of final total activity to amount of carbon source in nutrient media (productivity of digested carbon); - The widespread method for the detection of enzyme activity seems to be per volume of cultural filtrate (1.0ml, 0.1ml, 0.01ml, etc.) or centrifuged extract of microorganism’s biomass. To keep microbial strains in active state synthesizing needed metabolite/s in sufficient amount, the knowledge of detailed physiological characteristics for any particular strain is required. There are several preservation methods currently being in use to keep microorganisms active to synthesize needed enzyme: as long-term preservation in liquid nitrogen seems to be universal and the most widely spread method for microorganisms preservation; preservation under mineral oil prevents evaporation and significantly (in a number of cases completely) decreases metabolic power of the strains; according to the long-term practice of microbiologists the freeze drying method of microorganism preservation, for long period is the best. Even plasmid containing bacteria are preserved being lyophilized. 110

Among other less important methods for the preservation of microorganisms and long-term storage, the Silica method should be mentioned. In a great number of cases, the above-mentioned preservation methods proved positive results. Genetic engineering most intensively uses microorganisms to produce enzymes by cloning of the corresponding gene into the microorganism. The presence of introns may then prevent proper expression of the gene, but techniques have been developed to overcome this difficulty. Chymosin (E.C. 3.4.23.4), calf rennet, has been cloned in procaryotes or in yeast, one of the first cloned mammalian enzyme to be produced industrially by microorganisms.The handling of microorganisms from their preserved state to the environment where they should expose characteristic microbial activity is referred to as an inoculum development. The primary purpose of inoculum development is to provide sufficient microbial biomass for further sustainable growth in bioreactor. The growth of strain producers of enzymes starts in tubes or Petri dishes. Each fermentation step, prior to the final bioreactor, is used to prepare inoculums for the next bioreactor. The number of steps in the fermentation train to reach the main industrial fermentor depends on the type of final fermentor, size and specific strain characteristics. Initially, the strains are transferred from agar containing Petri dishes or tubes to shake flasks, with synthetic or natural nutrient medium. The following step starts in a small size bioreactor-fermentor (15-30 liters), then in increased capacity fermentor (100-500 litres) and finally to industrial reactor having volume above 10m3. The figure below shows an example of a fermentation train used in preparing the inoculums for main fermentation (Figure 1).

Figure 1. Enzymes producers microorganisms fermentation process.



1.3 Fermentation Process The development of entirely new processes or the improvement of existing processes requires the evaluation of a wide range of strains and cultivation conditions. Shake-flask fermentation studies have a cost advantage but there is no process control option (pH, nutrient addition, aeration). This often leads to the use of laboratory-scale fermentors (10L) agitated, aerated with adequate instrumentation and control. Most fermentation processes can be translated to production scale by use of laboratory-scale fermentors. However, pilot-scale fermentors are often necessary for downstream process scale-up. According to the calculations once suitable organisms has been found, either genetically improved or not, the next task of process is to define optimal input parameters. The rate of enzyme synthesis re is defined by: re=qe∙cx where re is the enzyme synthesis rate in units per liter per hour, qe the specific enzyme synthesis rate in units per gram of biomass per hour, and cx the biomass concentration in grams per liter. Growth can be expressed by a specific growth rate as the amount of biomass synthesized per unit biomass and unit time:

μ=

1 . dcx cx dt

where cx is the biomass concentration in grams per liter and t the time in hours. Specific enzyme synthesis rate and specific growth rate express the metabolic activities of the cell. These are abbreviated as synthesis rate qe and growth rate , as:

μ = μmax .

Cs Ks + Cs

where μmax is the maximum attainable growth rate per hour; Ks is the saturation, and cs the concentration of substrate, both expressed as per liter.


Theoretically primary selection of appropriate strain in combination with corresponding modification at the genetic level and fermentation process should lead to qualitative improvement of fermentation process capable of producing enzymes up to 20 kg m-3. Genetic regulation of enzyme synthesis is helpful in selection of improved strains and optimizing the fermentation process. While selecting the enzyme producing microbial strains, special attention should be paid to the investigation of possible induction of searching enzyme. The level of induction can dramatically increase the total quantity of enzymes produced by microorganisms; in case of an inducer existence up to 1000-fold increase over non-induced conditions in total activity can be observed in some cases. Compounds similar to the substrate are common for the induction of synthesis of extracellular enzymes degrading a large polymer to smaller molecular weight fragments. The majority of catabolic enzymes are well inducible molecules. For industrial scale production of enzymes two types of fermentation (methods of microorganism’s cultivation) are used: the submerged liquid fermentation (SLF) and solid-state fermentation (SSF). Depending on group of microorganisms, in a number of cases, their large scale cultivation is economically more efficient in case of using cheap components of nutrient media in combination with short fermentation cycles. Once when the appropriate strain is selected use of an anaerobic submerged culture in a stirred-tank represents the typical industrial process for extracellular enzymes production. The correct selection of strain and equipment determines the success in enzyme yield, in case if the composition of nutrient media is arranged in a most effective way to realize full energetic potential of nutrient media directed to produce maximal amount of enzyme. The SSF is a typical microbiological process in which substrates (the source of carbon which most often is solid) in sterile volume conditions and regulated moisture and temperature are used for the growth and development of microorganisms and

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realization of their biosynthetic potential directed to the accumulation of enzymes. The great majority of microorganisms undergoes cultivation by this technology and produces quite a large number of enzymes and small-molecular-weight metabolites. Microorganisms grown in solid state conditions in some cases exceed enzyme production as compared to the submerged fermentation; the reason being is that the natural habitat of many microorganisms is a solid state. Another interesting fact consists in revealing of novel isoenzymes (biopolymers degrading enzymes) produced by SSF as compared with deep cultivation technology, these results were confirmed by DNA sequential analysis. Especially good results SSF shows in case of the following hydrolytic enzymes production: pectnases, amylases, cellulases, proteases, lignin-oxidizing enzymes (laccase), etc. Evolved during microorganism’s growth CO2 affects the accumulation of enzymes sometimes increasing it up to 5-6%. For example, the induction of the majority of hydrolytic enzymes is induced in the SSF in greater amount. For industrial application of SSF technology, depending on the strain of microorganism, searching final metabolite, scale of production, etc., the following types of bioreactors are used: Tray Fermentors, Packed–Bed-Fermentors, Rotary Drum Fermentors. It must be stated that a large scale use of the SSF is mainly observed in Asian fermented food production. Technological problems associated with SSF exist with scaling up; hence the use of SSF for enzyme production is less frequent as compared to SLF. The physiological state of microorganisms during SLF process should be well controlled. Fermentation process proceeds in the following three levels: a flask level (50-200 ml, each flask) where the fermentation is carried out on shaker; a pilot plant level using fermentors of different volume (from 2 up to 1000 liters); and finally an industrial, economically profitable level. For enzymes production such level corresponds to the fermentors volume equaling 103m (10.000 litres) and more. The essence of scale-up process of enzyme producers is the selection of conditions providing physiological regulation of growing strain directed to expose 112

maximum of synthesis potential, accumulation in cells (intracellular enzymes) or excretion in grown liquid (extracellular enzymes), of needed enzyme. This includes the number of the factors such as: optimal nutrient medium, mass transfer ability, dissolved oxygen concentration, power consumption, etc. Such significant factors for bioprocess improvement must be determined in small-scale fermentors and only then reproduced in large-size fermentors. Undoubtedly, SLF is the most widespread and progressive fermentation technology for the industrial production of enzymes. The technology of SLF meets many requirements for fermentation objectives. This type of fermentation includes cultivating aerobic microorganisms, animals and plant cells. The production of enzymes by SLF is resulted in production of two main products: biomass of microorganisms containing the needed enzyme or other metabolite and filtrate of cultivated microorganisms containing quite often 60-90% of excreted through cell walls enzymes (extracellular enzyme) activity. This, cultivation technology for the industrial scale growth of all kinds of microorganism’s (fungi, bacteria, actinomycetes and yeasts) has proved good results. Fermentation process design is interdisciplinary and uses concepts and methodologies of both chemical engineering and microbial physiology to accomplish scale-up. In practice, scale-up effects are more pronounced for aerobic, i.e., aerated and agitated environment, than for anaerobic fermentations. Therefore, as a rule of thumb, in an aerobic fermentor, the constant oxygen transfer rate and concentration of dissolved oxygen are generally maintained in the scaleup. While thermodynamic and kinetic phenomena are independent of scale, momentum, mass and heat transfer are functions of scale. Mixing, aeration, and cooling are well controlled and uniform at 10 L scale, but not all transport parameters can be maintained in this way at large scale. Further scale-up complications arise from cell response to distributed values of dissolved oxygen, temperature, pH, and nutrients. The fermentation process starts with multiplication of strain maintained in Petri dishes or tubes with



corresponding microbial colony grown on agar and other components containing media. This nutrient media can also contain an inducer of required enzyme. In case of SLF, microorganisms are transferred in flasks that most often contain synthetic nutrient media. Further cultivation takes place in a special construction of bioreactor-fermentor provided with regulation of temperature, pH, level of supplied sterile dissolved oxygen, mixers. The availability of recombinants production technology over the past 20 years has a profound effect on the production of microbial enzymes. The host E.coli is a suitable organism for the expression of cloned enzymes. Species of Bacillus are appropriate bacterial hosts for the production of extracellular non-glicosilated enzymes. Species from Apergillus family are also well examined hosts for the expression of extracellular glycosylated enzymes. Once fermentation process is ended there are two products: microbial biomass and liquid cultural filtrate. Recovery of both intracellular and extracellular enzymes starts usually with filtration or centrifugation of cultural liquid. Extracellular enzymes are relatively easy to isolate and purify. Quite often they are the main portion of total secreted proteins. To concentrate the cultural filtrate containing extracellular enzymes, quite often the ultrafiltation is used. For this reason a semi-permeable membranes as a selective barrier retaining the protein molecules, bigger than pore size, while allowing the smaller molecules to permeate through the pores are used. In industry different types of ultrafilters are applied (stirred, aerated, fed-batch type bioreactor, tangential-flow units, etc.). Sometimes secreted in cultural liquid, enzymes are absorbed on ion-exchange resin column or ion-exchange media and after desorption concentrated in a small volume. Extracellular enzymes are easily precipitated by organic solvents (ethyl alcohol, isopropyl alcohol, and toluene) and in saturated ammonium sulfate water. Preparation of intracellular enzyme preparations requires more operations as compared with extracellular. The cells are often tougher and sizes range from 0.2-10 microns. The breakage of microbial cells presents greater problems than animal or plant cells. Microbial INDUSTURIAL ENGINEERING

intracellular enzymes are liberated by cell lysis and often require chemicals, ethylendiamintetraacetete (EDTA) and sodium dodecylsulfate (SDS). Enzymatic treatment of microbial cells (lysozyme) is also wide spread. Among other techniques for intracellular enzymes isolation freeze-thaw and mechanical stress methods are used. Quite often the primary enzyme preparations usually are commercially available crude enzyme preparations containing the main enzyme up to 40% of total protein. High resolution purification is required for small numbers of enzymes mainly used in medicine, scientific research, cosmetic, in some cases used as additives to food. To obtain high purity enzymes the following methods are used: Ionexchange chromatography, Affinity chromatography, Molecular weight dependent chromatography (gel filtration). The great majority of highly purified enzymes were subjected to the above mentioned methods. Chromatographic methods are known as low pressure, medium pressure, and high pressure techniques. Depending on pressure, the FPLC (Fast Performance Liquid Chromatography) and HPLC (High Performance Liquid Chromatography) are used to force the packed bed of absorbent particles. Enzymes purification can generally be divided on several broad sections: preparation of the source, collecting all available information about properties of enzymes, development of activity assay method, primary isolation (crude enzyme preparation), and method used for high state purification. Enzyme purification could be necessary in cases when its extra cost is justified by enzyme’s application. The level of purification dictates isolation technology choice.

1.4 Extremozymes Life may have been present on Earth about 3.8 billion years ago or even earlier. Multidisciplinary research, in paleobiology and evolution of early microorganisms and the microbiology of extremophiles on the Earth’s environments and under space conditions, enables the defining of strategies for the detection of extraterrestrial life potential. At present, only 1–2 % of the known microorganisms on the Earth have been commercially exploited CHAPTER 8

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revealing only very small amount of extreme extremophiles. However, recently renewed interest to extremophiles and success in the cloning and expression of their genes in mesophilic, fast growing hosts has increased the potential of extremozymes (enzymes from extremophiles) application. The industrial application of enzymes that can withstand harsh conditions has greatly increased over the past decade. This is mainly a result of the discovery of novel enzymes from extremophilic microorganisms. Recent advances in the study of extremozymes point to the acceleration of this trend. In particular, enzymes from thermophilic organisms have found the most commercial use to date because of their overall inherent stability. This has also led to a greater understanding of stability factors involved in adaptation of these enzymes to their unusual environments. The synthesis of polymer intermediates, pharmaceuticals, specialty chemicals and agrochemicals is often hampered by expensive processes that suffer from low selectivity and undesired byproducts. Mesophilic enzymes are often not well suited for the harsh reaction conditions required in industrial processes because of the lack of enzyme stability. For this reason, the use of biocatalysts in organic reactions represented only a small fraction of the potential industrial market in the past. Two decades ago, extremophiles were exotic organisms, explored by only a few research groups throughout the world. Now, although they still retain some of their eccentric status, they are often routinely used as the sources of novel enzymes at enzyme-discovery companies. The capabilities of extremophilic microorganisms have been the subject of many recent reviews, articles, and a new journal (Extremophiles; Springer–Verlag) is entirely devoted to the topic. Below are presented widespread enzymes from extremophiles and some of their applications. Most of the work has been devoted to thermophiles and hyperthermophiles, but other groups have received even more attention recently because of their biotechnological potential.

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1.4.1 Enzymes from Extremophiles Amylases. Glucose/fructose mixture (45–65°C); Xylanases. Paper bleaching (65–85°C); Proteases. Baking, brewing, (up to 650C) DNA polymerases. Genetic engineering (85°C) Low temperature acting enzymes proteases. Cheese maturation; Dehydrogenases. Biosensors Alkaline pH acting cellulases, Detergents. The extreme conditions may be high or low temperature, extremes of pH, high salinity, high heavy metal concentrations, very low nutrient content, very low water activity, high radiation, high pressure and low oxygen tension, etc. Enzymes from extremophiles (extremozymes) show activity and stability at extreme conditions of environment: high and low temperature and pH, low water activity, and high hydrostatic pressure. Aqueous/organic and nonaqueous media allow the modification of reaction equilibrium and enzyme specificity, creating pathways for synthesizing novel compounds. Used in combination with such media, extremozymes show great potential by their unique properties in aqueous media. Low temperature processes carried out by psychrophilic (cold-active) enzymes may enhance yields of heat-sensitive products and reduce energy consumption. Halophilic enzymes required saturation by 1M salts are effective in a number of processing treatments. Enzymes from thermophilic microorganisms are active and stable at elevated temperatures up to 130°C and almost as a rule are highly resistant to proteases and detergents. Extremophiles are adapted to survive in ecological niches such as at high and low temperatures, extremes of pH, high salt concentrations and high pressure. In a number of cases these microorganisms produce unique biocatalysts that function for a long time and under extreme conditions comparable with various industrial processes. In particular, the research is focused on the selection of strains extremophiles-producers of extracellular-polymer-degrading enzymes such as amylases,



pullulanases, cellulases, xylanases, chitinases, proteinases, laccase and some other enzymes such as esterases, glucose isomerases, alcohol dehydrogenases and DNA-modifying enzymes with potential use in food, chemical and pharmaceutical industries, in science and in environmental biotechnologies. The selection of extremophiles producers of stable enzymes requires the existence of microorganism’s collection accounting minimum hundreds of strains. It should also be taken into consideration that extremophiles, even being of extreme forms, do not always produce stable enzymes exceeding in stability their mezophilic analogues. Projects concerning the selection of extremophilic strains of different taxonomic groups and origin are carried out in dozens of laboratories in Europe, USA, Japan, China, etc., and companies are dealing with the production or exploitation of enzymes. The Durmishidze Institute of Biochemistry and Biotechnology of the Agrarian University of Georgia keeps collections of microorganisms (fungi, bacteria, yeasts, actinomycetes), which is the base of this publication. The collection is accounting over 10.000 strains, being isolated from different ecological niches and extreme environments of more than 15 soils and up to 30 soil/climatic zones of Georgia and other countries bordering the Caucasus. The existence of such collections in combination with over 30 years experience dealing with isolation, purification, investigation of metabolic specificities of collection strains allowed the creation of collection of a new group of extremophiles and extremotolerants accounting above 1000 strains. These collections being, first of all, carefully analyzed on the existence of hydrolytic, biopolymers degrading enzymes activities, allowed to select several extreme extremophilic strains of bacteria and fungi producing highly heat and alkali stable enzymes (amylase, cellulases, protease, glucoamylase, etc.). The results of selection of microorganisms for the last two-three decades have proved that stable forms of enzymes are more expedient to be searched among microorganisms possessing optimum growth at unusual, so called extreme conditions. INDUSTURIAL ENGINEERING

Such microorganisms as thermophiles, halopiles, acidophiles, alkaliphiles, psychrofiles, etc. are frequently capable to produce enzymes surpassing in stability and activity currently commercially available enzymes of mesophiles. Data indicating that enzymes from “wild” strains of fungi thermophiles considerably exceed in heat stability the same enzymes from mesophiles is shown below (Figure 2).

Figure 2. Heat stability of cellulases from thermophilic and mesophilic strains (activity of enzymes assayed by their action on filter paper at 60ºC) (easily previewed in a form of the chart).

1 – Trichoderma reesei “Onozuka R-10” (1,2,3-mesophiles); 2 – Sporotrichum pulverulentum; 3 – Aspergillus terreus; 4 – Aspergillus wentii; 5 – Aspergillus versicolor; (4,5-thermotolerants); 6 – Chaetomium thermophile; 7 – Allesheria terrestris (6,7-thermophiles); In spite of great success in molecular biology and genetic engineering one of the main aims in enzymes production is to carry out the primary selection of highly technological strains of microorganisms - producers of stable forms of enzymes with high stability against moderate and extreme conditions occurring in industrial processes during their usage in nonconventional biotechnologies. The existence of stable enzymes would allow the creation of competitive and low cost innovative technologies based on their use. The microbiological collections are good sources for the isolation of appropriate strains – producers of stable enzymes. In addition to other forms of extremophiles the great attention for last time has been paid to investigaCHAPTER 8

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tion of so called low temperature biotechnology, directly dealing with the selection of psychrophillic strains. Energy save biotechnologies based on psychrophiles use attracts more and more attention for their use in different biotechnologies. As a result of decades of experience in selection of regular and extreme forms of microorganisms in Georgia special map of extremophiles distribution has been created (Figure 3).

clusion into the structure of polymers; adsorption on polymers of different nature; the most common for immobilization are insoluble polymers, not affecting on the charge of the enzymes used. The immobilized enzymes are physically confined while during catalytic process they perform and at the end of reaction can be recovered from reaction mixture and used repeatedly. As a result of coupling, in spite of some decrease of specific activity, that takes place quite often, the half life of acting immobilized enzymes is significantly prolonged. That decreases the cost of enzymes application and makes the whole process more profitable.

1.4.3 Genetic and Protein Engineering of Enzymes

Figure 3. Map of extremophiles existence in Georgia.

The stability of enzymes against organic solvents, extreme temperatures and pH, high salt concentration has decisive importance even in regular industrial enzymatic reactions. To develop commercially profitable technologies of stable amylolytic, lipolytic, cellulolytic, xylanolytic proteolytic, pectolytic, lygnolitic, etc. enzymes production a broad selection of producers should be carried out followed by genetic improvement of strains producers, as well as purification, identification and characterization of the most stable enzymes. Moreover, morphological, physiological and biochemical properties of the selected extremophiles must carefully be investigated.

1.4.2 Immobilized Enzymes Industrial application of soluble enzymes is considered as wasteful technologies based on the considerations that enzymes cannot be recovered at the end of any processes they catalyze. The most appropriate way of effective, long term, repeated application of enzymes, is use of their immobilized forms. There are several well adapted methods of enzymes immobilization such as: chemical coupling on insoluble carriers of organic or inorganic nature; in116

Recombinant DNA technology has allowed the transfer of industrially important enzymes genes from one organism to another. Thus, enzyme being identified as a good catalyst could be doubled in host organism or cloned in more appropriate (well known, fast growing, nontoxic, etc.) another host microorganism according to the widely used following scheme, in case of unknown enzyme (conventional method): Growth of microorganism-enzyme producer

Isolation and purification of enzyme of interest

Determination of partial amino acids sequence

Synthesis of oligonucleotide probes

Identification of cDNA clones by hybridization with oligoprobes

Transforming created fragment of DNA in industrial host organism



For several reasons, nowadays genes of prospect enzymes are often cloned in nontoxic fast growing organisms. The special systems used for expression of cloned genes of pro- and eukaryotic organisms, are rather various: bacteria (Escherichia coli, Bacillus subtilis), yeasts (Saccharomyces cerevisae, Kluyveromices lactis, Pichia pasteris, Hasenula polymorpha), baculoviruses, insect cell cultures. Some representatives of aspergilli and namely strains of Aspergillus oryzae seem to be one of the best hosts among fungi. The genes of the most stable enzymes are cloned in yeasts and bacterial strains using corresponding expression systems. Future prospects of modern technologies for commercial purposes can be revealed in order to use the selected strains, producers of stable forms of cellulases, xylanases, lignin oxidazing enzymes for enzymatic preprocessing of plant raw materials (production of bioethanol, glucose/fructose syrup, single cell biomass, etc.). Hydrolytic enzymes mainly from mold origin – cellulases, xylanases, amylases, proteases, inulinase, lipases, pectinaes, laccases, being above 80% of all enzymes used in practice, are of great interest due to their demand in various areas of industry, agriculture, medicine and pharmacology. Availability of stable forms of the above mentioned enzymes could not only extend the areas of their usage, but also reduce the price of products with the application of these enzymes.

2. Ecological Potential of Plants 2.1 Higher Plants Organic Contaminants Detoxifiers Until recently, plants, which still occupy about 40% of the world’s land area, were considered as organisms just accumulating contaminants but having no potential to transform them into harmful compounds. According to the existing information, plants could only slightly transform toxic compounds, presumably oxidize, than conjugate INDUSTURIAL ENGINEERING

and deposit in vacuoles. Analysis of experimental data in the last two-three decades has revealed the visible ecological potential of plants. It has been exposed the deep degradation processes proceeding in higher plants, depending on the structure of contaminants quite often leading to mineralization or deep degradation of contaminants. As a result, enzymes carrying out partial/deep oxidation, conjugation and compartmentation processes have been revealed and characterised; the formation of anthropogenic contaminants conjugates with endogenous compounds has been shown. Although, there are still some unlearned steps in detoxification process carried out in plants closely related to the contaminants multistage degradation process, the authors are making attempts for the evaluation of different aspects of plants ecological potential from the modern understanding, revealing the criterion for the evaluation of deviations under the action of contaminants in ultra-structural architectonics of plant cells. Table 1 presents plants potential to absorb and metabolize contaminants examined by authors. In spite of difficulties in quantitative, as well as qualitative estimation, and having a tendency to be increased, the level of spread-out contaminants in many places of the planet significantly exceeds permissible standards. Most dangerous among these contaminants are considered as emergent because of their persistence, bioaccumulation, and toxicity along with our awareness of their prominent occurrence in the environment. In different ways, huge amounts of these hazardous substances or toxic intermediates of their incomplete transformations are accumulated in different niches of biosphere, significantly affecting ecological balance. Lately, many ecological technologies targeted to minimize the flow of toxic compounds into the biosphere and monitoring of their level or state have been developed. Despite some positive effect from the realization of these technologies (physical, chemical, mechanical etc), the intensive flow of toxic compounds to the biosphere is still increasing. The international character of this problem being determined by global migration of contaminants CHAPTER 8

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(migration between soil, air and water, geographical, biotic, etc.) leads to the distribution of toxic compounds of different structure and overall increase of the toxicity level. Nevertheless, the members of the plant kingdom assimilate toxic compounds, removing them from the environment, naturally providing long-term protection and monitoring against their environmental dispersal. Obviously, microorganisms and plants represent the main power of nature permanently defending the ecological balance. Plants being recently recognized as an important ecological tool and in order to properly evaluate their detoxification potential should be emphasized according to following features: - Higher plants simultaneously contact three main ecological niches: soil, water and air - A well-developed root system of higher plants determines the soil-plant-microbial interaction, representing a unique process by producing exudates, significantly affecting the overall plant metabolism - The large assimilating surface area of plant leaves (adaxial and abaxial) significantly exceeds in size the corresponding aboveground surface located under the plant, and permits the absorption of contaminants in large quantities from the air via the cuticle and stomata - The unique internal transportation system in both directions, distributing all the penetrated compounds throughout the entire plant - The autonomous synthesis of vitally important organics and extra energy by using intracellular potential during the prolonged remediation process - The existence of enzymes catalysing oxidation, reduction, hydrolysis, conjugation, compartmentation and other reactions of the multistage detoxification process - The large intracellular space to deposit heavy metals and conjugates of organic contaminants. - Functionalization and further transformation of organic contaminants by following conjugation, deep oxidation, etc. in plant cells. 118

In order to penetrate into a leaf, xenobiotics (contaminants) should pass through the stomata, or traverse the epidermis which is covered by film-like wax cuticle. Generally, stomata are located on the lower (abaxial) side of a leaf, and the cuticular layer is thicker on the upper (adaxial) side. Gases and liquids penetrate through the stoma into the leaves. The permeability for gases depends on the degree of opening of stomata apertures (4–10 nm) and for liquids, on moistening of the leaf surface, the surface tension of the liquid and morphology of the stomata. The majority of toxic compounds of law and average molecular weight quite easily penetrate into a leaf as solutions (pesticides, liquid aerosols, etc.). The contaminant penetration into the roots essentially differs from the leaves. Substances pass into roots only through unsuberized cell walls. Therefore, roots absorb substances much less selectively than leaves. Roots absorb environmental contaminants in two phases: in the first fast phase, substances diffuse from the surrounding medium into the root; in the second they gradually distribute and accumulate in the tissues. The intensity of the contaminants absorption process, characterized by various regulations, depends on the contaminant solubility, molecular mass, concentration, polarity, pH, temperature, soil humidity, etc. Nowadays, there are experimental data obviously demonstrating that plants activate a definite set of biochemical and physiological processes to resist the toxic action of contaminants by using following physiological/biochemical mechanisms: - excretion; - conjugation of contaminants with intracellular compounds following by compartmentalization of the conjugates in cellular structures; - decomposition of environmental contaminants (or their significant part) to standard cell metabolites or their mineralization. Commonly, plants gradually degrade penetrated through cell wall organic contaminants to avoid their toxic action. According to contaminants assimilating potential plants sometimes are differing



Hydrocarbons

Methane, Ethane, Propane, Butane

Organochlorine solvents

Chloroform

Alcohols

Pentane, Hexane, Cyclohexane

Benzene, Toluene, Napthalene

1,2-Benz(а)anthracene, 3,4-Benzpyrene, Dibenz(a,h)antracene, 3-Methylcholantrene

Methanol, Ethanol

Isopropanol, Butanol

Pentanol, Hexanol

Octanol, Benzyl alcohol

Phenols

Phenol, o-Cresol, m-Cresol, p-Cresol

Catechol, Hydroquinone, Methylhydroquinone, Resorcin

Pyrogallol, α-Naphthol, Fluoroglucine, Thymol, Guaiacol

Oxyhydroquinone, Toluhydroquinone, Thymohydroquinone, Durohydroquinone

Quinones

o-Benzoquinone, c-Benzoquinone

Toluquinone, Timoquinone

Duroquinone, Anthraquinone

2-Methyl-1,4-naphthoquinone, 2-Hydroxy-1,4-naphthoquinone

Aldehydes and Ketones

Formaldehyde

Acetaldehyde

Acetone

Organic acids

Formic acid, Acetic acid

Acetahydride, Propionic acid

Butyric acid, Valeric acid

Caproic acid, Benzoic acid

Nitroderivates

o-Nitrophenol p-Nitrophenol

2,4-Dinitrophenol p-Nitroanisole

Nitrobenzene Dinitrobenzenes

2,4,6-Trinitrotoluene (TNT) Hexahydro-1,3,5-trinitro-1,3,5triazin (RDX)

Amines

Aniline

N,N-Dimethylaniline

Benzidine

Atrazine, Simazine, Lindane

Carbaryl (Sevan)

Phenoxiacetic acid Pesticides (Herbi2,4-Dichloroacetic cides) acid (2,4-D) Drugs

Aminopyrine

2,4-Dinitro-o-Cresol (DNOC) Dichlorodiphenyltrichloroethane (DDT)

Ethylmorphine

Table 1. List of examined toxicants.

up to four orders of magnitude that allowed classifying plants as strong, average and weak absorbers of different structure contaminants. For instance, the most active assimilators uptake up to 10 mg of benzene per 1kg of fresh biomass per day, whereas the assimilation potential of the weak absorbers is measured in hundredths of mg (Table 2).

excretion is that the toxic molecule does not undergo chemical transformation, and being translocated through the apoplast, it is excreted from the plant. This pathway of xenobiotics (contaminants) elimination is rather rare and takes place at high concentrations of highly mobile (phloem-mobile or ambi-mobile) xenobiotics.

2.2 Organic Contaminants Transformation in Plant Cells

In the majority of cases, contaminants being absorbed and penetrated into plant cell undergo enzymatic transformations leading to an increase in their hydrophilicity - process simultaneously accompanied by contaminats toxicity decreasing. Below are presented successive phases of contaminant initial transformations in accordance with Sandermann’s “green liver” concept (Figure 4):

The fate of the entered plant cell contaminants depends on their chemical nature, external temperature, variety of plants and phase of vegetation, etc. The simplest pathway of entered the plant cell organic contaminants is excretion. The essence of INDUSTURIAL ENGINEERING

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PLANT CELL

Contaminant with functional group

Conjugation

Soluble conjugates of Contaminant in VACUOLE

Apple-tree (Malus domestica) Zelkova (Zelcova caprinifolia) Poplar (Populus canadensis) Ryegrass (Lolium perene) Lilac (Siringa vulgaris) Weeping willow (Salix) Catalpa (Catalpa bignonioides) Platan-tree (Platanus orientalis) Sophora (Sophora japonica)

Alder (Alnus barbata) Asp (Populus tremula) Elm (Ulmus filiacea) Ash (Fraximus excelsior) 0.1 – 1.0 Tea (Camellia sinensis L.) Persimmon (Diospyros lotus) Bay laurel (Laurus nobilis)

Gleditdchia (Gleditschia triacanthos) Kidney (Phaseolus vulgaris) Pine (Pinus) Pine (Pinus eldarica) Thuja (Tuja) Apricot (Prunus armenicana) Vine (Vitis vinifera)

Cypress (Cupressus sempervirens var. Pyramidalis) Geranium (PelargoFir (Picea abies) nium roseum) Mulberry (Morus alba) Privet (Ligustrum Lime-tree (Tilia cauxasica) vulgare) Reed (Phragmites Fig (Ficus carica) communis) Pomegranate (Punica Maize (Zea mays) granatum) 0.001 – Wild plum (Prunus Rhododendron (Rhododendron ponticum) 0.1 divaricata) Kiwi (Apteryx australis) Peach-tree (Persica Aloe (Aloe) vulgaris) Medlar (Mespilus Potato (Solanum germanica) tuberosum) Tomato (LycoperssiRose (Rosa) Platan-tree (Platanus) cum esculentum) Pussy-willow (Salix alba) Cherry-plum (Prunus vachuschtii)

Compartmentation

Conjugate of Contaminant with cell compounds

Functionalization is a process whereby a molecule of a hydrophobic organic xenobiotic acquires a hydrophilic functional group (hydroxyl, carboxyl, amino, etc.) as a result of enzymatic oxidation, reduction, hydrolysis, etc. Due to the introduction of functional group, the polarity and corresponding reactivity of the toxicant molecule is enhanced. This promotes an increase of intermediate’s affinity to enzymes, catalyzing further transformation. Conjugation takes place as a basic process of phytoremediation and is determined by the formation of chemical coupling of the contaminant to the endogenous cell compounds (proteins, peptides, amino acids, organic acids, mono-, oligo-, polysaccharides, lignin, etc.), so forming peptide, ether, ester, thioether or other type covalent bonds. Intermediates of the contaminant initial transformations or those contaminants which themselves possessing functional groups capable of reacting with intracellular endogenous compounds, are all susceptible to conjugation. Commonly, the main part of the toxicant molecules undergoes conjugation and only a small amount is deeply degraded (0.1-2%, depending on structure of contaminants). Conjugation is a widespread defence mechanism in higher plants, especially in cases when the penetrated contaminant concentration is exceeding the plant transformation (decomposition) potential. Increased amounts of deep degradation to regular plant sell metabolites, or CO2 and water, most often is achieved in the case of linear, low molecular weight structures of contaminants. The toxicity of the conjugates compared to parent compounds is significantly decreased due to creat-

Plants

Maple (Acer campestre) Oleaster (Elaeagnus angustifolia) Locust (Robina pseudoacacia) Wild pear (Pyrus caucasica) Walnut (Juglans regia) 1.0 – 10.0 Almond-tree (Amigdalus communis) Cherry-tree (Cerasus avium) Amorpha (Amorpha fructicosa) Cherry-tree (Cerasus vulgaris) Chestnut (Castanea sativa)

Insoluble conjugates of Contaminant in CELL WALL

Figure 4. The main pathways of organic contaminant transformation in plant cells.

120

Amount of absorbed aromatic hydrocarbon by 1 kg of fresh leaves, for 24 hours, in mg.

Functionalization

Plants

Organic Contaminant

Deep Oxidation

Strong absorbers

Excretion

CO2

Average absorbers

Nontransformed Organic Contaminant

Weak absorbers


Table 2. Plants according to leafs potential to assimilate benzene and toluene.



ing the new compound containing large non-toxic part. Conjugates are kept in the cell for a certain period of time without causing visible pathological deviations in from cell homeostasis. The conjugate formation also gives the plant cell extra time for the internal mobilization, and the induction of enzymes responsible for contaminants further transformation. Relatively quickly, after the termination of plant incubation with the contaminant, conjugates are no longer found in plant cells. Some attempts have been made by authors (unpublished data) to estimate different plant (soybean, ryegrass) cells’ potential to accumulate conjugated benzene in their cells in the case of toxicant saturation. In spite of incomplete information, it was supposed that for genetically non-modified plants, it could be, as a minimum, several molecules of contaminant conjugates per one of plant sell. Although conjugation is the most widely distributed pathways of plant self-defence, it cannot be assumed as energetically and physiologically advantageous for metabolic processes in plants. Firstly, the formation of conjugates leads to the depletion of vitally important cellular compounds, and secondly, unlike deep degradation, the formation of conjugates maintains the contaminant basic molecular structure, and hence results only in partial and provisional decreasing of its toxicity. Compartmentation in most cases is the final step of conjugates processing. Soluble conjugates of toxic compounds (coupled with peptides, sugars, amino acids, etc.) are accumulated in the cell structures (primarily in vacuoles), while insoluble conjugates (coupled with lignin, starch, pectin, cellulose, xylan) are moved out of the cell via exocytosis into the apoplast and accumulated in the cell wall. The compartmentalization process is analogous to mammalian excretion, essentially removing the toxic part from metabolic tissues. The major difference between detoxification in mammals and plants is that plants do not have a special excretion system for the removal of contaminant conjugates from the organism. Hence, they use a mechanism of active transport for the removal of the toxic residues away from the vitally important sites of the INDUSTURIAL ENGINEERING

cell (nuclei, mitochondria, plastids, etc.). This active transport is facilitated and controlled by the ATP-dependent glutathione pump and is known as “storage excretion”. The above described pathway of toxic compound processing, i.e., functionalization → conjugation → compartmentalization, is well illustrated by the processing of anthropogenic contaminants of different structures. One of such examples demonstrating the transformation of organochlorine pesticides is the hydroxylation of 2,4-D followed by conjugation with glucose and malonyl residues and deposition in vacuoles (Figure 5). COOH CH2 O CH2 COOH Cl

Cl 2,4-D

O CH2 COOH Cl

I

O C O Cl CH2

II

Cl OH 4-Hydroxy-2,5-D

CH2 COOH O Cl

O O

OH

III

In vacuole

HO OH O-�-D-Glucoside of 4-hydroxy-2,5-D

Figure 5. 2,4-D transformation for deposition in vacuoles.

Anthropogenic organic toxicants decomposition processes are closely related to many aspects of higher plants cellular metabolism. In prolonged and multifunctional detoxification processes quite a few enzymes are actively involved. According to catalyzed reactions they are directly or indirectly participating in detoxification process.

2.3 Enzymes Transformations of contaminants during functionalization, conjugation and compartmentation are of enzymatic nature. It is remarkable that due to their unusual flexibility in the absence of contaminants, in plant cell these enzymes catalyze reactions typical for regular plant cell metabolism. Based on multiple literature data the following enzymes directly participate in the transformation process of anthropogenic contaminants: - Oxidases, catalyzing hydroxylation, dehydrogenation, demethylation and other oxidative reactions (cytochrome P450-containing monooxygenases, peroxidases, phenoloxidases, ascorbatoxidase, catalase, etc.) CHAPTER 8

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- Reductases, catalyzing the reduction of nitro groups (nitroreductase) - Dehalogenases, splitting atoms of halogens from halogenated and polyhalogenated xenobiotics - Esterases, hydrolyzing ester bonds in pesticides and other organic contaminants. The first step of contaminates transformation in majority of cases is carried out by oxidative enzymes, the most often contaminants oxidation is performed by the following metabolically active enzymes having the various metabolic functions: Cytochrome P450-containing monooxygenases (EC 1.14.14.1) are mixed-function enzymes located in the membranes of the endoplasmic reticulum (microsomes). Monooxygenase system contains redox-chain for electron free transport, the initial stage of electron transfer is a nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (EC 1.6.2.4); the intermediate carrier-cytochrome b5, and the terminal acceptor of electrons-cytochrome P450. When NADPH is used as the only source of reductive equivalents, the existence of an additional carrier, a nicotinamide adenine dinucleotide (NADH)-dependent flavoprotein is required. NADH may also be oxidized by the NADPH-dependent redox system. In the latter case cytochrome b5 is not required. The cytochrome P450-containing monooxygenases use NADPH and/or NADH reductive equivalents for the activation of molecular oxygen and incorporation of one of its atom into lipophilic organic compounds (XH) that results in formation of hydroxylated products (XOH). The second atom of oxygen is used for the formation of a water molecule (Figure 6). Plant cytochrome P450-containing monooxygenases play an important role in the hydroxylation of organic contaminants. The enzymes participate in the reactions of C- and N-hydroxylation of aliphatic and aromatic compounds, N-, O-, and Sdealkylation, sulpho-oxidation, deamination, Noxidation, oxidative and reductive dehalogenation, etc. The resistance of plants against herbicides is mediated by their rapid intracellular transforma122

tion into hydroxylated products and subsequently conjugated to carbohydrate moieties in the plant cell wall. For examples, N-demethylation and ringmethyl hydroxylation of the phenylurea herbicide chlorotoluron in wheat and maize is cytochrome P450-dependent processes. For some phenyl urea herbicides in the Jerusalem artichoke cytochrome P450-mediated N-demethylation is sufficient to cause significant or complete loss of phytotoxicity.

NADPH NADH

2e-

XOH + H2O

XH

O2

2e-

Reductase

b5

N N

2e-

Fe

2+

N

N

P450

XH - nonpolar xenobiotic XOH - hydroxylated xenobiotic

Figure 6. Microsomal monooxygenase system.

Peroxidase. In higher plants, peroxidase activity increases in response to stress. The great catalytic versatility of the peroxidase is its predominant characteristic and, therefore, no single role exists for this multifunctional enzyme. - The peroxidase is defined by the following reaction: - RH2 + H2O2 → 2H2O + R - The peroxidases catalyze a number of free radical reactions. Alternatively, the compound that is directly oxidized by the enzyme further oxidizes other organic compounds, including xenobiotics. This notion is based on the wide ubiquitous distribution of this enzyme in plants (the isozymes of peroxidase in green plants occur in the cell walls, plasmalemma, tonoplasts, intracellular membranes of endoplasmic reticulum, plastids and cytoplasm), and the high affinity and wide substrate specificity of plants peroxidases to organic xenobiotics of different chemical structures. The participation of plant peroxidases in hydroxylation reactions of xenobiotics has



been widely discussed. For example, peroxidases from different plants are capable of oxidizing N,N-dimethylaniline, 3,4-benzpyrene, 4-nitroo-phenylendiamine, 4-chloroaniline, phenol, aminoflourene, acetaminophen, diethylstilbestrol, butylated hydroxytoluene, hydroxyanisoles, benzidine; horseradish (Armoracia rusticana) peroxidase oxidizes tritium-labelled [C3H3] TNT. Phenoloxidases, group of the copper-containing enzymes (other names-tyrosinase, monophenol monooxygenase, phenolase, monophenol oxidase, etc.) are spread within the plant cell organelles catalyzing both monooxygenase and oxygenase reactions: the o-hydroxylation of monophenols (monophenolase reaction) and the oxidation of o-diphenols to o-quinones (diphenolase reaction). Currently accepted enzyme nomenclature classifies hydroxylating phenol oxidase as monophenol monoxygenase (EC. 1.14.18.1) and o-diphenols oxidizing phenol oxidase as catechol oxidase (EC 1.10.3.1). Plant phenol oxidases appear to be a group of specific enzymes, oxidizing wide range of o-diphenols, such as DOPA (dihydroxyphenylalanine), catechol, etc, but unable to convert m- or p- diphenols to the corresponding quinons, Substrate specificity of catechol oxidase from Lucopus europaeus and characterization of the bioproducts of enzymatic caffeic acid oxidation. The active center of phenol oxidases contains two cooper atoms and exists in three states: “met’, “deoxy” and “oxy”. Phenoloxidases actively participate in the oxidation of xenobiotics of aromatic structure. As it has been demonstrated phenoloxidase from spinach, analogously to many other plants, oxidizes aromatic xenobiotics (benzene, toluene), by their hydroxylation and further oxidation to quinone. In a number of the cases, if the xenobiotic is not a substrate for the phenoloxidase, it may undergo co-oxidation in the following manner: the enzyme oxidizes the corresponding endogenous phenol by forming quinones or semi-quinones or both, i.e. compounds with a high redox potential. These compounds activate molecular oxygen by forming oxygen radicals, such as superoxide anion radical (O2–.) and hydroxyl radical (.OH), that gives compounds the capacity INDUSTURIAL ENGINEERING

for the further oxidation of xenobiotic. The formation of these radicals enables phenoloxidase to participate in contaminants degradation processes also by co-oxidation mechanism presented Figure 7 . OH

OH OH

R

O O

o-Diphenoloxidase

O

R Semi-quinone

o-Diphenol

...

R o-Quinone

+ O2 HO hydroxyl radical _ O2 . superoxide anion-radical

[O] Oxygen active species

OH

OH

O OH

Benzene

Phenol

Catechol

o-Quinone

OH

O O

C

C

O OH

...

Cis-cis-muconic acid

Figure 7. Enzymatic oxidation of o-diphenols (upper) by phenoloxidase and non-enzymatic co-oxidation of benzene (lower).

Analogously, nitrobenzene is oxidized to m-nitrophenol, and the methyl group of [C3H3] TNT is oxidized by phenoloxidase from tea plant. The information confirming participation of this enzyme in the oxidative degradation of xenobiotics in higher plants is sparse, despite the fact that participation of phenoloxidase should definitely be expected while xenobiotics degradation. Laccase of basidial fungi, analogous to higher plant phenoloxidase, have been better explored. Laccase degrades different aliphatic and aromatic hydrocarbons, and actively participates in the enzymatic oxidation of alkenes. Crude preparations of laccase isolated from the white rot fungus Trametes versicolor oxidizes 3,4-benzopyrene, anthracene, chrysene, phenanthrene, acenaphthene and some other PAHs. The intensity of oxidation of these antropogenic contaminants is increased in the presence of such mediators as: phenol, aniline, 4-hydroxybenzoic acid, 4-hydroxybenzyl alcohol, methionine, cysteine, reduced glutathione, and others compounds-substrates of laccase. These data indicate that in the cases of fungal laccase and plant o-diphenoloxidase, the oxidation of hydrocarbons is carried out by a co-oxidation mechanism. CHAPTER 8

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Conjugation reactions of contaminants in plant cell are catalyzed by transferases: glutathione Stransferase (GST), glucuronozyl-O-transferase, malonyl-O-transferase, glucosyl-O-transferase, etc. Compartmentation of intermediates of contaminants transformation-conjugates takes place under the action of ATP-binding cassette (ABC) transporters. Depending on the structure of the contaminant some other enzymes may also be involved in their degradation process. Prolonged in time cellular decomposition of contaminants involves participation of enzymes providing plant cell with extra energy needed for the defence processes, induction of the enzymes, and provision of cells by vitally important secondary metabolites. Enzymes involved in these and similar processes obviously indirectly participate in the detoxification of contaminants. The correlation between the penetration of organic contaminants (alkenes, aromatic hydrocarbons, polycyclic aromatic hydrocarbons) in plant cells and the corresponding changes in the activities of enzymes participating in energy supply (malate dehydrogenase) and nitrogen metabolism (glutamate dehydrogenase, glutamine synthetase) has been revealed. As it has been shown the activities of the enzymes are highly affected by xenobiotics concentration, exposure time and mode of illumination. Ecologically the most advantageous pathway of organic contaminants transformation in plants is their deep oxidative degradation. In higher plants mainly the following enzymes are responsible for this process: cytochrome P450-containing monooxygenese, peroxidase and phenoloxidase. To correctly evaluate the universality of the action of these enzymes, responsible for the degradation of different structure organic contaminants, some of their specificities should be emphasized (Table 3).

2.4 Contaminants Action on Plant Ultrastructure To evaluate the ecological potential of plants, the data proving the responses at the level of cellular ultrastructure under the action of contaminants, as the most precise indications of plants exploitation, 124

should also be emphasized. Undoubtedly, penetration of even a small concentration of contaminants into plant cells leads to invisible, but most often measurable deviations in cell metabolic processes such as induction of enzymes, inhibition of some intracellular metabolic processes, change the level of secondary metabolites, etc. The existence of plant cell contaminants in increased concentrations provokes clearly noticeable deviations in cellular ultrastructure under the action of contaminants, as the most precise indications of plants ultrastructural organization. It has been shown that the complex of changes and alterations in the main metabolic processes of plant cell elicited by organic pollutants (pesticides, hydrocarbons, phenols, aromatic amines, etc.) is connected with the deviations of cell ultrastructural architecture.

Figure 8. Electron micrographs showing the penetration and movement of 14C-labelled nitrobenzene (0.15 mM) in a maize root apex cell. The xenobiotic penetrated through the plasmalemma, moved to the cytoplasm, and thereafter translocated into vacuoles. 1 – x 48 000; 2 – x 36 000; 3 – x 50 000; 4 – x 30 000

The sequence and deepness of the destruction in plant cell organelles are determined by the variety of plant, chemical nature, concentration and duration of the contaminant action, etc. This course of events has been experimentally demonstrated by



authors in a number of various higher plants exposed to different 14C-labelled toxic compounds. Due to the penetration of contaminants in plant cells changes in ultrastructural organization has been shown. Apparently, the negative effects of toxic compounds on cell ultra-structure, depending on its concentration, could be divided in two types, being different for each contaminant and plant:

seldom, in the cytoplasm and mitochondria. As a result of prolonged exposure the amount of a label significantly increases in the nucleus, at the membranes of organelles, in tonoplasts, and further in vacuoles, i.e. xenobiotic becomes distributed in most of subcellular organelles, but ultimately, there is a tendency of contaminants primary accumulation in vacuoles.

- metabolic which is digested by the plant in spite of insignificant negative effect;

2.5 General Considerations

- lethal, leading to indigestible deviations and to the plant death. Figure 8 shows maize root apex cells exposed to 14C-nitrobenzene action, its penetration across the plasmalemma and localization in subcellular organelles. Studies of penetration of 14C–labelled xenobiotics into the plant cell indicate that the labelled compounds at the early stages of exposure (5–10 min) are detected in the cell membrane, in the nuclei and nucleolus (in small amounts) and,

Enzyme

Obviously plants, as remediators, for a long time act most effectively at low and shallow contamination of soil and air, when no significant changes in cell ultrastructure take place. Planting of almost any kind of vegetation, including agricultural vegetation is beneficial for the environment. However, in order to make the exploitation of the most ecological potential of each particular plant, the selection should be carried out according to the plants potential to assimilate/accumulate toxic compounds of different structure.

Physiological function

Existence in cell

Localization

Specificity to toxicants

Cytochrome P450 containing monooxygenase

Participation in a number of intracellular synthesizing reactions

Small amount, inductive nature

Endoplasmatic reticulum, cytosole

Very high affinity to nonpolar toxicants

Peroxidase

Hormonal regulation, lignification, response on stress, removing of peroxides

Large amount, inductive nature

Cell wall,vacoules, cytosole, tonoplasts, plastids, plasmalemma

Phenoloxidase

Oxidative transformation of phenols, lignification, cell defence reactions

Large amount, presents in latent form too, inductive nature

Chloroplasts, cell wall, cytosole, tonoplasts

Affinity to aliphatic compounds

Affinity to aromatic compounds

Limiting factors

Stability

NADPH, NADH

Labile, inactivating during substrate oxidation

Hydrogen peroxide or organic hydroperoxides

Stable

Endogenous phenols

Stable

Table 3. Plants oxidative metalloenzymes. INDUSTURIAL ENGINEERING

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Phytoremediation is a unique clean-up strategy. The realization of phytoremediation technologies implies the planting on a contaminated area with one or more specific, previously selected plant species with the potential to extract contaminants from soil. A precise survey of the vegetation on site should be undertaken to determine what species of plants would have the best growth at the contaminated site. Based on a number of experimental results including the use of labeled xenobiotics and electron microscopic observations, the deep degradation of anthropogenic contaminants in plants could be considered as narrow but permanently acting pathway having much less potential than conjugates formation process (especially in case of contaminants saturation). Transgenic plants have also been studied in connection with degradation of some particular contaminants. For this purpose the widely distributed explosive TNT (trinitrotoulene) has generally been chosen. In order to increase the degradability of TNT and similar compounds, the transgenic plants contained the gene of bacterial enzyme (pentaeritrole tetranitrate reductase, EC 1.6.99.7) were received. Transgenic tobacco has been analysed for its ability to assimilate the residues of TNT and trinitroglycerine. Seedlings of the transgenic plants extracted explosives from the liquid area much faster, accomplishing denitration of nitro groups, than the seedlings of common forms of the same plants, in which growth was inhibited by the contaminants. Transgenic tobacco differed substantially from the common plant by its tolerance, fast uptake and assimilation of significant amounts of TNT. Analogous experimental results have been obtained with other plant species. There are dozens of publications concerning successful improvement of plant detoxification abilities by cloning the genes of transferases and oxidases, which intensively participate in contaminants transformation processes. Obviously, the attempts to improve artificially ecological potential of higher plants will be continued and the results will be more substantial from the viewpoint of their eventual practical realization. The positive effect of these investigations could be 126

much more impressive if all aspects of the quite complicated and multistage detoxification process would be better elucidated with regard to plant physiology and biochemistry. Such information would allow the creation of more rational and effective strategy for the gene engineering potential application. The cost of phytoremediation technologies. Bioremediation is a completely natural process based on the joint detoxification action of plants and microorganisms. Phytoremediation technologies are economically competitive, compared with existing conventional ones. Dozens of scaled up examples have demonstrated the superiority of plant-based remediation technologies, mainly due to the following reasons: phytoremediation, being a natural, solar energy-driven process, does not require any additional energetic or significant material or other input; phytoremediation takes place in situ and requires no digging or hauling; little mechanical equipment is needed to operate the phytoremediation process. The cost components for the implementation of phytoremediation technologies include: - detailed characterization of the polluted site (soil type, water content, type of contaminant(s), concentration of contaminant(s) in the soil, etc.); - selection of appropriate plants and consortia of microorganisms; - the corresponding irrigation system; - capital cost, materials, monitoring, including required instrumentation, indirect costs, etc.; - operation and maintenance (labour, materials, chemicals, laboratory analyses, etc.). Phytoremediation offers cost advantages, but it should be underlined that the time needed for full remediation is typically lengthy. Table 4 gives estimates of the costs of phytoremediation as compared with existing conventional technologies. Plants solely or in combination with specially selected microorganisms (or their consortia), are very promising detoxifiers allowing to create ecologically friendly technologies around or along hotbeds of



Phytoremediation

Conventional Treatment

Contaminant and matrix

Application

Estimated cost

Application

Estimated cost

Lead in soil (1 acre)

Extraction, harvest, and disposal

$ 150,000– 250,000

Excavate and landfill

$ 500,000

Solvents in groundwater (2.5 acres)

Degradation and hydraulic control

$ 200,000 for installation and initial maintenance

Pump and treat

Total petroleum hydrocarbons in soil (1 acre)

In situ degradation

$ 50,000– 100,000

Excavate and landfill or incinerate

Projected savings 50–65%

$ 700,000 50% cost saving annual by the 3rd year operating cost $ 500,000

80%

Table 4. Estimated cost savings through the use of phytoremediation rather than conventional treatment, according to EPA data.

contamination. Ecotechnologies based on the use of microorganisms and plants represent the most modern way of remediation potential realization. Elaboration of a new ecological concept, unifying experience accumulated for last 3-4 decades and based on effective use phytoremediation/remediation (plants/microbial) joint potential should be highly beneficial for the whole world, by increasing its ecological potential.

3. Bacterial Viruses Against Crop Pathogens 3.1 Bacterial Diseases of Plants There is a continuing need in safe, residue-free food production. A major threat to food production is plant diseases and absence of a proper effective disease management. Plant diseases caused by bacterial pathogens can account for major economic losses to agricultural production. Fire blight of pear and apple, caused by Erwinia amylovora was first bacterial disease of plants discovered in 1877 by T. J. Burrill. Since that several bacterial diseases of plants, both annual and perennial have been described and their causative pathogens identified. Generally, phytopathogens belong to the genera Erwinia, Pectobacterium, Pantoea, Agrobacterium, Pseudomonas, Ralstonia, Burkholderia, Acidovorax, Xanthomonas, Clavibacter, Streptomyces, Xyllella, Spiroplasma, and Phytoplasma.


Bacteria become active and cause problems when there are suitable conditions for their multiplication. Such conditions could be determined by factors such as high humidity, crowding, and poor air circulation, plant stress caused by irregular or overwatering. Deficient or excess of nutrients are also conductive for the bacterial diseases development. Bacterial diseases are spread in many ways – rain, wind, birds or insects. People can also spread bacterial diseases by using infected pruning tools, by improper disposal of infected plant material, improperly managing plants in the winter, or introducing infected plants in an area. Bacteria get inside a plant through natural opening, such as stomata or wounds. Bacteria can survive in the soil and crop debris, and in seeds and other plant parts that serve as a source of disease. In many cases, they can be in a latent form and not detectable for a long time. A bacterial disease may cause a variety of symptoms: blights, cankers, galls, leaf spots, overgrowths, specks, scabs, or wilts. Generally, the common name of the disease is a combination of the symptom or appearance and its location on the plant, like bacterial leaf spot caused by Pseudomonas cichorii and fire blight in pears and apples, caused by Erwinia amylovora. In contrast to viruses that live inside plant cells, bacteria grow in the spaces between cells, producing toxins, special proteins or enzymes that damage the plant cells. Agrobacterium causes genetic modification of cells, producing cancer-like growths called galls. As a result of bacterial diseases caused by phytopathogenic bacteria, the loss of different kinds of vegetables in the field as well as post-harvest is high and varies depending on disCHAPTER 8

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ease and geographic location. When the conditions are beneficial for phytopathogen multiplication, the loss may be very high significantly affecting country’s economics. Crop diseases caused by bacteria need to be controlled to maintain the quality and abundance of food. Bacterial diseases are difficult to control; usually it is better to prevent the spread rather than cure a plant. However in agricultural applications there is a need for both - prevention and treatment.

3.2 Plant Disease Control Different approaches may be used to prevent, mitigate or control plant diseases . The application of disease- and pathogen-free transplants, repeated digging, burning of plant wastes, three years or longer crop rotation, prevention of surface wounds on plants are in arsenal of cultural practice. Challenges in bacterial-incited diseases control include low effectiveness or in some cases ineffectiveness of cultural practice. Application of pesticides against plant pathogens and overuse of chemical fertilizers together with good agronomic practices have contributed significantly to the spectacular improvements in crop productivity and quality over the past century. However, excessive use and misuse of agrochemicals has led to environmental pollution and consequently to considerable changes in public attitudes towards the use of pesticides in agriculture. Development of alternative methods for pest control and diseases is of paramount importance. Plant disease management greatly relies on application of chemicals such as bactericides: antibiotics and copper-based compounds. Extensive application of copper compounds resulted in copper resistance in many plant pathogenic bacteria, which is associated with plasmids and chromosomes. Besides the resistance, extensive use of the bactericides may cause phytotoxicity and soil contamination, especially when applied at high concentrations to overcome the resistance and unfavorable weather conditions. After discovery of antibacterial activity of Penicillium notatum mould by Alexander Fleming (1929), the era of antibiotics began. Antibiotics, microbial toxins can poison or kill other microorganisms and thus became as part of a management strategy for 128

various bacterial diseases. The antibiotic streptomycin has been used to control phytopathogens since 1950s. Just after several years of application against bacterial spot of tomato and pepper, phytopathogens developed resistance to streptomycin, which limited its efficacy for managing these diseases . Streptomycin has also been used for many years for the management of a number of other bacterial plant pathogens. The efficacy of streptomycin for control of fire blight of apple and pear lasted much longer than for bacterial spot of tomato and pepper because the streptomycin resistance was associated with a plasmid which required acquisition by sensitive strains. Streptomycin applied against Pseudomonas syringae pathovars for many years also resulted in subsequent development of resistance in these bacteria. The antibiotic resistance is not the only challenge for the use of these chemicals against phytopathogens. Another serious concern with antibiotics is spread of resistance genes to other bacteria, including other pathogenic or nonpathogenic bacteria present in the environment. Widespread emergence of resistance among common pathogenic bacteria has driven demand to develop alternatives to conventional antibiotic therapy. Diverse microorganisms excrete other metabolites that can interfere with pathogen growth and/or activities. Many microorganisms produce and release lytic enzymes that can hydrolyse a wide variety of polymeric compounds, such as chitin, proteins, cellulose, hemicellulose, DNA. Microorganisms that show a preference for colonizing and lysing plant pathogens (by means of antibiotics or lytic enzymes they produce) might be classified as biocontrol agents. Phytoncides and phytoalexins are biologically active substances of plant origin that kill or inhibit the growth and development of bacteria, microscopic fungi, and protozoa. Phytoncides play an important role in plant immunity and in the interrelationships of organisms in biogeocoenoses. The antimicrobial potency and range of phytoncides vary greatly among different plant species and study on their potential against phytopathogens of vegetables could be of interest. Several alternative control methods of bacterial diseases have been investigated in recent years. Among them



is application of the systemic acquired resistance (SAR) inducers. Systemic acquired resistance is a biochemical state of the plant in which plant develops greater resistance to different pathogens. Several substances that specifically induce SAR, such as acibenzolar-S-methyl (ASM), (known and Actigard, Syngenta NC) and harpin (Messenger, Eden Bioscience, WA) have shown activity against bacterial spot in tomato in Florida, Alabama, North Carolina, Ohio and Ontario, Canada. The SAR inducers have shown success for managing several other bacterial diseases, including bacterial speck of tomato and pepper, Xanthomonas leaf blight on onion and fire blight on apple but were ineffective with other pathosystems. In addition, in some cases negative effects on plants yield were reported. One of the alternative ways of bacterial diseases prevention is the application of genetically modified plants. However, in many countries there is consumer reluctance to use them as food. Biological control of plant disease control is important strategy both from environmental and economic points of view. Biological control of disease is the employment of natural enemies of pests or pathogens in the eradication or control of their population Some biological approaches to control plant bacterial diseases were based on application of nonpathogenic, saprophytic bacteria, non-pathogenic bacteriocin-producing Agrobacterium radiobacter strains that inhibit closely related pathogenic strains, and plant growth-promoting rhizobacteria (PGPR), to suppress pathogen populations. These approaches to biological control achieved varying levels of success, and need additional research to improve their reliability under field condition.

(phages) are viruses that infect bacterial cells. These obligate intracellular parasites multiply inside bacteria by using of some or all of the host biosynthetic machinery. Bacterial viruses can be found in every environment where their bacterial hosts are present. Bacteriophages population number in the world (aquatic systems and soil) has been estimated in the range of 1030-1032, represented by different genera and species. Over 5500 different bacteriophages have been discovered to date, each of which being able to infect one or several types of bacteria. Phages are obligatory parasites of a bacterial cell. They exhibit several life cycles, mostly: lytic and lysogenic. The lytic phages are of interest in phage application as antimicrobials. Investigations of several isolated phages with antimicrobial activities revealed that these are lytic tailed phages represented by three families of order Caudovirales: Myoviridae, Siphoviridae and Podoviridae. They differ with each other according to morphology: Myoviridae have the biggest hexagonal capsid head of size around 150nm and contractile tail (Figure 9); Siphoviridae have small capsid head of 60-60nm and a long noncontractile tail; Podoviridae with a small capsid head and short tail.

3.3 Bacteriophages as Biological Control Agents Bacterial viruses or bacteriophages, or phages being natural enemies of bacteria offer complementary approaches to conventional antibiotics and other antimicrobial agents, and they can fight bacterial infections either in humans, animals and agricultural crops. Bacterial viruses or bacteriophages INDUSTURIAL ENGINEERING

Figure 9. Structure of a typical miovirus bacteriophage structure. 1 - icosohedral head; 2 - DNA; 3 - protein coat; 4 - collar; 5 - sheath; 6 - baseplate; 7 - tail fiber.

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The stages of the lytic cycle include: Adsorption highly specific phage attachment to a specific receptor on the surface of host bacterial cell; Infection of the DNA - transfer of phage nucleic acid into the bacterial cell through pores that are formed in the host cell wall as a result of phage lysozyme activity; Direction of bacterial metabolism to phage DNA replication and encoded phage protein synthesis; Assembly and packing of phage particles; Bacterial cell lysis and phage progeny release (Figure 10).

disease. By application of co-inoculating potato tubers with pathogen and phage, the researchers succeeded in preventing rotting of the tubers. In around three decades after the first works on using phages against plant bacterial diseases it was reported that few phage particles completely blocked tumor induction by a highly virulent strain of Agrobacterim tumefaciens after 21 h of inoculation. Later, in 1969 Civerolo and Keil applied foliar phage-treatments 1h before inoculation of the peach seedlings by Xanthomonas pruni to fight the pathogen on peach seedlings and significantly reduced bacterial spot severity (86% to 100%). In 70ies, most of the published research concerning phages specific to bacterial plant pathogens was oriented to typing of phytopathogens, mostly E. amylovora strains.

Figure 10. The lytic cycle of bacteriophage.

Bacteriophage-host system is highly specific. Most of the phages adsorb and lyse a single species of bacteria. Bacteriophages as potential agents for control of plants bacterial diseases were first proposed in the 1920s, soon as they were discovered by Twort in 1915 and d’Herelle in 1917. Mallman and Hemstreet were pioneers who observed in 1924 inhibition of bacteria X.campestris pv. campestris, causing cabbage rot from filtrate of the liquid collected from the decomposing plant. The first phages isolated from environmental sources were against plant pathogens Pectobacterium carotovorum subsp carotovorum and Agrobacterium tumefaciens. It was demonstrated by Kotila and Coons in 1925 that bacteriophages isolated from the soil suppressed growth of Pectobacterium carotovorum subsp atrosepticum, the causal agent of potato blackleg 130

The possibility of application of bacteriophage in the epidemiology of E. amylovora-associated fire blight was firstly reported by Erskine. It followed by E. amylovora specific phage jEa1 successful application to prevent/treat fire blight in apple seedlings artificially inoculated with E. amylovora. Fire blight symptoms in pear fruit inoculated with E. amylovora was attenuated by treatment with the enzyme polysaccharide depolymerase encoded by jEa1. Dozens of several E.amylovora specific bacteriophages were isolated later from various fruit and soil samples collected at sites displaying fire blight- and sites around Niagara, the Royal Botanical Gardens in Hamilton, Ontario. Molecular characterization of the phages with PCR and Restriction Fragment Length Polymorphism (RFLP) revealed close relation of some of them to jEa1. However a study of the phages host ranges revealed that not all of them were able to lyse efficiently some E.amylovora strains, and also that some phages were capable of lysing the epiphytic bacterium Pantoea agglomerans. Kozloff et al., received US patent in 1983 for the idea to increase frost-resistance of plants by using phage to reduce population of E.herbicola and Pseudomonas syringae on plant leaves, contributing to tissue damage caused by exposure to low temperatures. Another US patent was



granted to Jackson in 1989 for the use of phage preparations for eliminating naturally occurring P. syringae from contaminated bean culls as well as reducing the severity of disease symptoms in bean leaves artificially infected with the phytopathogen. Next, phage preparations targeting against select agent Ralstonia solanacearum and X. campestris pv.vesicatoria responsible for the bacterial wilt and bacterial spot, two main diseases of tomato plants were developed. After 2 weeks of inoculation with the R. solanacearum, 60% of tomato plants treated with phage had 12% less defoliation as compared to untreated plants. The authors of the Chapter have isolated and studied bacteriophages, specific to endemic strains of X. vesicatoria bacterial strains, causing tomato bacterial spot in Georgia in 2005-2008. Phages pure lines were isolated from bacterial spot diseased tomato and sewage collected from different tomato production regions of the country. Physical, chemical and biological properties and their potential for tomato bacterial spot control have been studied. All phages were characterized by quite short period of intracellular propagation (the maximum time of adsorption varied from 18 minutes to 24, and latent period from 36 to 45 minutes), productive cycle and strong virulence.

The efficacy of 7x107p.f.u/ml mixture of the three pages have been studied on tomato seedlings foliages of which were artificially inoculated with 109 c.f.u/ml culture of X. vesicatoria virulent strains in greenhouse. Spraying of phage with artificially infected plants simultaneously with inoculation or after 24 hours completely was hindered initiation of a bacterial spot disease. Treatment with phage a week later reduced the severity of the disease (Figure 11). Similar results were obtained in case of application of the mixture of phages on tomato seedlings with artificially infected green fruits (Georgian National Patent 4860). The first field trials with phages were conducted by Thomas in 1935 against Stewart’s disease of corn (Zea mays). He treated corn seeds infested with the pathogen, Pantoea stewartii, with phages isolated from diseased plant material. This seed treatment was quite effective and resulted in a reduction of disease incidence from 18% (untreated) to 1.45%. As it was already mentioned, the discovery of Alexander Fleming was followed with boom in wide application of antibiotics, including in agriculture that further led to the decline of interest in phagotherapy. Lately, there has been a renewed interest in phages due to concerns over environmental contamination. Phages are natural components of the biosphere; they can readily be isolated from wherever bacteria are present. No adverse effects to humans, wildlife or the environment are expected from phages. Due to their high specificity it is assumed that they are non-toxic to the eukaryotic cells. The main advantages are that they are specific to the disease-causing bacterium, and are eliminating only target bacteria without damaging other, possibly beneficial members of the indigenous flora.

Figure 11. Phage treatment of artificially infected with X.vesicatoria tomato seedlings. 1-Intact plants; 2 - The bacterial-control plant; 3 - Phage sprayed at the moment of infection; 4 Phage sprayed after a week of Infection. INDUSTURIAL ENGINEERING

Use of phages can also be coupled with the application of antagonistic bacteria for increased pressure on the pathogen; or they can be used to promote a desired strain against other members of the indigenous flora. They are self-replicating and selflimiting. Phage treatment, integrated with other practices, such as plant activators, recent formuCHAPTER 8

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lated phages in combination with other biological control agents and SAR inducers is currently used in greenhouses and fields sad (Jones et al., 2007). Phage preparations are fairly easy and inexpensive to produce and can be stored at 4◦C for months without significant reduction in titer. Application can be carried out by standard farm equipment. Since phages are not inhibited by the majority of agrochemicals, they can be tank-mixed with many agrochemicals without significant loss in titer.

3.4 Challenges in Using Phages for Disease Control

isolates within a species, are spontaneously derived from their wild-type parent phages, and are so named because they lyse not only parent wild-type bacteria, but also phage-resistant mutants originating from parent bacteria. Despite recent interest in biological control of plant bacterial diseases, the interactions between phages, bacteria, plants, and influence of environmental factors on the system are still not well understood. Until recently, the role of populations of bacteriophages in plant biocenoses has not been adequately studied. Despite many researches with phages, plant bacteriophages are less studied in the world. Though there were successes at the early stages of bacteriophage against plant pathogenic bacteria, phage-therapy did not turn into a practical antibacterial strategy for controlling plant pathogenic bacteria due to some problems and first of all, with efficacy and reliability. Resistance to phages, availability of suitable phage, strain variation, persistence of phages in the phyllosphere and rhizosphere are main challenges in phage therapy. Besides, phages narrow spectrum of activity against specific bacterial species is a disadvantage as compared to antibiotics, which have broader spectrum activity.

The probability that bacteria mutate and become resistant to individual phages is a real concern when considering phages for use as biological control agents. Bacteria have evolved several adaptive mechanisms protecting themselves from viral infection. The mechanisms of bacterial resistance to phages include: blocking phage adsorption, inhibiting the phage genome injection to bacterial cells, degradation of phage DNA by Restriction-Modification defense system, blocking phage replication, transcription, translation or virions assembly by Abortive Infection system (Abi), etc. Unlike antibiotic resistance, anti-phage resistance problem is relatively easy to overcome. It could be realized by isolation of novel active phages from environmental sources or progressive adaptation of the viruses to resistant host population. Phages evolve simultaneously with bacteria following changes among resistant clones of bacteria. Application of cocktail of phages prevents possible resistance development in common phage therapies. Similarly to antibiotics, the composition of several phages against one type of bacterial pathogen may increase in activity spectrum and may have synergistic effect as in case of antibiotics in the treatment.

In spite of this, phage application as a part of biological control strategy is based on its lytic activity, prescreening with different criteria for selection of a proper phage has been considered. For example, to fight Xanthomonas campestris pv pruni, that cause bacterial spot of plum and peach, a lytic phage with the broadest host range was selected from available eight phages. In case of X.citri it was able to interact with host bacterium and multiply on it. A unique strategy for controlling fire blight of pear, by selecting phages that lysed the target organism, Erwinia amylovora and its antagonistic phyllosphere bacterium, Pantoea agglomerans was employed.

A method to reduce the likelihood of phage-resistant bacterial mutants emerging in a cropping system was developed by Jackson. He used a mixture of different wild-type phages, including host range (h-) mutant bacteriophages. The h-mutant phages, capable of attacking an extended range of bacterial

Great genetic diversity of bacterial strains causing a bacterial disease of a certain plant is a major factor for identifying suitable phages for biocontrol. For example, a devastating plant pathogen species causing bacterial spot of tomato and peper Xanthomonas campestris pv vesicatoria has now been separated

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into four different ones. Moreover, phage specificity may vary to different bacterial strains within a species. Twenty six bacteriophages were used to type approximately 100 X. vesicatoria strains isolated from various countries in the Caribbean including Central America, and identified at least 26 different phage lysis patterns. For effective biocontrol the amount of phage is considered to be of certain titer of target bacteria. It is important for persistence of phages in the plant phyllosphere and rhizosphere. Several environmental factors were analyzed as potential inhibitors of the disease control by phage in phylosphere and rizosphere. Balogh et al., observed that phage mixtures applied at 106 or 108 PFU/ml concentration provided similar levels of control of bacterial spot to tomatoes inoculated with108 cfu/ml of Xantomonas perforans, but at 104 PFU/ml was ineffective. One critical factor relating to the phyllosphere that should be taken into consideration is that the phage comes in contact with its host. Both laboratory and field studies have demonstrated that several factors as high temperatures, high and low pH and sunlight, rain are critical and determinant of the short-lived persistence of phages on plant leaf surfaces. These factors in addition to UV-A and UV-B spectra of sunlight the most destructive factors to viruses incommon, are the major limiting factors for phage therapy in the phyllosphere. Bacteriophage ability to persist in the phyllosphere is also limited by exposure to copper bactericides. In field studies copper caused significant phage reduction if applied on the day of phage application, but not if applied 4-7 days in advance. Ambient temperature had a pronounced effect on nonformulated phage but not on formulated phages. Desiccation caused a significant but only slight reduction in phage populations after 60 days, whereas fluorescent light eliminated phages within 2 weeks. The protective formulation developed succeeded in elimination of the reduction caused by both factors. Phage populations 109 PFU/g applied on tomato leaf surfaces during the early afternoon hours when UV radiation was highest after 6 hours INDUSTURIAL ENGINEERING

was not detectable. But, phages applied later, close to sunset resulted in improved persistence of the viruses on the treated leaf surfaces. Among the factors identified that can hinder success of disease control in the rhizosphere are: low rate of diffusion through the heterogenous soil matrix; phages can become trapped in biofilms and reversibly adsorb on soil particles, such as clay; physical refuges can protect bacteria from coming into contact with phages; low soil pH can inactivate phage. Protective formulations were developed to increase longevity of phages on plant surfaces in the field. It was experimentally demonstrated that phage applied to soil surrounding tomato plants were detected at reasonable level in the stem and upper leaves after two days, indicating on translocation of absorbed from rhizosphere phage to aboveground parts. The persistence of phage population was better in plants with undamaged, as compared to with damaged roots. Phage was shown to persist in rhizosphere with varying level, and decline between ten and a hundred-fold over a 14-d period.

3.5 Strategies for Optimization in Using Phages for Disease Control Strategies considered for optimization of bacteriophage applications as biological control agents include: improvement of phage efficacy, and minimization of the occurrence of phage-resistant mutants. Several considerations exist to improve phage efficacy: population density and accessibility of the target bacterium; timing of phage application; the ability of the phage to infect and replicate in the target environment; phage density and persistence in phyllosphere or rhizosphere; rates of virion degradation and the presence of adequate moisture to promote phage diffusion. For foliar pathogens the harshness of the leaf surface environment greatly limits bacteriophage survival. Studies have shown that the timing of bacteriophage applications is essential to extend the CHAPTER 8

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persistence of high bacteriophage populations in close proximity to the target bacterium to encourage biological control. It was observed that once bacteria reached the intercellular spaces, they were inaccessible to phage. A significant reduction of fire blight on apple blossoms was achieved when the phage mixture was applied at the same time as the pathogen, Erwinia amylovora. In contrast, disease reduction was not significant when phages were applied a day before inoculation. The effect of timing on the efficacy of phage treatment in greenhouse trials with two pathosystems: black rot of cabbage, caused by Xanthomonas campestris pv campestris and bacterial spot of pepper, caused by X. campestris pv vesicatoria was very important. The greatest disease reduction occurred when phages were applied at the day of inoculation in both pathosytems. The time of day when phages are applied also may affect efficacy. With sunlight irradiation being the single most detrimental factor reducing phage persistence in the tomato canopy application of phages when they are not exposed immediately to direct sunlight prolonged their residual activity. More effective control of tomato bacterial spot was achieved when applying bacteriophages in the early evening, immediately preceding sunset, in comparison with morning applications. Another factor that one might consider when choosing when to apply phages, (and what application volume to use), is the availability of free moisture on the leaves, which is important for interaction of phages with their target. The following approaches are proposed for the increase of phage persistence on plant: development of formulations, existence of host population on leaf surface to promote phage multiplication. In addition to environmental factors, the abundance of host bacteria causes fluctuations in phage numbers. On leaf surfaces, where high host populations persist, phages persist at significantly higher levels than on surfaces without the host. For this purpose avirulent strain to which phage is active could be used. 134

Strategy for minimization of the occurrence of phage-resistant mutants is very important for optimization in using phages for disease control. In field trials, tomato bacterial spot control with the mixture of four phages including wild-type and h-mutant phages when applied twice weekly to plants provided significantly better disease control and produced greater yield of extra large tomato fruits than the standard copper-mancozeb treatment. As it was already mentioned bacterial strains may vary within a species in their sensitivity to bacteriophage. So, phage selection for field use requires careful monitoring of bacterial strains in the field for their natural resistance to deployed phages. To reduce the evolution of resistant bacteria, further developments are needed. They may include application of phages in combination with other antimicrobials, such as antibiotics, cycling through different phage mixtures, and engineering phages to directly target phage-resistance mechanisms. Selection of a suitable phage is a critical factor in ensuring success of phage therapy in agriculture. For this, in vitro assays only are not adequate predictors of biological control ability. For example, plaque size, antibacterial activity or phage multiplication rate for eight Xanthomonas perforans phages, was not correlated with disease control efficacy. Therefore, actual plant bioassays are unavoidable in order to gauge biocontrol activity. Persistence on leaf surfaces is a major limiting factor in using phage therapy for disease control in the phyllosphere. Several strategies have been evaluated for increasing phage persistence, including the use of protective formulations, application scheduling for sunlight avoidance and co-application of bacterial hosts for in vivo phage propagation. In several studies solar protective compounds were identified that increased biocontrol efficacy not only for bacteriophages, but also for entomopathogenic viruses and biopesticides of protein origin. Balogh identified compounds (skim milk alone or in combination with sucrose) that, when mixed with phage, extended the persistence of phage on the phyllosphere. Such protective formulation enhanced the efficacy of phage treatment on tomato foliage.



3.6 Phages as Part of Integrated Disease Management Strategies None of the practices, including the use of pathogen-free seed or planting material, the deployment of genetic host resistance, and the appropriate cultural and sanitation practices as was discussed above, are enough for the control of bacterial diseases. There are only a few chemicals, bactericides with limited efficacy suitable for crop protection. Therefore, there is a need to identify complimentary strategies to enhance the control of plant pathogenic bacteria. Some of the integrated management strategies were already discussed above, including co-application of an avirulent strain of the pathogen R. solanacearum, with a phage that was active against both virulent and avirulent strains. Analysis of efficacy of various combinations of unformulated phages, biocontrol agents, including strains of plant growth promoting bacteria, bacterial antagonists, SAR inducers (harpin, ASM) and copper hydroxide in greenhouse experiments suggest advantage of integrated disease management. Phage treatment in combination with ASM or with copper-mancozeb for the control of Xanthomonas leaf blight of onion resulted enhanced disease control in both cases. However, no improvement in the control of citrus canker or citrus bacterial spot was observed with the combination of bacteriophages with copper-mancozeb. One of the best studied phage control of bacterial disease is tomato bacterial spot, caused by Xanthomonas campestris pv. vesicatoria. Mixture of four phages active against two predominant races of the pathogen applied by Flaherty et al., effectively controlled the disease in greenhouse and field experiments. Balogh et al., applied the phage with protective formulation and thus increased phage persistence on tomato leaves. Obradovich et al., formulated phages combined with other biological control agents and plant resistance inducers, as a part of integrated disease management approach. Phages can be used effectively as part of integrated disease management strategies. With the aim to INDUSTURIAL ENGINEERING

develop a comprehensive phage-based integrated management strategy for bacterial spot disease control in commercial tomato fields, several combinations of treatments that effectively controlled tomato bacterial spot in the greenhouse were tested in field trials performed in north and central Florida during three consecutive seasons. Although copper-sensitive strains were used, the application of formulated phages twice a week was either more effective or equally as effective as the standard copper-mancozeb treatment. Phage-treated plants produced significantly more marketable fruit than plants not receiving phage. However, integration of phage treatments and ASM provided an additional reduction in disease pressure and resulted in more efficient foliar disease control than ASM, phage, or copper-mancozeb alone. Phage-based integrated management of tomato bacterial spot is now officially recommended to growers of tomato in Florida and relevant bacteriophage mixtures (Agriphage from OmniLytics Inc., Salt Lake City, UT EPA Registration #67986-1) are commercially available. Agriphage represents a mixture of active bacteriophages specific to Xanthomonas campristrispv. vesicatoria and Pseudomonas syringe, was registered first times in 2005 (www.omnilytics.com). The use of phages for disease control is a fast expanding area of plant protection with great potential to replace the chemical control measures now prevalent. The relative ease of preparing phage treatments and low cost of production of these agents make them good candidates for widespread use globally. However, the efficacy of phages, as is true of many biological control agents, depends greatly on prevailing environmental factors as well as on susceptibility of the target organism. Great care is necessary during development, production and application of phage treatments. In addition, constant monitoring for the emergence of resistant bacterial strains is essential. Phage-based disease control management is a dynamic process with a need for continuous adjustment of the phage preparation in order to effectively fight potentially adapting pathogenic bacteria. In addition to being used to kill bacteria for bicontrol of plant diseases, or as agents for processed foods, bacteriophages have numerous applications. CHAPTER 8

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3.7 Other Application of Bacteriophages The intensive research on phage biology has led to increasing potential phage application in different aspects of human activity. These are: phage therapy proteins; phage typing; bacteria detection; disinfection of medical tools and devices; food decontamination, and drug delivery. FDA recently approved Microphage, Inc. blood culture test that uses phage infection for detection of methicillin-resistant Streptomyces aureus. Phage typing methods are used for expression of luciferase genes delivered by modified phages, fluorescently labelled phages, and traditional plaque formation. Besides the diagnostics these products can help the development of phage therapy, which needs the rapid and accurate identification of bacterial targets and their susceptibility to specific phages, which is crucial for the effective therapy. Phage-based products were successfully developed by several companies being approved by EPA, USDA, and FDA. Such products have established a favorable regulatory precedent in which individual components of phage cocktails can be tailored towards bacterial targets. Products targeted at Listeria monocytogenes represent one of the first examples of phage cocktails to obtain Generally Recognized as Safe (GRAS) status from the FDA. These products are designed to be used as sterilizing agents for processed foods (ListShieldTM and LISTEXTM P100). Further products are in development against other bacterial pathogens, including Escherichia coli strains (O157:H7) and Salmonella enterica. Bacteriophages have greatly contributed to the development of molecular biology. Due to their simple structure, small numbers of genes, phages are reliable experimental systems for genetic engineering and exploring molecular biological processes. Not only bacteriophages, but phage-derived products remain useful research tools in molecular biology. For example, T4 DNA ligase, l phage-derived cloning vectors, M13 derived phagemits are widely used for the preparation of single stranded templates and nucleic acid probes. 136

Bacteriophages are used in antibody library technology, a useful tool for displaying antibodies of interest on the surface of bacteriophages, to obtain the corresponding antigen specific antibodies.

3.8 Engineered Bacteriophages Increasing threat posed by antibiotic resistant bacteria, bacterial viruses or bacteriophages have regained attention of biomedical scientists. The engineering of bacteriophages opens new era in many directions of phage therapy, including as adjuvants for human antibiotic therapy. A bacteriophage was engineered to overexpress proteins and attack gene networks that are not directly targeted by antibiotics. The engineered bacteriophage can enhance the killing of antibiotic-resistant bacteria, persister cells, and biofilm cells, reduce the number of antibiotic-resistant bacteria that arise from an antibiotic-treated population, and act as a strong adjuvant for other bactericidal antibiotics (e.g. aminoglycosides and β-lactams. Using novel synthetic biology technologies, presented a methodology to engineer enzymatic active phages that are both capable of killing the bacteria in species-specific manner by lysis and dispersing the exopolysaccharide (EPS) matrix because they have been also engineered to express the most effective EPS-degrading enzymes. Genetic conjugation methodology has been developed to improve the design of engineered phage and allow further exploitation of these particles as functional nanobiomaterials for various applications.

3.9 Bacteriophages as Potential Bioterrorism Agents and AntiTerrorism Tools. bacteriophages could be used as bioterrorism agents and tools, as well as counter-measures against bioterrorism. For example, lysogenic bacteriophage often carrying virulence genes can alter bacterial virulence. The cause of E.coli outbreak in Germany



in 2010 was EHEC, which had become pathogenic by obtaining a Shiga toxin-containing lysogenic bacteriophage. Thus, lysogenic bacteriophage can integrate deadly virulence genes into a chromosome of host harmless bacteria and turn them into a deadly killer. Consequently, lysogenic bacteriophages containing virulence genes and/or broadspectrum drug-resistance genes could be used as tools to turn non-pathogenic bacteria into pandrug resistant bacteria which are resistant to all known antimicrobial agents. Such lysogenic phages could be directly used as biological weapons that could be dispersed in an appropriate environment to generate highly pathogenic drug-resistant bacteria killers. The bacterial viruses could be used as effective Anti-Terrorism Tools. One of the most effective solutions to pandrug-resistant bacteria may simply be bacteriophage. Since bacterial viruses are the largest biological group in nature no matter how drugresistant or how virulent a bacterium is, it should be possible to find a bacteriophage that can kill it.

Review Questions and Answers Q1. How to detect the microbial strains potential to synthesize enzyme (any)? A1. The producer should be grown on a previously selected nutrient media (from literature) containing the substrate or its similar compound as a constituent part of nutrient media. Q2. How to detect whether the enzyme is accumulated as intracellular microbial metabolite or penetrates through cell walls and belongs to extracellular enzymes? A2. The strain should be grown on selected nutrient media and activities of searchable enzymes should be determined in both: biomass extract received in corresponding buffer and cultural filtrate. If the main part of activity is found in cultural filtrate (approximately 40-80%) than it could suggest that enzyme is extracellular. Q3. How to determine the productivity of strain enzyme producer?


A3. Usually activities of enzymes are expressed in units of cultural filtrate or biomass extract solution most often in ml. That being multiplied on the whole amount of solution we are getting the total activity of the strain. To detect the productivity of any particular strain the activity could be expressed: being related to the whole amount of biomass; activities could be expressed per mg of DNA; or per mg of intracellular protein. Q4. What would be the sources for the extramophiles isolation? A4. Extremophiles growing under extreme of conditions could be isolated together with mezophiles characterized by optimal growth in normal conditions from regular sources. But the most desirable sources for isolation of extremophiles are unusual places such as: alkali and acidic lakes and soils, hot water sources, gazers, hot and cold places, poor or very poor soils, salty and polluted soils, lakes, rivers, etc. Q5. What would be the methodology of extremophilic enzymes detection? A5. The optimal conditions for the detection of searchable enzyme activity should be determined, which might be differing from regular one. The conditions of the activity measurement and further search could be directed for precise analysis of buffer system, pH, temperature, salts concentration, etc. Q6. What advantages would have the use of enzymes from extremophiles? A6. The enzymes from extremophiles in some critical conditions most often expose increased stability especially in prolonged enzymatic reactions such as wooden biopolymers deconstruction. They are also more resistant against inhibition by reaction products and intermediates occurring during enzymatic processes. Due to these feathers’ they represent a unique agents for the creation of novel more effective biotechnologies. Q7. What are the criteria for the selection of plants as phytoremediators? A7. Plants to be selected for the remediation of areas polluted by organic toxicants should posses the CHAPTER 8

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activities of the following oxidative enzymes: monooxigenase system, peroxidase, phenoloxidase, to perform initial oxidation of organic contaminants. Q8. What kinds of deviations are created by organic contaminants after their penetration in plant cells? A8. Architectonic structure of plant cell undergoes deviations as a result of contaminants penetration. Such deviations could be metabolic which would be avoided by plants internal potential or in case of contaminants high concentration they could be lethal leading to plants death. Q9. What is the strategy of the polluted sites remediation? A9. First of all the detailed characterization of polluted sites should be carried out, and then based on received information the strategy of clean up should be created based on the type of contaminant(s), concentration of contaminant(s); selection of appropriate plants and consortia of microorganisms; corresponding irrigation system and, finally, capital cost of the whole biotechnology. Q10. What are the approaches for plant disease control? A10. Cultural practice and chemicals are used to prevent, mitigate or control plant diseases. The application of disease- and pathogen-free transplants, repeated digging, burning of plant wastes, three years or longer crop rotation, prevention of surface wounds on plants are in arsenal of cultural practice. Antibiotics and copper-based compounds are chemical pesticides, bactericides applied in agriculture for disease management. Q11.What chemicals are used as bactericides to fight plant bacterial diseases? A11. Antibiotics and copper-based compounds are used to fight plant bacterial diseases. Q12. What are adverse effects of using antibiotics and copper-based compounds in agriculture? A12. Extensive application of copper compounds and antibiotics resulted in copper and antibiotic 138

resistances in many plant pathogenic bacteria. Besides, extensive use of the bactericides may cause phytotoxicity and soil contamination, especially when applied at high concentrations to overcome the resistance and unfavorable weather conditions. Q13. What are the stages of the lytic cycle of bacteriophage. A13. The stages of the lytic cycle include: Adsorption - highly specific phage attachment to a specific receptor on the surface of host bacterial cell; Infection of the DNA - transfer of phage nucleic acid into the bacterial cell through pores that are formed in the host cell wall as a result of phage lysozyme activity; Direction of bacterial metabolism to phage DNA replication and encoded phage protein synthesis; Assembly and packing of phage particles; Bacterial cell lysis and phage progeny release. Q14. Main advantages of bacteriophage application as biological control agents. A14. Phages are natural components of the biosphere; they can readily be isolated from wherever bacteria are present. No adverse effects to humans, wildlife or the environment are expected from phages. Due to their high specificity it is assumed that they are non-toxic to the eukaryotic cells. The main advantages are that they are specific to the disease-causing bacterium, and are eliminating only target bacteria without damaging other, possibly beneficial members of the indigenous flora. Q15. What are the main challenges in using phages for disease control? A15. Resistance of phytopathogens to phages, availability of suitable phage, strain variation, persistence of phages in the phyllosphere and rhizosphere are main challenges in phage therapy. Q16. What are the strategies for optimization in using phages for disease control? A16. Strategies considered for optimization in using bacterial viruses as biological control agents include: improvement of phage efficacy, and minimization of the occurrence of phage-resistant mutants. Several considerations exist to improve phage



efficacy: population density and accessibility of the target bacterium; timing of phage application, the ability of the phage to infect and replicate in the target environment; phage density and persistence in phyllosphere or rhizosphere; rates of virion degradation and the presence of adequate moisture to promote phage diffusion. Minimization of the occurrence of phage-resistant bacteria pathogens could be achieved by application of mixture of phages including wild-type and h-mutant ones.

5. Govardhan CP, Margolin AL. Extremozymes for industry: by nature and design. Chemistry Industry 1995;17:689–92.

Q17. What are the advantages of integrated disease management strategy?

8. Tsao DT. Phytoremediation. Advances in biochemical engineering and biotechnology. Springer 2003.

A17. Analysis of efficacy of application of various combinations of unformulated phages, biocontrol agents, including strains of plant growth promoting bacteria, bacterial antagonists, SAR inducers (harpin, ASM) and copper hydroxide in greenhouse experiments suggest advantage of integrated disease management expressed in enhanced efficacy of disease control. Q18. What are the prospects of engineered bacteriophages? A18. The engineering of bacteriophages opens new era in phage therapy. The engineered bacteriophage can enhance the killing of antibiotic-resistant bacteria, persister cells, and biofilm cells, reduce the number of antibiotic-resistant bacteria.

Further Readings 1. Demain A, Davies J. Industrial Microbiology and Biotechnology American Society for Microbiology. Edited by Demain A, Davies J. 1999;830. 2. Kvesitadze, GI, Bezborodov AM. Introduction in Biotechnology. Journal of Analytical Chemistry 2002;284. 3. Hough DW, Danson MJ. Extremozymes. Elsevier Science 1999;3:39–46. 4. Niehausá F, Bertoldoá MC, KaÈhlerá G. Antranikian. Extremophiles as a source of novel enzymes for industrial application. Applied Microbiology and Biotechnology - Springer 1999;51:711-729.


6. Van den Burg B, Eijsink VGH Selection of mutations for increased protein stability. Current Opinion in Biotechnology 2002;13:333-337. 7. Horikoshi K: Alkaliphiles: some applications of their products for biotechnology. Microbiology and Molecular Biology Reviews 1999;63:735750.

9. Kvesitadze G, Khatisashvili G, Sadunishvili T, Evstigneeva ZG. Metabolism of anthropogenic toxicants in higher plants. Journal of Analytical Chemistry 2005;199. 10. Kvesitadze G, Khatisashvili G, Sadunishvili T, Ramsden JJ. Mechanisms of detoxification: the basis of phytoremediation. Springer 2006;262. 11. Sandermann H. Higher plant metabolism of xenobiotics: the “green liver” concept. Pharmacogenetics 1994;4:225–241. 12. Schuler MA. Plant Cytochrome P450 monooxygenases. Critical Reviews in Plant Sciences1996;15:235-284. 13. Morant M, Bak S, Moller BL, Werck-Reichhart D. Plant Cytochromes P450: tools for pharmacology, plant protection and phytoremediation. Current Opinion in Biotechnology 2003;2:151–162 14. Niku-Paavola ML, Viikari L. Enzymatic oxidation of alkenes. Journal of Molecular Catalysis 2000;10:435-444. 15. Jones JB, Jackson LE, Balogh B, Obradovic A, Iriarte FB, Momol MT. Bacteriophages for Plant Disease Control. Annual Review of Phytopathology 2007;45:245–262. 16. Jones BJ, Vallad GE, Iriate FB, Obradovich A, Wernsing MH, Jackson LE, Balogh B, Hong JG, Momol T. Bacteriophage 2012;2:208–214. 17. Balogh B, Jones JB, Momol MT, Olson SM. Persistence of bacteriophages as biocontrol

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agents in the tomato canopy. Proc Int Symp Tomato Dis, 1st, Orlando, FL.ISHS Acta Hortic 2005:695:299-101. 18. Sadunishvili TA, Giorgobiani NSh, Amashukeli NV, et al,. Strategy of biological control of phytopathogenic bacteria in Georgia. Annals of Agrarian Science 2012;10:62-66. 19. Kutter E, Sulakvelidze. Bacteriophages: Biology and Applications. Boca Raton, London, New York, Washington: CRC Press 2005;500.

21. Flaherty JE, Jones JB, Harbaugh BK, Somodi GC, Jackson LE. Control of bacterial spot on tomato in the greenhouse and field with H-mutant bacteriophages. HortScience 2000;35:882–4. 22. Momol MT, Jones JB, Olson SM, Obradovic A, Balogh B, King P. Integrated management of bacterial spot on tomato in Florida. Institute of Food and Agricultural Sciences - University of Florida 2002.

20. Gill JJ, Abedon ST. Bacteriophage ecology and plants. http://www.apsnet.org/publications/apsnetfeatures/Pages/BacteriophageEcology.aspx

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BIOREFINERY AND BIOENGERGY APPLICATIONS

CHAPTER 9 BIOREFINERY AND BIOENERGY APPLICATIONS Haiyan Yang, Kun Wang, Run-Cang Sun


1. Structure and Recalcitrance of Biomass ........................................................................ 144

2. Biorefinery Technologies .............................................................................................. 146

2.1 Biochemical Processes ................................................................................................. 146

2.1.1 Pre-treatment Under Alkaline Conditions .......................................................... 2.1.2 Acid-Catalyzed Pre-treatment And Steam Pre-treatment ..................................... 2.1.3 Ionic Liquid Pre-treatment ................................................................................. 2.1.4 Biological Pre-treatment ..................................................................................... 2.1.5 Alternative Pre-treatments ..................................................................................

146 147 149 149 149

2.2 Thermal Chemical Processes ........................................................................................ 150

2.2.1 Pyrolysis .............................................................................................................. 150 2.2.2 Liquefaction ........................................................................................................ 151 2.2.3 Gasification ......................................................................................................... 151 2.2.4 Combustion ........................................................................................................ 152

3. Conclusions .................................................................................................................. 152

Acknowledgements ................................................................................................................ 152 Summary Box ........................................................................................................................ 152 Review Questions and Answers .............................................................................................. 153 References .............................................................................................................................. 153


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Summary

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ossil oil is the primary source for energy and chemicals in the world, but the availability of conventional oil is becoming geographically restricted. Concerns about global warming, soaring cost of gasoline and national security issues, have rekindled interest in producing liquid fuels from renewable resources. Bioenergy, derived from biomass, is one of the most important alternatives to mitigate greenhouse gas emissions and substitute for fossil fuels (Figure 1). Biomass is a biological matter, which is considered as the only renewable supply of carbon for liquid fuels and chemicals with an estimated annual production of 1011 to 1012 tons. In recent, biomass contributes to 14% of the world’s primary energy demand. Furthermore, the International Energy Agency (IEA) data indicates that the electricity generation from

solid biomass in the European Union (EU) had been growing at an average rate of 2.5% per year over the last decade. Generally, the sources for biofuels production include energy crops, agricultural and woody residues, organic waste and vegetable oils. Among these materials, only willow and miscanthus are suggested as perennial energy crops combining high yields with low inputs. As compared to the cost of producing a tone of specially cultivated energy crops, the use of agricultural and woody residues may result in a lower overall cost in the biofuels process. The annual yield of agricultural residues ranges from 1-10 Mg DM ha-1, and about 15% of the residues are available for energy generation to date. Woody residues are from wood industry (such as sawdust and wood chips) and forest. In 2005, 1.4 billion m3 of wood obtained from the forest were used for

Figure 1. Sustainable technology in an integrated biorefinery (adapted from Stöcker, 2008).


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fuel production. Trees provide potentially higher calorific values for biofuels production than agricultural residues due to their high lignocellulosic energy conversion factor of 16 (1-1.5 for corn and 8-10 for sugarcane), as well as high growth ability in marginal agricultural land, reducing competition for space with food crops. Although only a small proportion of liquid biofuels are forest-based today, the development of an economically viable process for producing cellulosic liquid biofuels could lead to the widespread use of forest biomass in the biofuel processing. Organic wastes from paper industry, animal fats and byproducts, recycled cooking oil, municipal solid waste (MSW) and other sources are also considered as sources for energy production. Life Cycle Assessment (LCA) suggested that only biofuels from waste resources combines favorable competitiveness with sustainable. In EU, around 80% MSW are biodegradable and can be considered as an alternative sustainable source of biofuels. In Northern Ireland, the potato peelings from chip plants have been identified as a potential fuel source. Scotland is one of the first in UK to use waste cooking oil as feedstock for biodiesel. Recently, castor bean is being introduced as a biodiesel crops in Brazil, and various perennial shrubs shearing seeds with high oil content are being promoted in India. However, the utilization of biomass is still ineffectively. Share of biomass energy to meet world energy demand is expected to rise in future by boosting technological development for biomass utilization. Normally, bioenergy is converted from biomass via two main types of process: bio-chemical/biological and thermo-chemical processes. The bio-chemical platform attacks much attention, which enzymatically hydrolyzes the cellulose and subsequently ferments to ethanol or butanol. The thermo-chemical approach thermolytically transforms biomass into gaseous or liquid intermediate chemicals that can be further upgraded to transportation fuels or chemicals. The objective of this review is to introduce the biorefinery technologies for conversion biomass into energy.

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1. Structure and Recalcitrance of Biomass Lignocellulosic biomass comprises three main types of carbon-based polymer: cellulose, hemicelluloses and lignin (Figure 2). Plant cell wall hold onto these polymers with a tight grip, providing the complex structural and chemical mechanisms of biomass for resisting the microbial and chemical assaults. The factors contributing to the recalcitrance of biomass include cellulose crystalline structure, limited accessible surface area, protection of cellulose by lignin and hemicelluloses etc. Cellulose is a high molecular weight linear homopolysaccharide composed of D-glucopyranose units linked by β-1,4-glycosidic bonds, with the polymerization degree (DP) of 500 to 15,000. The basic building unit of the cellulose skeleton is an elementary fibril, which is formed from insoluble microfibrils. These microfibrils are considered to be a bundle of 36 parallel cellodextrin chains held together by intermolecular hydrogen bonds. In addition, the hydroxyl groups in cellulose macromolecules result in various properties of cellulose, such as crystalline patterns, stiffnesses and solubilities in solvent. In nature, cellulose is supposed to be a combination of two crystalline forms: cellulose Iα and cellulose Iβ, together with amorphous cellulose in cell wall. Hemicelluloses, comprising the non-cellulose cell wall polysaccharides, represent an immense renewable resource of biopolymers. The term of hemicelluloses was originally proposed by Schulze to designate polysaccharides extractable from plants by aqueous alkaline solution, in comparison to cellulose. Commonly, hemicelluloses refer to non-starch polysaccharides except pectin in association with cellulose in the cell wall of higher plants. Structurally, hemicelluloses provide the matrix in which the crystalline cellulose elementary fibrils are embedded. Yan et al. have observed that some hemicelluloses chains line perpendicularly to the direction of cellulose microfibrils with diameters of about 5-10 nm, linking the cellulose microfibrils together to form a network structure in the straw cell wall. Moreover, hemicelluloses link to lignin


with α–benzyl ether linkages, and to acetyl units and hydroxycinnamic acids with ester linkages. In plant cell walls, lignin cross-links with carbohydrates and acts as a resin that holds the lignocellulose matrix together, conferring strength and rigidity to the system. Lignin is a three dimensional amorphous polymer consisting of methoxylated phenylpropane. It is generally accepted that there are three basic phenol derivatives that make up almost all types of lignin found in nature: p-coumaryl alcohol, coniferyl and sinapyl alcohol. Each monolignol produces p-hydroxyphenyl, guaiacyl and syringyl residues in the polymer correspondly. The The basic units are linked by the interunit C-C bonds (e.g. β-5, 5-5, β-β) and aryl ether linkages

with aryl-glycerol and β–aryl ether (e. g. β-O-4 and 4-O-5). The monolignols also link with other cellwall polymers to form complex three-dimensional networks. The various linkages between monolignols give lignin a great diversity. In addition, the composition of lignin varies with plant sources and the fine structure of it remains unknown. basic units are linked by the interunit C-C bonds (e.g. β-5, 5-5, β-β) and aryl ether linkages with arylglycerol and β–aryl ether (e. g. β-O-4 and 4-O5). The monolignols also link with other cell-wall polymers to form complex three-dimensional networks. The various linkages between monolignols give lignin a great diversity. In addition, the composition of lignin varies with plant sources and the fine structure of it remains unknown.

Figure 2. Structure of lignocellulose (adapted from Rubin, 2008).


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2. Biorefinery Technologies 2.1 Biochemical Processes First generation bioethanol refers to biofuels manufactured from edible feedstocks, including sugar, starch and oil crops, by conventional technologies. However, there are still some concerns related to the fact that the extensive use of food crops for the first generation biofuels production can introduce deforestation, global warming and threats to biodiversity. Therefore, the production of second generation biofuels from non-edible biomass has interesting possibilities. Second generation biofuels can supply a larger proportion of fuel with greater environmental benefits than the first generation biofuels. However, the natural resistances of plant cell wall to microbial and enzymatic deconstruction make the second generation biofuels not available on a fully commercial scale. Pre-treatment of lignocellulosic biomass is a crucial prerequisite for breaking the intact structure of materials and increasing the amenability of cellulose to enzymatic attack in the cellulose-to-ethanol processes (Figure 3). Currently, a number of pre-treatments involv-

ing biological, chemical, physical and thermal approaches have been investigated with the goal of reducing costs and accelerating commercial application of biofuels.

2.1.1 Pre-treatment Under Alkaline Conditions Pre-treatments accomplished under alkaline condition increase the digestibility of the biomass by altering the lignin composition, and thus the lignin content of material closely relates to the efficiency of alkaline pre-treatment. During the alkaline pretreatment, the disruption of lignin varies with pretreatment temperature. At media condition (below 140 °C), ester linkages joining the phenolic acids can be cleaved to form a carboxylic salt and an alcohol. Comparatively, the cleavage of ether bonds linking the lignols occurs at a much higher temperature. Although the disruption of lignin structure makes the carbohydrates more accessible, the released monomeric lignin compounds were considered as a group of potential inhibitors for enzymes and microorganisms. Generally, the suitable agents for alkaline pre-treatment are found to be sodium, potassium, calcium and ammonium hydroxides.

Figure 3. The role of pre-treatment in the conversion of biomass to fuels (adapted from Hsu et al., 1980).

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These agents can cause swelling of biomass, which increases the internal surface area of the biomass, and decreases both the degree of polymerization and crystallinity of cellulose. Of these four, calcium hydroxide (lime) is the lowest cost agent. Lime pretreatment is operated for hours to weeks at room temperature, and the pre-treatment time can be shorted by increasing the temperature to reduce the reaction time. The efficiency of lime-treatment is affected by the structural features of substrate, such as acetylation, lignification and crystallization. Removal of acetyl groups from hemicelluloses can reduce the steric hindrance of hydrolytic enzymes and enhance the carbohydrate digestibility. Removal of lignin increases enzyme effectiveness by eliminating nonproductive adsorption sites and by increasing access to cellulose and hemicelluloses. Delignification efficiency of pre-treatment is highly dependent on temperature, the presence of oxygen, and the type of biomass. Thus, lime pre-treatment is less effective on woody biomass than herbaceous plants or agricultural residues in the same condition due to the higher lignin content of wood. Ammonia pre-treatment has been used to improve cellulose digestion and remove lignin with the recyclability of ammonia. Ammonia fibre expansion (AFEX) pre-treatment uses lignocellulosic biomass with liquid ammonia under high pressure and then rapidly releases pressure. Although the temperature for AFEX pre-treatment is much lower than steam explosion pre-treatment, it is still an important variable in the AFEX process. At higher temperature, more ammonia vapours flash and therefore, greater disruption of the biomass fiber structure probably occurs. After AFEX pre-treatment, cellulose is decrystallized, hemicelluloses are degraded to oligomers, and the structure of lignin is altered. The small amount of residual ammonia in the solid residue is served as the nitrogen source for microorganisms in the subsequent fermentation process. In order to achieve a considerable substrate digestibility, various AFEX parameters (temperature, ammonia loading, moisture content of biomass, and residence time) have been optimized for different types of substrates. The optimal pre-treatment con-

ditions for corn stover were found to be temperature 90 °C, ammonia/dry corn stover mass ratio 1:1, moisture content 60%, and incubation time 5 min. Higher temperature (~ 100 °C) and moisture content (80%) was needed to pretreat switchgrass, than for corn stover. The effective pre-treatment for miscanthus grass is conducted at 160 °C with 2:1 (w/w) ammonia-to-biomass ratio and 230% biomass moisture content for 5 min. Generally, greater than 90% conversion of cellulose and hemicelluloses to fermentable sugars can be achieved for a variety of herbaceous and agricultural residues after AFEX pre-treatment. Comparing with grass and agricultural residues, AFEX pre-treatment of hardwood requires much harsher condition to obtain equivalent sugar yield upon enzymatic hydrolysis. However, AFEX is not a very efficient technology for lignocellulosic biomass with relatively high lignin content. For example, hydrolysis of AFEXpretreated aspen chips was reported as below 50%. Another type of process utilizing ammonia is the ammonia recycle percolation (ARP) method. Ammonia in aqueous solution at high temperature breaks down lignin via ammoniolysis reaction but has virtually no effect on carbohydrates. Kim and Lee have reported that the ARP process can remove up to 85% of lignin from corn stover. The major challenge for ARP is to reduce liquid loading to keep energy costs low. Kim et al. have reported a low liquid ARP pre-treatment process, which achieved 86% digestibility of biomass via enzymatic hydrolysis at enzyme loading of 7.5 FPU/g glucan. However, the ARP is also ineffective in the pre-treatment of high lignin containing lignocellulosic biomass. In addition, the environmental concerns with the stench of ammonia is a negative factor for pilot, as well as industrial scale applications.

2.1.2 Acid-Catalyzed Pre-treatment and Steam Pre-treatment Dilute acid pre-treatment has been successfully developed for pre-treatment of lignocellulosic materials. In general, acid pre-treatment is conducted with mineral acids, such as H2SO4 or HCl, at temperature of < 200 °C. With acid pre-treatment strategies,


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cellulose and hemicelluloses are degraded via hydrolysis, releasing polysaccharides as fermentable sugars. The removal of hemicelluloses in turn facilitates the accessibility of cellulose to enzymes. Although little amount of lignin is dissolved, various studies indicate that the lignin macromolecule is structurally disrupted, increasing cellulose susceptibility to enzymes. The high reaction rate and significant improvement of carbohydrate digestibility make acid pre-treatment a potential process for large scale application. As much as 80.3% glucose can be released by enzymatic hydrolysis from the photoperiod sensitive sorghum after the pre-treatment with 1.0 % H2SO4 at 160 °C for 40 min. Huang et al. obtained 87% of the theoretical ethanol by fermentation of rice straw acid hydrolyzate using an enhanced inhibitor-tolerant strain Pichia stipitis. However, dilute acid pretreatment degrades carbohydroates into mono- and oligomeric sugars, and further into acetic, formic and levulinic acids, furfural, and 5-hydroxymethylfurfural (5-HMF). In addition, a variety of aromatic and aldehydic compounds are released from lignin decomposition. These compounds are toxic to fermenting microorganisms. Luo et al. identified more than 35 potential inhibitors to S. cerevisiae in dilute nitric acid hydrolyzate of hybrid poplar. In order to overcome the inhibitory effect of the degradation products, biological, physical and chemical methods have been employed to detoxify. Although the dilute acid pre-treatment is an efficient approach for enhancing biomass digestion, the major limitation of dilute acid pre-treatment is the corrosion of acid, which mandates expensive materials of construction. At high temperature and pressure, the acetyl group released from the biomass generates acetic acid, acidifying the reaction medium and mediating the hydrolysis of cellulose and hemicelluloses. Thus, steam explosion, conducting at 160-240°C and 0.7-4.8 MPa, is thought to be a kind of acidic pretreatment. After steam explosion, the digestibility of cellulose is improved by the disruption of lignocelluloses structure and depolymerization of lignin. Previous study reported that steam explosion pretreatment and subsequent enzymatic hydrolysis of sunflower stalk can release 16.7 g glucose from 100 148


g raw material. In order to enhance the effectiveness of the pre-treatment, H2SO4, CO2 or SO2 is added as catalyst. Cara et al. comparatively studied the production of ethanol from steam-pretreated olive tree with and without chemical impregnation. Results showed that the maximum ethanol yield (7.2 g ethanol/ 100 g raw material) was obtained from water pre-impregnated and steam pretreated solids. Sassner et al. produced bioethanol from Salix after steam pre-treatment with H2SO4 impregnation. A total of 55.6 g glucose and xylose per 100 g raw material was obtained under the optimal conditions (pretreated at 200 °C for 4 or 8 min after impregnation with 0.5% H2SO4 for 90 min), and an overall theoretical ethanol yield of 79% was achieved by simultaneous saccharification and fermentation (SSF) of the pretreated slurry. Oxaolic acid was also used to impregnate biomass before explosion pre-treatment, which could improve the ethanol production from eel grass at high solid concentration. However, steam explosion is more effective for the pre-treatment of hardwood and agricultural residues than for soft-wood. In addition, the lignin-carbohydrate matrix cannot be completely deconstructed by steam explosion, which may result in the risk of condensation and precipitation of soluble lignin components. Liquid hot water (LHW) is similar to steam explosion but uses water in the liquid state at elevated temperature instead of steam. The optimum temperature for most of lignocellulosic biomass ranges between 160-200°C. In this process, hot water cleaves hemiacetal linkages and liberates acids, which accelerate acid hydrolysis of cellulose and hemicelluloses. These acids further catalyze the hydrolysis of oligosaccharides to monomeric sugars. Thus, monitoring and controlling the pH value between 4 and 7 can minimize hydrolysis of carbohydrates and avoid formation of the monosaccharide degradation products. Although the low temperature, low inhibitor formation and low cost of solvent are advantages for large scale application of LHW, the large volumes of water needed is a disadvantage of LHW.


2.1.3 Ionic Liquid Pre-treatment Ionic liquids (ILs) are generally defined as the melt at or below 100°C, affording liquids exclusively composed of ions. ILs are attractive candidates for a wide range of applications due to their low vapour pressure, non-inflammability, thermal and chemical stability. The use of ILs to biomass refinery recently starts to attract a great deal of attention. The most successful cations for biorefinery are based on the methylimidazolium and methylpyridinium cores with allyl-, ethyl-, or butyl- side chains, and the most promising anions are found to be chloride, acetate and formate. The proposed dissolution mechanism of cellulose in ionic liquids suggests that the oxygen and hydrogen atoms of the cellulose form electron donor-acceptor (EDA) complexes with the charged species of the IL. This interaction results in the separation of hydroxyl groups of different cellulose chains, leading to dissolution of the cellulose in the IL. After dissolution in and regeneration from the IL, more accessible surface area and binding sites of cellulose are exposed, and thus rendering a higher enzyme-adsorption capacity than the untreated one. Previous research suggests that the initial hydrolysis rate and reducing sugar release of microcrystalline cellulose via enzymatic hydrolysis were improved 50-fold and 1.5-fold, respectively, after IL treatment. However, the residual IL in the regenerated cellulose may lead to the inactivation of cellulase and inhibition of microorganisms. The need to develop biologically friendly ILs is crucial. In addition, many challenges such as high cost, deficient toxicological data and limited knowledge about basic physic-chemical characteristics still remain for the successful and widespread implementation of ILs for biorefining.

2.1.4 Biological Pre-treatment Biological treatment with various types of rot fungi is a safe and environmentally friendly method. It is advocated as a process that does not require high energy input for lignin removal from a lignocellulosic biomass. The ability of fungi to degrade lignocellulose is thought to be associated with a mycelia growth habit that allows the fungi to consume the

lignocelluloses as carbon source. In the biological pre-treatment process, brown rots mainly attack cellulose, and white and soft rots attack both cellulose and lignin. Among all the microorganisms, white rot fungi are most effective for pre-treatment of lignocellulosic biomass. Because of the insolubility of lignocellulose, fungal degradation occurs at exocellularly. Two types of cellularly enzymatic systems are responsible for degradation: the hydrolytic system, which produces hydrolases that responsible for polysaccharides degradation; and a unique oxidative and extracellular ligninolytic system, which degrades lignin and opens phenyl rings. The degradation of lignin by whit-rot fungi is an oxidative process and phenol oxidases are the key enzymes. Of these, lignin peroxidases (LiP), manganese peroxidases (MnP) and laccases have been thoroughly studied. LiP and MnP oxidize the substrate by two consecutive one-electron oxidation steps with intermediate cation radical formation. LiP degrades non-phenolic lignin units whereas MnP generates Mn3+ acting as a diffusible oxidizer on phenolic or non-pehnolic lignin via lipid peroxidation reactions. Laccase catalyzes the one-electron oxidation of phenolics and other electron-rich substrate, which has broad substrate specificity. The reaction of the aforementioned enzymes can be regulated by carbon and nitrogen sources. Previous literature suggested that the delignification of corn stover by the white rot fungus Ceriporiopsis subvermispora reached 39.2% after 42 days cultivation, and the digestibility increased from 22% (for untreated samples) to 67%. White rot fungus Phanerochaete chrysosporium can degrade 42% Klason lignin in corn fiber and 30 % total lignin in wheat straw in 3 weeks, respectively. However, the loss in carbohydrate during fungal growth and the long cultivation period reduce the overall productivity of biological pre-treatment.

2.1.5 Alternative Pre-treatments Apart from the aforementioned leading technologies, some new technologies are adopted in lab to pretreat the biomass to improve its digestibility. Ul-


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trasound is a sound wave with frequency above the normal hearing range of humans, and it has been applied widely in various biological and chemical processes. When ultrasound is applied in a medium such as liquid or slurry, it generates a series of compression and rarefaction waves, and subsequently generate powerful hydro-mechanical shear forces in the bulk liquid. The usage of high-power ultrasound in biomass pre-treatment has the potential to disintegrate the particle size of biomass, to improve mass transformation and to expose a much larger surface area to enzymes. Previous studies have demonstrated that high power ultrasound treatment could enhance starch-protein separation, increase the oil extraction from soybeans by 11.2%, and improve the ethanol production from recycled paper via SSF by as much as 20%. In addition, Khanal et al. have reported that ultrasound pre-treatment not only increased the glucose release from corn by three-fold, but also enhanced the activity of enzyme. Microwave radiation pre-treatment is a promising approach that utilizes thermal and non-thermal effects caused by microwaves in aqueous environment. These thermal and nonthermal effects can accelerate chemical, biological and physical processes. Previous studies suggest that microwave heating could disrupt the silicon-containing waxy surface and disrupt the recalcitrant structure of lignocellulose. Keshwani and Cheng predicted that the effect of microwave-assisted alkali pre-treatment on the changes in chemical composition of biomass would boost the process simulation and economic assessments of bioethanol production from lignocellulosic materials. Several studies have reported that microwave-based pre-treatment enhanced the sugar yields from lignocellulosic biomass. Zhu et al. reported a glucose yield of 65% and total carbohydrate conversion of 78% from microwave pretreated rice straw via enzymatic hydrolysis. After microwave pre-treatment, Keshwani et al. achieved a sugar yield of 80-85% from switchgrass. In addition, the sugar yield from switchgrass increased to 70-90% after microwaveassisted alkali pre-treatment.

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2.2 Thermalchemical Processes Thermalchemical biomass conversion provides a number of possible roots to produce useful fuels and chemicals from the lignocellulosic feedstock. The thermalchemical processes have higher efficiencies than biochemical/biological processes in terms of the lower reaction time and the superior ability to destroy most of the organic compounds. Generally, the thermalchemical processes can be classified into four fundamental sub-categories: pyrolysis, liquefaction, gasification and combustion. Among these four technologies, pyrolysis and liquefaction are the two major processes to produce bio-oils.

2.2.1 Pyrolysis Pyrolysis involves fundamental chemical reactions in the absence of oxygen. It is considered as an industrially realized process for biomass to convertion into solid charcoal, bio-oil and gases. Once the biomass reaches high temperature (above 300°C), it is thermally depolymerized to small compounds, which are vapors in the reactor but condense to liquid mixture (bio-oils) at room temperature. Thermal gravity analysis of biomass suggests that there are three stages for a typical pyrolysis process: prepyrolysis (between 120 and 200 °C), main pyrolysis and continuous char devolatilization. Depending on the reaction temperature and residence time, pyrolysis can be divided into fast pyrolysis and slow pyrolysis. Conventional slow pyrolysis occurs under a low temperature and a slow heating rate for a relatively long vapor residence time, which has been used to produce charcoal for thousands of years. At the temperature of about 400 °C, charcoal represents the largest component in wood products, containing approximately 80% carbon and 12-15% volatile components. Although the yield of charcoal decreases with the increment of temperature, the carbon content of the charcoal increases dramatically. In addition, the chemical composition of biomass is another factor closely relating to the yield of charcoal. A higher yield of charcoal can be achieved from the biomass feedstock with higher lignin content and lower hemicelluloses content. The term of


fast pyrolysis is a process with high heating rate and short residence time, which favours the formation of liquid products rather than solid chars. In fast pyrolysis, the yield of bio-oils can reach 80 wt. % of the dry feed. In the pyrolysis of biomass, the breaking down of hemicelluloses, cellulose and lignin occurs at 200-260 °C, 240-350 °C and 280-500 °C, respectively. The degradation of biomass includes various reactions, such as hydrolysis, dehydration, isomerization, dehydrogenation, aromatization, retro-condensation and coking. Thus, the products from biomass pyrolysis consist an aqueous phase (low molecular weight light organo-oxygen compounds) and a nonaqueous phase (high molecular weight insoluble aromatic organic compounds). The techno-economic analysis suggests that fast pyrolysis is typically competitive with biological and gasification technologies in terms of reducing biofuels cost. However, the instability, the low energy intensity and the relatively low heating value (26 MJ/kg, the heating value of petroleum fuel oils is 42-45 MJ/kg) of the bio-oil may be crucial for the successful commercialization of pyrolysis oils.

2.2.2 Liquefaction Hydrothermal liquefaction is a process for producing clean bio-oil from biomass in the presence of solvent at medium temperature (250-450 °C) and high pressure (5-25 MPa). In the liquefaction, lignocellulosic biomass is decomposed into unstable and reactive small molecules. These fragments can then repolymerize into oily compounds with a wide range of molecular weight distribution. Generally, removal of oxygen is the overall object in producing fuels from biomass. Ideally, oxygen heteroatom should be removed as the fully oxidized compounds without losing heating value. The removal of oxygen occurs most readily by dehydration and decarboxylation, which removes oxygen in the form of fully oxidized compound, water and carbon dioxide, respectively. Furthermore, the dehydration and decarboxylation also lead to the formation of new molecular rearrangements. Apart from these reactions, solvolysis results in the micellization of biomas and

depolymerization leads to degradation of biomass into smaller molecules. When hydrogen is present, hydrogenolysis and hydrogenation of functional groups occur. For comparison, bio-oilis from liquefaction have a greater heating value (30-36 MJ/ kg) than that from fast pyrolysis, due to the lower oxygen content and moisture, and the larger proportion of organic compounds. However, the solubilization of depolymerized material may introduce viscous tarry lumps in the bio-oils from liquefaction and cause handling difficulties. Thus, a number of organic solvents have been used for liquefaction to reduce the viscosity of bio-oils. Water is used as one of the most common solvents due to its low cost. In thermal regions of liquefaction, water can behave as organic compounds and is miscible with organic species, introducing an effective extraction of plant products. In order to reduce the reaction temperature, enhance reaction kinetic, and improve the yield of desired products, the use of catalyst is also a critical factor. The main catalysts include alkalis, acids, salts, metals, and heterogeneous catalysts. Specially, alkaline catalysts are able to enhance the yield of heavy oil and decreasing the formation of residues; while acid catalysts are capable of decreasing the reaction temperature and time by hydrolysis of cellulosic components, and also have the potential to condense lignin materials, consequently increasing the amount of insoluble residue.

2.2.3 Gasification Gasification can be viewed as a special form of pyrolysis, carryied out at high temperature to convert solid or liquid carbonaceous material into combustible gases (H2, CO, CH4) in the presence of oxidants (air, steam, O2 and CO2). Based on the target products, gasification has fallen into three general categories: high temperature gasification (T > 500°C) for hydrogen-rich gases, moderate temperature process (~ 500°C) for methane-rich gas and low temperature catalytic gasification processes. In the high gasification process, when the temperature and pressure are increased to or above 374 °C and 22.1MPa, high concentrations of H+ and


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OH- can be released from water, providing a perfect environment for acid- or base-catalyzed reaction. As the temperature is increased to higher than 600 °C, water acts as both catalyst and reactant, which transfers the carbon atoms in biomass to form CO2 and CO, and the hydrogen atoms in both of biomass and water to form H2. Previous study has reported that the biomass can be nearly completely converted into a H2-rich product in the vicinity of 600 °C in supercritical water. However, the low economical efficiency caused by high temperature and pressure has become the greatest obstacle to the development of supercritical water gasification. Lower temperature (~ 400°C) favours the production of methane and catalysts become the main factor to affect the distribution of products in this process. In order to improve the efficiency of biofuels production, integration of the biomass gasification and Fischer-Tropsch (FT) synthesis is a promising route. Biomass is used as the feedstock for producing synthesis gas used in production of FT liquids. The heating value of the syngas is closely related to the oxidants used in biomass gasification, such as gas from air-blown and oxygen-blown gasification with heating values approximately 5 MJ/Nm3 and 15 MJ/Nm3, respectively.

2.2.4 Combustion Combustion is the most widely used process for biomass conversion due to its low costs, straightforward and commercially available. However, only 12% of the global energy required is generated by combustion of biomass. In developing countries, around 35% of the energy is originated from biomass combustion, but the total contribution of biomass to the primary energy is 3% in industrialized countries. Thus, combustion technologies deserve attention to improve efficiency and to remain competitive with the other options. Generally, there are three stages in the combustion of biomass, including drying, pyrolysis and reduction, and combustion of volatile gases and solid char. The combustion of volatile gases contributes to 70% of the total heating value of biomass. Although combustion technologies are the 152


most frequently applied process for solid biomass fuels, it need to be further improved to maximize efficiency, safety and simplicity. In comparison to fossil fuels, the high moisture content and oxygen content of biomass are the distinct disadvantages of using biomass as a fuel. In addition, the fouling and corrosion of combustor caused by the alkali metals and some other elements in the biomass are also considered to be detrimental. In order to overcome the disadvantages of biomass fuels, co-firing biomass and coal has been considered as a promising option for bioenergy utilization, reducing the occurrence of fouling, corrosion, and the emission of CO2 and other toxic gases (SOx and NOx).

3. Conclusions An increasing use of biofuels would contribute to the sustainable development by reducing greenhouse gas emission and the use of nonrenewable resources. Lignocellulosic biomass is a cheap and abundantly available source for transportation fuels production. The lignocellulosic biofuels platforms include bioethanol of biochemical platform in which biomass is enzymatically hydrolyzed to sugar and subsequently fermented into ethanol, and three main products of thermochemical platform in which biomass is gasified and upgraded to hydrogen, methanol or Fischer-Tropsch liquids. Further improvements in the biochemical and thermochemical platforms for processing lignocellulose have the potential to reduce the costs of biofuels.

Acknowledgements This work was supported by the grants from the State Forestry Administration (201204803), Natural Science Foundation of China (31110103902), Ministry of Science and Technology (973-2010CB732204).

Summary box Lignocellulosic biomass, containing polymers of cellulose, hemicelluloses and lignin, has been considered as the alternative resource for fuels pro-


duction. Currently, a variety of bio-chemical and thermo-chemical technologies have been proposed for the production of biofuels from lignocellulosic biomass. In the bio-chemical platform, the shadow of hemicelluloses and lignin, the crystalline structure of cellulose, and limited accessible area are the main factors hindering the enzymatic digestibility of cellulose in the lignocellulosic biomass. Thus, efficiency pre-treatment is the vital prerequisite to overcome the natural resistance of lignocellulosic biomass. Unlike the biochemical platform, there is a greater diversity of opinion on how the thermochemical platform should be configured. Generally, there are three main products of thermochemical platform in which biomass is gasified and upgraded to hydrogen, methanol or Fischer-Tropsch liquids.

Review Questions and Answers Q1. What are the main components of lignocellulosic biomass? A1. The lignocellulosic biomass mainly contains polymers of cellulose, hemicelluloses and lignin. Q2. What are the main factors contributing to the recalcitrance of lignocellulosic biomass? A2. The main factors include the shadow of hemicelluloses and lignin, the crystalline structure of cellulose, and limited accessible area. Q3. What are the main technologies in pre-treatment of biomass for improving cellulose digestibility? A3. The leading technologies in pre-treatment include lime pre-treatment, ammonia fiber expansion (AFEX), ammonia recycle percolation (ARP), dilute acid (DA) pre-treatment, hot liquid water (HLW) pre-treatment and steam explosion (SE). In addition, ionic liquid and microorganisms may also be applied in pre-treatment. Q4. How many sub-categories of thermo-chemical technologies exist for biofuels production? A4. Generally, the thermalchemical processes can be classified into four fundamental sub-categories: pyrolysis, liquefaction, gasification and combustion.

Q5. What are the main products of the thermochemical platform? A5. There are three main products of thermochemical platform in which biomass is gasified and upgraded to hydrogen, methanol or Fischer-Tropsch liquids.

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26. Klass D. Biomass for renewable energy, fuels and chemicals. San Diego: Academic Press 1998. 27. Kuhad RC, Singh A, Eriksson KEL. Microorganisms and enzymes involved in the degradation of plant fiber cell walls. Advances in Biochemical Engineering / Biotechnology 1997;5745–125. 28. Kumar P, Barrett DM, Delwiche MJ, Stroeve P. Methods for pre-treatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Industrial & Engineering Chemistry Research 2009;48:3713–29. 29. Kuttruff H. Ultrasonics fundamentals and applications. Essex England: Elsevier Science Publishers Ltd.1991. 30. Luo CD, Brink DL, Blanch HW. Identification of potential fermentation inhibitors in conversion of hybrid poplar hydrolyzate to ethanol. Biomass & Bioenergy 2002;22:125–38. 31. McMillan JD. Pre-treatment of lignocellulosic biomass. Edited by Himmel HE, Baker JO, Overend RP, Enzymatic conversion of biomass for fuels production. Washington, DC: American Chemical Society 1994;292–324. 32. Milne TA, Agblevor F, Davis M, Deutch S, Johnson D. A review of the chemical composition of fastpyrolysis oils from biomass. Edited by Bridgwater AV, Boocock DGB, Development in thermal biomass conversion. London: Blackie Academic and Professional 1997;409–25. . 33. Mohan D. Pittman CU, Steele PH. Pyrolysis of wood/biomass for bio-oil: a critical review. Energy Fuels 2006;20:848–89. 34. Mussatto SI, Roberto IC. Alternaives for detoxification of diluted-acid lignocellulosic hydrolyzates for use in fermentative process: A review. Bioresource Technology 2004;93:1–18. 35. Palmqvist E. Fermentation of lignocellulosic hydrolysates: inhibition and detoxification. Ph.D. thesis, Lund University, Sweden.


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46. Veringa HJ. Advanced technique for generation of energy from biomass and waste. https://www. ecn.nl/fileadmin/ecn/units/bio/Overig/pdf/Biomassa_voordelen.pdf 47. Wright MM, Brown RC. Comparative economics of biorefineries based on the biochemical and thermochemical platforms. Biofuels Bioprod Bioref 2007;1:49–56. 48. Yan L, Wan L, Yang J, Zhu Q. Direct visualization of straw cell walls by AFM. Macromolecular Bioscience 2004;4:112–8. 49. Zakzeski J, Bruijnincx PCA, Jongerius AL, Weckhuysen BM. The catalytic valorization of lignin for the production of renewable chemicals. Chemical Reviews 2010;10:3552–99. 50. Zhang LH, Xu CB, Champagne P. Overview of recent advances in thermo-chemical conversion of biomass. Energy Conversion and Management 2010;51:969–82.

43. Stöcker M. Biofuels and Biomass-To-Liquid Fuels in the Biorefinery: Catalytic Conversion of Ligno-cellulosic Biomass using Porous Materials. Angewandte Chemie International Edition 2008;47:9200-11.


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CHAPTER 10 NANOBIOTECHNOLOGY Mine Altunbek, Ertuğ Avcı, Mustafa Çulha


1. Introduction to Nanobiotechnology ............................................................................. 159

2. Nanomaterials Used in Nanotechnology: A Nanomedicine Perspective ........................ 160

2.1 Quantum Dots ........................................................................................................... 160

2.2 Gold Nanoparticles ..................................................................................................... 163

2.2.1 Synthesis and Modification of AuNPs ................................................................. 163

2.3 Superparamagnetic Iron Oxide Nanoparticles ............................................................. 164

2.1.1 Synthesis and Modification of ODs .................................................................... 161

2.3.1 Synthesis and Modification of SPION ................................................................ 165

3. Challenges of Nanomedicine ........................................................................................ 166

3.1 Requirements for Efficient Diagnostic and Therapeutic Nanoparticle ......................... 166

4. Nanobiosensors ............................................................................................................ 168

5. Self-Assembly ................................................................................................................ 169

5.1 Evaporation Induced Self-Assembly ............................................................................ 169

5.2 Programmed Self-Assembly ......................................................................................... 170

5.3 Geometry (Shape) Driven Self-Assembly ..................................................................... 170

6. Conclusions .................................................................................................................. 170


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Summary

T

his chapter is intended to provide a brief explanation of nanobiotechnology in broad terms and “nanomedicine” more specifically, giving examples of some materials, engineered nanostructures and devices in this field. First, three types of nanoparticles used in imaging and theranostics will be introduced. There are many nanoparticles and nanocomposite structures used in nanomedicine, but for the sake of the brevity, the number is restricted to three. Some practical and problematic issues in the field will be discussed. Finally, nanobiosensors and self-assembly concepts will be discussed.

1. Introduction to Nanobiotechnology Nanobiotechnology is referred to as an interdisciplinary field aiming at the use of nanomaterials for the solution of problems encountered in biotechnology and medical-related fields. Nanotechnology is an emerging field aiming to construct novel tools and devices by manipulating matter in the size range of 1 to 100 nm. Materials reveal unique novel properties as their size get smaller to the electron confinement phenomenon. In addition, the nanometer-sized materials significantly deviate from Newtonian behavior as their size or at least one dimension is in the nanometer regime. Although their unique physicochemical properties can be used to construct novel tools and develop novel materials for daily use, they can also be used in areas where biology is involved. In order to understand the extent of nanotechnology into biology, one should take a look at the size regime in living cell operations and cellular components. Table 1 shows the size comparisons of the sizes of components of a cell and selected nanomaterials (NMs). As seen in Table 1, there is a distinct size relationship between the cellular components and NMs. A living cell is very complex biomolecular machinery involving billions of molecular events and interacNANOBIOTECHNOLOGY

tions in a very short time and a volume defined by the cell dimensions. In a cellular pathway, the molecular machinery involves a few nanometer sizes of molecular assemblies and proteins. The biomacromolecules and their assemblies are transported from one region to another. One can easily realize that the cellular molecular machinery operates in the nanometer scale. One can also realize that the size and unique physicochemical properties of NMs can be used to make new tools and devices for the solution of problems challenging humans today. This may indeed open new paths and may bring new opportunities to solve many problems in biotechnology, biomedical applications and medicine.

Endoplasmic Reticulum

Components of a cell

Nanomaterial

Animal Cell (10 μm)

AuNPs (13 nm)

Lysosome

Ribosomes Mitochondrion

Nucleus Golgi Body

DNA

Vacuole

Mitochondrion (0.5-1 μm)

SWCNTs (1-5 nm)

Diameter of DNA (2.5 nm)

BNNTs (100 nm)

Diameter of a RNA and an Oligonucleotide (1.5 nm)

QDs (2-4 nm)

Table 1. The size comparison of components of a cell and selected nanomaterials.

In recent years, progress in the preparation of NMs and better understanding of their interactions with living systems have made the creation of functional, commercial materials and devices possible. In broad terms, nanobiotechnology aims at the use of biological materials (oligonucleotides, peptides, proteins, microorganisms, and cells) with the help of nanotechnology to produce novel NMs and nanoarchitectures for use in biotechnology and medical-related fields. Since nanobiotechnology concept refers to a very broad area, it is difficult to cover all subfields. Therefore, this chapter mainly focuses on the use of NMs CHAPTER 10

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in medicine and biomedical applications. Nanomedicine focuses on medically related, patient-centric nanotechnologies. It encompasses production and application of nanostructures and devices for diagnosis and treatment of diseases. To realize this goal, a wide range of functionalized nanomaterials have been produced such as liposomes, quantum dots, carbon nanotubes, polymeric nanoparticles, polymeric micelles, and inorganic nanoparticles in recent years. In addition, novel nano and microdevices have been developed using top-down, bottom-up approaches and microfluidics. The development of nanomaterials for medicine is aimed at imaging, diagnosis and therapeutic purposes. The functionalization of nanostructures with diverse chemistries and biological materials enables production of multifunctionalized nanoparticles (NPs) and nanostructures, which combine therapeutic and diagnostic functions in a single formulation (theranostics). The combination of a variety of nanomaterials can be used for development of more efficient delivery systems. The strategies for imaging of diseased areas and site specific release of drugs relies on development of nanomaterials, which release therapeutic molecules upon sensing a specific feature of the target location, such as its temperature, pH, redox balance, or the presence of a specific enzyme at that site. In addition, nanoparticles/nanocarriers loaded with imaging and therapeutic agents can be stimulated externally by heat or radiation when they reach their target location.

2. Nanomaterials Used in Nanotechnology: A Nanomedicine Perspective Nanotechnology may provide unique opportunities in medicine and biomedical applications by improving the conventional approaches or developing new strategies for targeting, delivery, imaging and therapeutic applications. The most important factors with the use of NPs in medical application are biocompatibility and stability in biological systems. Once these two conditions are assured, the multiple functions of NPs can be adapted for specific actions 160


through their size, shape and surface chemistry. The specific modifications with specific targeting moieties, therapeutic molecules or contrast agents via direct conjugations, linker chemistry or physical interactions methods can be achieved without disturbing the functions of the agents. The selection strategy of targeting modifier depends on recognized changes indicating diseased areas in the body such as reactive oxygen species (ROS), temperature, or a specific receptor, hypoxia, acidic or basic conditions. In this section, the mostly used nanomaterials in biomedical applications including the quantum dots (QDs), gold nanoparticles (AuNPs) and iron oxide nanoparticles (IONs) will be characterized depending on their properties and modifications.

2.1 Quantum Dots Quantum dots (QDs) are 2-10 nm semiconductor nano-crystals that are composed of cadmium-selenide/sulphure/telluride, indium phosphide (CdSe, CdS, CdTe, InP) core and usually a zinc sulfide shell (ZnS). The semi-conductive materials have a bandgap energy, which is a term used for the minimum energy level required to excite an electron to a high energy level from ground state. When the electron relaxes to the ground state after excitation, the bandgap energy is converted into an emitted photon called fluorescence. Depending on the size and the composition of the QDs, the bandgap energy changes and therefore fluorescence wavelength can be adjusted to by varying bandgap though changing the size and composition of the semiconductor material. The strong bright and photostable fluorescence properties make QDs attractive in biological applications including labeling of proteins and nucleic acids in several applications as alternatives to conventional molecular labels. The emission spectra of QDs are compared to organic dyes on Figure 1. As seen, QDs (1a-c) have broader absorption spectra providing a clear separation between excitation and emission wavelengths. Their emission wavelength can be adjusted by changing the size of QDs. However, the distinction between excitation and emission wavelengths of organic dyes is very poor (1d-e) and upon multiplex detec-

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tion or imaging the interference between emission wavelengths of different dyes is also another problem. The other advantages of QDs over fluorescent dyes and proteins are high quantum yield and high resistance to photobleaching, which refers to the decomposition of fluorophore molecules under exposure to the excitation light. Figure 2 shows a representative depiction of QD structure and their emission wavelengths depending of particle size.

Figure 1. Comparison of spectra of QDs and organic dyes. Absorption and emission spectra of QDs (a–c) and organic dye molecules (d–f ). The size is color coded: blue < green < black < red. Reprinted by permission from Macmillan Publishers Ltd: Nature Methods. Copyright © 2008.

Figure 2. A typical QD with surface modifications (a). QDs are composed of a cadmium selenide (CdSe) core and a zinc sulphide (ZnS) shell surrounding the core. A polymer and NANOBIOTECHNOLOGY

hydrophilic coating is necessary for stability. Finally, a functional group carrying molecule to provide further functionalization with a biomolecule is bound to the surface. Size dependent change of emission wavelength CdSe QDs (b). Emission wavelength is narrow, symmetrical and reaches into the near-infrared as size increases. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

2.1.1 Synthesis and Modification of QDs The synthesis of QDs is performed at high temperatures in the presence of long alkaline chain surfactants such as trioctylphosphineoxide (TOPO) and hexadecylamine (HDA). The high boiling point surfactants adhere onto the surface of QDs and block the un-controlled growth of the particles. However, the hydrophobic surfaces and leakage of cadmium ions Cd2+ from the nanoparticle cause toxic effects, which limit the applications of QDs in biology and medicine. The surface modifications strategies are developed to increase their biocompatibility, prevent release of toxic ions and increase the dispersibility in aqueous media. The hydrophobic surface of QDs can be turned into hydrophilic by using two different strategies. In the first strategy, the affinity of hydrophilic thiol (-SH) containing heterobifunctional molecules such as mercapto silanes or mercaptoacetic acid, onto QD surfaces can be used to exchange with hydrophobic TOPO or HAD ligands. The other approach is the adsorption of an amphiphilic polymer onto the QDs, which contain a hydrophobic side, interacting with hydrophobic surface of the QDs and a hydrophilic part, providing water solubility. The thickness of the ligand-polymer coating is usually 1-2 nm. The surface of the QDs has been modified with water-soluble biomolecules including peptides, DNA, antibodies, proteins and carbohydrates. During modifications, the important factor is to keep the QDs properties intact. For example, the modification can cause particle aggregation and decrease in the quantum yield or complete loss of fluorescence properties. In addition, modification with an unstable modifier can lead to a naked QDsurface, which causes the release of toxic ions of QD-elements to the environment. CHAPTER 10

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Based on their fluorescence properties, the potential use of QDs in medicine has been investigated in in vitro and in vivo imaging studies. The main focus is to use QDs in imaging to monitor cancer development, tumor progression, and angiogenesis through modifications with specific markers. Bhang et al. modified the surface of the QDs with hyaluronic acid (QDs-HA) that was highly expressed in cancer and tumor cells during angiogenesis. Figure 3 shows the schematic representation of the HA modification on the QDs surface. In vitro studies showed that QDs-HA specifically bound to the HeLa (cancer cell model that highly expresses HA binding receptor) compared to the human dermal fibroblast cells (HDF-normal cells). In addition, the cytotoxicity of the HA modified QDs decreased compared to the non-modified forms, which is also important for in vivo studies. The particle size was found to be another important factor for the transportation of the nanoparticles through the lymphatic capillaries. For example, the effective size for the delivery of the QDs-HA was found to be 60 nm in the lymphatic vessel, where HA binding lymphatic vessel endothelial receptor 1 (LYVE-1) is expressed. Fluorescence microscopy images were obtained from the sections of the tissues 30 min later after QDs and QDs-HA subcutaneous injection. The specific binding (yellow spot) of QDs-HA (red) to LYVE-1 receptor (green) is showed on Figure 4.

Figure 3. Schematics of synthesis procedure of hyaluronic acid coated QDs. Copyright © 2009, American Chemical Society.

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Figure 4. Fluorescence microscope images of mouse ear tissues where HA-QDs (a,b) and blank QDs (c) are injected. Red fluorescence from QDs (a). Fluorescence images (b and c) are merged to overlay the red (QD), green (LYVE-1), and blue (DAPI) signals. Bright yellow co-localization areas originating from QD and LYVE-1 are indicated by arrowheads in (b). Copyright © 2009, American Chemical Society.

The stability against photobleaching makes QDs appropriate for long term monitoring applications in cancer diagnosis and prognosis assays. In a study, QDs were used for the evaluation of cancer cell coordination and tumor invasion by monitoring the transport of wheat germ agglutinin (WGA) modified QDs between cancer cells. The entire transport mechanism of WGA between A549 cells through membrane nanotubes was demonstrated by monitoring of QDs emission and shown to be important for drug development in medicine. QDs were investigated as biosensors for the early detection of Parkinson’s disease (PD) and its progression. PD is a neurological disease and the inhibition of the mitochondrial NADH:ubiquinone oxidoreductase (complex I) in the enzymatic reaction of the electron transport chain is used in diagnosis in the early stages. In a study, QDs were modified with ubiquinone for the diagnosis of PD. Depending on the reduction or oxidation state of ubiquinone, the emission from the ubiquinone modified QDs was enhanced or quenched. The in vitro study showed that in the oxidized state, ubiquinone functions as a favorable electron acceptor, and this results in the effective quenching in the fluorescence indicating early stage of PD while in the presence of NADH, ubiquinol on the surface of QDs was reduced state forming complex I and the fluorescence enhancement of the QDs was observed. In addition to the fluorescence properties of the QDs, the multifunctional features can be added through surface modifications, including differ-

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entiation of cancer tissues from healthy ones, real time monitoring of drug behavior and early detection of diseases.

amined through light microscope that is modified with a dark field condenser. b

a

c

2.2 Gold Nanoparticles

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d

0,5

520

530

550 13 nm 25 nm 50 nm

Absorbance

Gold nanoparticles (AuNPs) show unique size and shape dependent properties upon their interaction with electromagnetic radiation. When the frequency of the electromagnetic radiation overlaps with the oscillation frequency of the conduction electrons of the AuNPs, absorption of electromagnetic radiation occurs, which results information of surface plasmons. Interestingly the region of the electromagnetic radiation, which strongly interacts with the gold nanostructures, falls into the visible region of the spectrum. As the size of the AuNPs increases, the absorbed wavelength of the visible light shifts to longer wavelengths due to the dampening of the electron system of the nanostructure. While the absorbed photons excite the surface plasmons, some are converted to heat. The unabsorbed light interacting with the NPs is efficiently scattered. Each of these processes can be used in medical and biomedical applications. When size and shape tunability and biocompatibility are considered, they become attractive novel materials to be used in biomedical imaging applications. When compared to conventional fluorescence dyes, which are used as probes or labels in biomedical applications, the plasmonic AuNPs are more advantageous. Firstly, the intensity of scattered light is greater than with fluorescence dyes. For instance, the scattered light from an 80 nm AuNPs is equal to 106 scattered photons from a fluorescent dye. Second, the color of emitted light and the intensity can be adjusted by changing the size and shape of the AuNPs. Figure 5 shows the TEM images of citrate reduced AuNPs with different sizes and their UV/Vis spectra. As it is seen, when the particle size gets larger, surface plasmon resonance changes and absorption spectra shifts to the longer wavelength. Third, the AuNPs have long lasting emissions compared to the fluorescence dyes. The easy imaging system also makes NPs favorable over fluorescence dyes in biological applications, as well. The sample can easily be ex-

0

400

450

500

550

600

650

700

750

800

Wavelength (nm)

Figure 5. TEM images of citrate reduced AuNPs with 13 (a), 25 (b) and 50 (c) nm sizes and UV/Vis spectra (d).

The use of AuNPs in medical applications goes back to around 1925. At that time, gold salts were used during clinical testing of heavy materials to treat rheumatoid arthritis. In 1960 the British Rheumatism Council approved a clinically efficient gold therapy. The applications of AuNPs are widespread. Given that AuNPs are less toxic to living organisms, they are used in biomedical and cellular visualization applications with the properties of absorption and emission of visible light. In addition, targeted drug delivery and gene therapy applications by AuNPs are possible through surface functionalization with the specific ligands. Further, AuNPs can heat the surrounding environment by converting the absorbed light energy into heat. Depending on those properties, AuNPs are intended to be used as multifunctional materials for targeted cancer therapy. The possibility of synthesizing AuNPs at smaller size is another advantage, which can be used as a vehicle to enter the cells easily.

2.2.1 Synthesis and Modification of AuNPs AuNPs have been synthesized by various methods with different sizes and shapes due to their practical uses in the field of nanobiotechnology. The easy CHAPTER 10

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and practical way to synthesize AuNPs is to use the sodium three-citrate reduction method with an average size of 13 nm. To change the particle size, citrate concentration can be varied. For example, less citrate concentration is used for larger sizes of particles compared to the 13 nm of AuNPs. Other reducing agents are also available such as ascorbic acid and sodium borohydride. The AuNPs can be modified with a molecule possessing a thiol group due to the affinity of thiol (-SH) to noble metal surfaces. Further modification can be pursued through –OH, -COOH or -NH2 on the thiol-containing molecule. The surface functionalization with biological molecules may enable their use in biomedical applications. The AuNPs are indeed proposed for many biomedical applications in the literature. Thomas et al. used AuNPs as non-viral gene therapy vehicles in their study. The AuNPs modified with polyethylamine (PEI) directly targeted to the nucleus could transfect monkey kidney (COS-7) cells six times better than the PEI modified could do alone. During in vitro and in vivo studies, the viability of the cells is important for the effective use of nanomaterials in biomedical applications. Even different sizes and surface modifications can create different toxicity in different cell lines. For instance, the cell viability during transfection of COS-7 cells with PEI modified AuNPs was 80 %. Further surface modification of AuNPs with dodecyl-PEI decreased the cell viability from 80 % to 70 %. Another study carried out by Tkachenko et al. showed the effects of peptide– BSA modified AuNPs in three different cell lines, HeLa, 3T3/NIH and HepG2. While peptide–BSA modified AuNPs could enter HeLa cells and escape endosomes, they could not escape within the endosomes of 3T3/NIH cells. On the other hand, NPs could not be taken up within the HepG2 cells. Additional cytotoxicity studies also demonstrated the different cellular response to modified AuNPs with the same dosage and time period treatment. 20 % of HeLa cells were dead after transfection whereas the death rate was 5 % in the 3T3/NIH cells. In another study, modified AuNPs were used to monitor apoptosis levels in vitro. The detection of 164


apoptosis is important for the diagnosis of diseases including cancer, neurodegenerative diseases and immune disorders. The AuNPs surface was modified with the peptide linker (DEVD) that is recognized by caspase-3 enzyme. The strong scattering property and high photo-stability of AuNPs enables the monitoring of caspase-3 activated apoptosis at a single live cell level during 2 hours under visible light without photobleaching. The photo-thermal effect of AuNPs was also used in controlled drug release for therapeutic applications. For this purpose, the AuNP surface was modified with both folate, which binds to the receptor expressed highly in tumor cells, and siRNA, which recognizes NF-KB involved in tumor development by Li et al. The folate was coupled with polyethylene glycol (PEG) to increase the circulation of the NPs inside the body. The possible release of the siRNA from the AuNPs upon irradiation in near infrared region (800 nm) showed the down-regulation of the NF-KB gene and apoptotic enhancement on HeLa cells in vitro. In vivo micropositron emission tomography (PET)/computed tomography (CT) imaging also demonstrated the tumor targeted therapy by the specific uptake of the AuNPs through folate modification. However, the modified AuNPs also observed in liver, spleen, kidney and lung. Since the NPs were activated through near infra-red irradiation, there was no significant down-regulation of the NF-KB gene.

2.3 Superparamagnetic Iron Oxide Nanoparticles Superparamagnetic iron oxide nanoparticles (SPION) are safe magnetic nanoparticles consisting on Fe3O4 and γ-Fe2O3 core and organic or inorganic polymer shell and they have been approved for the clinical use. They are used in a variety of biological applications such as cell sorting, drug delivery and imaging. Surface modifications improve the SPION applicability as therapeutic or targeting agents to localize in specific tissue and real time monitoring of the treatment response through their magnetic property, which is used in magnetic resonance imaging (MRI).

NANOBIOTECHNOLOGY

2.3.1 Synthesis and Modification of SPION The SPION to be used in the biomedical area must be in appropriate size, surface charge and magnetization. The stability and duration of their magnetic properties in their environment are also important parameters for SPION. Fe3O4 and γ- Fe2O3 have similar crystalline structures but the magnetic property of Fe3O4 is 10 times greater than γ-Fe2O3 and Fe2+ ions are present in the structure of Fe3O4 nanoparticles. Since Fe2+-O bond is longer than Fe3+-O, Fe3O4 are weaker and dissolution in acidic environment is faster than γ-Fe2O3. The response of 10 nm size SPION NPs to magnetic field was found to be inefficient. However, when these 10 nm NPs were brought together forming nano-clusturs about 30-180 nm with a help of polymers, the magnetic properties of SPIONs were improved. There have been many literature reports of uniformly formed SPIONs nano-clusturs with polymers. The molecular weight and amount

of polymers are indicated as important factors for the magnetization. The other important factor is the size of the particle, which must be 10-100 nm for the in vivo therapeutical applications to escape clearance from the body before reaching the target. SPION surfaces are modified with macromolecules to prevent their oxidation in air, which results in loss of magnetism. The surface modifications also enable the SPIONs to direct them to the targeted area. SPION have been modified with hydrophilic polymers such as polyethylene glycol (PEG), dextran, heparin, chitosan and polyvinyl alcohol (PVA) for the preparation of stable particles. The polymer coatings also increased the retention of SPIONs in the body by escaping reticulo-endothelial system (RES) clearance. The SPIONs have been engineered for many medical applications. It is known that, in tumor tissues, the extracellular environment is at around pH 6.5-7.0 since the rapidly growing cells cause an increasing rate of glycolysis and lactic acid formation. In a study, a fluorescent dye and its quencher were entrapped into pH

Figure 6. pH responsive micelle used in vivo pH imaging in tumor and brain pH-responsive MPEG-PAE block copolymer chemical structure(a). Design of pH-dependent fluorescence recovery system in acidic condition prepared from ‘nano-flash’(b). Non-invasive real-time fluorescent imaging of tumoral pH in MDA-MB-231 tumor-bearing mice (c). Illustration of the pH-sensitive Fe3O4-encapsulated PEG-PAE micelle mechanism (d). Fe3O4-encapsulated PEG-PAE micelles precititation depending on pH in aqueous media at 37 0C(e). MRI of the rat brain with Fe3O4-PEG-PAE micelles (f ). Reprinted from Nanoprobes for biomedical imaging in living systems, 6, Koo H, Huh MS, Ryu JH, et al.. Nano Today 204-20, Copyright © 2011 with permission from Elsevier. NANOBIOTECHNOLOGY

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sensitive methyl ether-b-(poly lactic acid-co-poly (β-amino esters)) (MPEG-b-(PLA-co-PAE)) block copolymers, which disassemble in acid conditions. Figure 6 shows the schematic representation of the co-polymer and its pH sensitive activation and imaging. These co-polymers were activated in acidic tumor tissue selectively and provided non-invasive tumor diagnosis and imaging in vivo. The same strategy was applied with Fe3O4 NPs to monitor ischemic regions in the brain by Ko et al. The released Fe3O4 NPs caused efficient accumulation in ischemic regions of the rat brain and allowed the imaging via MRI. SPIONs were also used to monitor targeted realtime treatment monitoring. Jon et al. modified surface of the SPION with a CG-rich specific aptamer recognized by the prostate specific membrane antigen on tumor cells. The CG-rich sequence was loaded with the doxorubicin cancer drug allowing the selective release of the drug to the tumor cells. In vivo imaging during 48 h showed precise signal drop from the tumor and indicated selective targeting of SPION to the tumor and delivery of doxorubicin to the tumor.

3. Challenges of Nanomedicine The major aim in imaging and diagnostics is to achieve a strong signal-to-noise ratio using as little agent as necessary in the shortest time. In addition, rapid clearance from the body is desired to decrease the risk of possible adverse effects. On the other hand, during therapies, high amount of nanocomplex (maximum tolerated dose) for prolonged durations is used to achieve higher therapeutic success rates. In order to overcome this dilemma and to adjust concentration and residence time of nanocomplexes and therapeutic/diagnostic agents, three approaches can be proposed. The first one can be the use of two-step theranostics. Nanocarriers are firstly loaded with a diagnostic agent and given to the patient, and then identical nanocarriers are loaded with therapeutic agent and administered to the patient. The second approach is to develop theranostic nanocomplexes in which the ingredients of the complex can be used initially 166


as diagnostic tool, and then as a therapeutic agent after application of an external trigger. The third approach is incorporation of therapeutic agents in a biocompatible diagnostic agent, which can reside in the body for prolonged times without causing any harm, especially during its degradation. All three approaches have both advantages and disadvantages and should be carefully considered for effective therapy. After taking a quick glance on the general issues on the way to produce a theranostic nanocomplex, we should take a closer look at the parameters for its development.

3.1 Requirements for Efficient Diagnostic and Therapeutic Nanoparticle The requirements and parameters for the preparation of efficient delivery and release nanoparticle/ nanocarriers can be listed as choice of the nanomaterials, stability in blood circulation, suitable size, shape, and surface chemistry, suitability and availability of biological molecules for targeting on the nanocomplex surface, endosomal escape routes, escape from RES system, and prolonged circulation. Low toxicity of the nanocomplex is also another issue. All parameters listed here are interrelated and should be taken into consideration wisely during preparation of a nanocomplex and its application for imaging and theranostic purposes. The first parameter is the choice of the nanomaterial to be functionalized. It is important because chemical composition determines the surface chemistry, surface energy, redox status, photoactivation potential and charge of the nanocomplex. In addition, some surface defects which determine available surfaces for further functionalization, and surface groups which define surface reactivity are directly related to the chemical composition. The nature (chemistry) of nanomaterial chosen as a delivery and release nanocomplex and its surface chemistry determines its hydrophilicity, stability, solubility, and dissolution profile. Dissolved ions from the nanomaterials can be toxic and can have adverse effects on the organism before reaching the target tis-

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sue. Furthermore, a nanocomplex should be stable enough to be able to circulate in harsh conditions through vessels, and should endure some enzymatic attacks in blood. It should have some molecules or ligands used for escape from macrophages. Its overall surface should be hydrophilic, because hydrophobic ones are filtered and removed by RES (composed of blood-associated macrophages and other phagocytic cells in liver and spleen) at a higher rate than hydrophilic ones. Overall charge of the complex is also extremely important. Positively charged nanocomplexes are more toxic than negatively charged ones. Size of the nanocomplex is important. Very small ones (less than 10 nm) are filtered by the kidney and nanocomplexes larger than 100 nm are filtered by the liver and spleen. In addition, size should be appropriate for extravasation (hydrodynamic size of 30-100 nm). Nanocomplexes should also include specific targeting molecules, antibodies specific to a region of interest. If endosomal escape is needed, it should have some chemical groups used for endosomal escape. Type of cell entry route should also be arranged by adding specific ligands for specific entry routes. Material to be released from the rest of the nanoagent in the cell should be linked to the nanoagent with appropriate chemicals which can be cleaved by remote actuation or local pH changes. There are several barriers for NPs to travel to the targeted destination when they are injected into the body. First encountered barriers are at the time of administration. There are several nanoparticle administration types: nanoparticles can be inhaled, can be given orally, or can be injected by intravenous, or intraperitoneal ways. The body has some sort of barriers for non-self things delivered by each of these routes, and mechanism of action depends on the tissue type. For pulmonary delivery, a major barrier is the size of the nanoparticles. For efficient delivery, NPs must have a certain size range to be able to penetrate alveolar cell layers. Since nanoparticles given orally encounter harsh conditions beginning from the mouth to gastrointestinal tract, they must have stability, especially for enzyme attacks and pH changes. The NPs administered intravenously also encounter many problems. Blood NANOBIOTECHNOLOGY

is a crowded environment, and cells of the immune system (e.g., macrophages, monocytes) survey the blood and eliminate non-self things. Opsonins (e.g., immunglobulins, some complement proteins) bind to nanoparticles in blood. These molecules are identified by cells of RES system, and phagocytosised. In addition, enzymatic degradation of some nanoparticles in blood is another problem. Therefore, prolonged circulation of nanoparticles is a major challenge and there are on-going studies to overcome this elimination from the circulation. Nanoparticles administered by intraperitoneal injection are also rapidly cleared via the lymphatic system. For intravenously administered NPs, there are some other barriers in RES system, namely organ level barriers. In liver and spleen, NPs larger than 100 nm are filtered and removed from the circulation. In addition, the kidney also has a filtration function: NPs smaller than 10 nm are filtered in in the glomerulus. Blood vessels are also barriers for proper penetration of nanoparticles. The gaps between endothelial cells are narrow restricting nanoparticle escape from the blood. The blood vessels in tumor tissue have enhanced permeability, and this phenomenon facilitates delivery of NPs to tumor tissue. Moreover, brain, the chief organ of the body, is highly protected and delivery of NPs to the brain is a huge challenge. The tight junctions between endothelial cells form the blood brain barrier and prevents passive access. Beside the organ level barriers, cellular level barriers also create problems for efficient delivery and these barriers are also challenging as organ level barriers. The cell membrane prevents diffusion of materials larger than 1kDa. There are certain endocytic entry mechanisms and each mechanism is related to different subcellular events or pathways determining the fate of nanoparticles in the cell. Size limits for each entry route are as follows: the large particles are internalized by phagocytosis, the particles larger than 1 μm are internalized by macropinocytosis, and the mean diameter of nanoparticles for caveolar-mediated endocytosis, clathrin-mediated endocytosis, and clathrin-independent caveolin-independent endocytosis are 60 nm, 120 nm, and 90 nm, reCHAPTER 10

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spectively. The NPs internalized via clathrin mediated endocytosis are destined for lysosomal compartments, whereas those internalized via a caveolin mediated process are not. Therefore, studies are ongoing for functionalization of NPs for endosomal escape not to encounter lysosomal degradation. For each internalization route, endosomal escape and targeting of nanoparticles to interested subcellular localizations are huge challenges and are being heavily studied. For example, in nuclear targeting, the NPs should be able to escape from endosome, escape from some sort of enzyme attacks and pass through nuclear pores.

4. Nanobiosensors A biosensor typically consists of a biological recognition element, a physical or chemical transducer for detection of biological molecules, and signal processing electronics (Figure 7). Biosensors can be classified according to the type of material to be analyzed and the signal transduction mechanisms employed. The main types are optical, electrochemical, calorimetric, and acoustic. These categories may also overlap. The main issues for efficiency of a typical biosensor are the limits of detection, sensitivity, and specificity. Since most of the biological structures are at the nanoscale, nanosizing a sensing element can improve the sensor performance. When it comes to categorization of nanobiosensors, we can set the criteria as the nature of nanomaterials used for sensing and detection. The NPs such as noble metal based NPs and carbon nanotubes (CNTs) are versatile nanostructures for optical and electrical applications and are heavily used for detection and sensing purposes.

platforms have been developed using aggregation induced color change of AuNPs. The small AuNP colloids (10-20 nm) have deep reddish color. Upon aggregation their absorption band shifts to longer wavelengths due to interparticle plasmon coupling, and color of the colloid turns into purple or blue. This color change can be observed by the naked eye. Figure 8 shows the color change of AuNPs as they form aggregates in their suspension with the addition of an analyte. In addition, due to AuNPs’ high extinction coefficients, limits of detection as low as the nanomolar level can be possible by just using a UV/Vis spectrophotometer. AuNP-based colorimetric assays provides simplicity and sensitivity, therefore they are widely used for quick detection of DNA and protein markers for cancer and other diseases. For example, in the pioneering work by Mirkin and co-workers single stranded DNA conjugated AuNPs used for the detection of nucleic acid hybridization. That study opened a way for DNA-AuNP interaction related studies including several for single nucleotide polymorphism (SNP) detection, determination of enzyme activities, and so on. AuNPs are also used in electrochemical sensors for the identification of glucose, xanthine, and hydrogen peroxide.

Figure 8. Color change of AuNP colloidal suspension as AuNPs form aggregates. Figure 7. Components of a nanobiosensor.

Among them, AuNPs have attracted interest of researchers due to their unique physical, optical, and catalytical properties. Many sensing and detection 168


Carbon nanotubes (CNTs) are the other widely used nanomaterial for biosensor development. They are classified according to their number of concentric cylindrical carbon layers. They can be either single walled (SWNTs) or multiwalled (MWNTs). SWNTs have been extensively em-

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ployed for biosensing applications due to their unique one-dimensional structure, distinct electrical and spectroscopic properties, rich chemical and robust mechanical properties. Up to now, they are used for DNA hybridization assays, detection of SNPs, DNA sequence detection, detection of small molecules such as adenosine 5’-triphosphate and glucose, reactive oxygen/nitrogen species, hydrogen peroxide sensing, nitric oxide sensing, and protein/ biomarker detection. Other than AuNP and CNT based nanobiosensors, nanowire based biosensors have also been developed. Silicon nanowires doped with boron have been used for the detection of biological and chemical species. ZnO nanowires coated with gold electrodes have been used for detection of hydrazine using amperometric responses. There are many other nanomaterials besides those mentioned here that have been used in biosensing applications. Due to limits of space, we have discussed only several of them to introduce the nanobiosensor concept. Readers may refer to other comprehensive reviews for additional information.

5. Self-Assembly During the past two decades, nanoparticles possessing different size and shape properties have been synthesized. However, their organization at the nanoscale remains a big challenge. Bringing the potential of nanobiotechnology to reality by creation of nanoparticle based tools and devices can be realized by making well-defined multi-dimensional nanostructures. Organization of nanoparticles can be achieved either using “top-down” or “bottomup” approaches. In the first one, lithography based methods are developed to pattern the NPs on surfaces. The latter approach is based on self-assembly. In nature, self-assembly is used to create complex biological structures such as cells and their components. Being inspired by nature, researchers seek novel ways to develop multidimensional nanostructures using atoms, molecules, and NPs for applications in nanoelectronics, sensing, catalysis, biodiagnostics and so forth. On the other hand, creation of these structures requires an in depth understanding about their chemical nature. As the size of particles NANOBIOTECHNOLOGY

get smaller, non-covalent interactions such as Van der Waals, electrostatic interactions, dipole-dipole interactions, and hydrogen bonding become more predominant factors and play important roles in the final shape of the assemblies. In addition to these factors, surface wettability at the interfaces, surface free energy are other key parameters. In order to manipulate NPs to create nanobiotechnological platforms and tools, one should know how to control all these parameters. In this section, the most used self-assembly approaches will be briefly discussed.

5.1 Evaporation Induced SelfAssembly Drying-mediated assembly of NPs is the simplest method of assembling them on surfaces. Effects of weak attractive forces between nanoparticles in solution become more dominant as the solvent evaporates, forcing nanoparticles to assemble. Mechanism of this phenomenon was shown first time by Denkov et al. Self-assembly of materials on surfaces via drying of a sessile drop of a colloidal suspension has been used for assembly of polystyrene latex particles, oligodeoxynucleotides, proteins and cells. While this assembly method is simple and no complex equipment or microfabrication steps are required, it has been shown that by varying parameters such as particle and surfactant concentration, solvent type, composition of the suspension, hydrophilicity/hydrophobicity of the surface, geometry of the surface, and temperature, it is possible to produce a variety of architectures. On the other hand, in order to use this technique efficiently, controllable distribution of particles in suspensions during drying is very important. However, it is hard to obtain desired patterns due to three convective mechanisms inside the drying droplets. First one is the radial outward flow, which carries particles toward the pinned wetting line resulting in a “coffee ring phenomenon”. The second mechanism, which affects the distribution of particles on surfaces after evaporative deposition, is Marangoni flow generated due to a surface tension gradient caused either by concentration gradient or by a temperature gradient. The third mechanism involves DLVO interCHAPTER 10

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actions which include the force between charged surfaces interacting through a liquid medium. By manipulating these three convective mechanisms inside the droplet, a variety of nanostructures can be formed. In addition, additional external fields (e.g., magnetic, electric, flow) during drying of the droplet can be used to direct the assembly of nanoparticles in order to produce platforms for detection and sensing. In a recent study, detection of a peptide mimic of the malaria biomarker pf HRPII using coffee ring phenomenon was shown as a model for disease diagnosis. In the absence of biomarkers, both green fluorescent polystyrene particles and red fluorescent polystyrene particles migrate to the edge resulting in a yellow colored ring. On the other hand, in the presence of biomarker, green particles assemble with magnetic iron oxide nanoparticles, and with the help of a magnet at the center of the droplet, this assembly creates a green spot at the center. The ring color also changes to yellow. As a result, a detection limit of 200-300 nM was successfully attained.

5.2 Programmed Self-Assembly Since DNA has intrinsic self-assembly ability, patterned multidimensional surfaces and nanostructures have been developed using hybridization of nucleotide sequences. Creation of DNA origami structures has a wide range of applications such as building blocks of higher organization self-assembled structures, nanorobotics, and drug delivery systems. These DNA assembly motifs are a most versatile method for creating structures of varying complexity and design. They can be used for assembly of plasmonic particles into different structures with useful optical properties.

5.3 Geometry (Shape) Driven SelfAssembly Anisotropic, non-spherical nanostructures with different shapes can be created using facet selective interactions. By differential chemical modifications of the facets of nanoparticles, they can assemble in different dimensions. 170


6. Conclusions Nanobiotechnology is an evolving interdisciplinary field with many possible applications in several fields. The diversity of the NMs and complexity of the living world also brings challenges and opportunities. The impact of nanotechnology has already been felt and many nanotechnology based products are already in our daily lives. Although this field provides many alternative solutions to the challenges human being faced, the introduction of novel materials with unique properties also brings new problems to deal with. At the current stage, the NPs with known properties and biocompatibility are interfaced with living systems. Due to their initial promising results with some real life applications, SPIONs, AuNPs and QDs are included in this chapter. As the level of understanding of their behavior in biological systems is increased, more effective tools and devices from this emerging field will be seen. At the moment, there is a significant effort for the development of multifunctional NPs through surface modifications for the targeting, delivery and imaging applications. There is also a great concern about the safety of these novel materials and a new field called nanotoxicology investigating the safety of these materials has already been emerging. Although this chapter mostly covers the nanomaterials and their applications in medicine, it is important to note that there are many other applications and issues related to the use of these novel materials. One of them is the development of nanobiosensors in order to obtain enhanced sensitivity and specificity. Many types of nanobiosensors have been proposed recently, and with progress in nanobiotechnology field, the major aim is the production of highly sensitive, lowcost, high-throughput, and multiplexed sensing devices. There is a long way to go but when the recent enormous advancement in the field within a decade is considered, it is highly possible that more functionalized and efficient nanobiosensors for detection of diseases and pathogens will be produced in the near future. In addition, self-assembly of nanomaterials and biological molecules opens up new ways to develop more complex nanoarchitectures

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and progressing to mimic nature’s way to produce biocompatible nanomaterials and nanostructures for drug delivery, sensing and screening devices, and theranostics. As the complex interactions of molecules and nanomaterials at atomic level are revealed, well-defined novel assembled nanostructures will be produced in the near future.

Review Questions and Answers Q1. What are the properties of nanomaterials to be used in medical applications? What are the requirements to adopt NPs in a specific action? A1. The NPs used in medical applications must be biocompatible and stable in biological systems. The multiple functions of NPs can be adapted for specific action through their size, shape and surface chemistry. The specific modifications with specific targeting moieties, therapeutic molecules or contrast agents via direct conjugations, linker chemistry or physical interactions methods without disturbing the functions of the agents can be achieved. The selection of the targeting agent placed onto the NPs is based on the changes in the diseased area of the body such as reactive oxygen species (ROS), temperature or a specific receptor, hypoxia, acidic or basic conditions. Q2. What are the advantages of QDs and AuNPs over fluorescent dyes? A2. The QDs are strongly bright providing high quantum yield and photo-stable nanomaterials. The QDs have broader absorption spectra providing a clear separation between excitation and emission wavelengths, which allows multiple detection or imaging compared to organic dyes. The emission wavelength of the QDs can be adjusted by changing their size while using the same excitation wavelength. The AuNPs also have advantages over fluorescent dyes. For example, the scattered light from one AuNP is 106 times greater than the photons scattered from a fluorescence dye. The color of scattered light and the intensity can be adjusted by changing the size and shape of the AuNPs. Q3. What limits QDs to be used in medical applications? What should be the strategies to make QDs biocompatible in medical applicaNANOBIOTECHNOLOGY

tions? What should be taken into consideration during this process? A3. The toxic effect through hydrophobic surfaces and leakage of cadmium ions Cd2+ from the nanoparticle limits the applications of the QDs in biology and medicine. The surface modifications strategies should be used to increase their biocompatibility, prevent release of toxic ions and increase the dispersibility in aqueous media. The hydrophobic surface of the QDs can be tuned into hydrophilic by using two different strategies. In the first strategy, the affinity of hydrophilic thiol (-SH) containing heterobifunctional molecules such as mercapto silanes or mercaptoacetic acid, onto QDs surface can be used to exchange with hydrophobic TOPO or HAD ligands. The other approach is the adsorption of an amphiphilic polymer onto the QDs, which contain a hydrophobic side, interacting with hydrophobic surface of the QDs and a hydrophilic part, providing water solubility. During modifications, the important factor is to keep the QDs properties intact. For example, the modification can cause particle aggregation and decrease in the quantum yield or complete loss of fluorescence. In addition, modification with an unstable modifier can lead to a naked QD-surface, which causes the release of toxic QD-elements ions to the environment. Q4. How are AuNPs modified and how can you prove the modification? A4. The AuNPs can be modified with a molecule possessing a thiol group due to the affinity of thiol (-SH) to the noble metal surfaces. Further modification can be pursued through –OH, -COOH or -NH2 on the thiol-containing molecules. The modification can be monitored from their UV/Vis spectra. As the particle size gets larger, surface plasmon resonance absorption maximum shifts to the longer wavelengths. Q5. What makes SPIONs valuable in biomedical applications? A5. The magnetic properties and ease of surface modifications with targeting moieties make SPION useful in targeted delivery and real time monitoring of specific tissue to observe the treatment response. CHAPTER 10

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Q6. What are the general parameters for the preparation of nanocarriers with efficient delivery and release? A6. First of all, nanocarrier should be biocompatible and have stability in blood circulation. It should have minimum toxicity. In addition, its size and shape should be suitable (e.g. should not be filtered by kidney and spleen easily). Surface chemistry should be wisely designed in order to escape from RES system and endosomal uptake. It should possess suitable chemistry to release its cargo only at the targeted site. Q7. What can be the goals of designing biosensors at nanometer scale? A7. The major issues for biosensors are to be able to lower the limit of detection, and to attain enhanced sensitivity, and specificity. Production of nanobiosensors using nanomaterials can enable us to realize these goals. Q8. What is the “self-assembly” concept in nanobiotechnology? What may it offer us? A8. Cells and living organisms in nature are formed by self-assembly of molecules in most intelligent and elegant ways. Therefore, during production of nanomaterials and nanostructures, it would be wise to follow nature’s footprints. Besides, it also offers us to have in depth understanding of chemical nature of nanomaterials. During last two decades, researchers have made enormous progress, but there are many mysteries to solve.

Further Readings 1. Berg P, Baltimore D, Boyer HW, et al,. Potential Biohazards of Recombinant DNA Molecules. Science 1974;185: 303. 2. National Center for Biotechnology Information. National Center for Biotechnology Information. http:// www.ncbi.nlm.nih.gov. Accessed April 13, 2014. 3. Cruz-Coke R. Ethical principles in human scientific research. Revista Médica de Chile 1994; 122:819-24. 4. Rinčić I, Amir MFJ. The Invention of Bioethics and Beyond. Perspectives in Biology and Medicine 2011;54:550–56. 5. Selgelid MJ, ‘Moderate Eugenics and Human En172


hancement. Medicine, Health Care and Philosophy 2014;17:3-12 6. Snežana B. The Declaration of Helsinki: The Cornerstone of Research Ethics. Archive of Oncology 2001; 9:179–84. 7. ELSI Research Program. ELSI Planning and Evaluation History. http://www.genome. gov/10001754.%20Accessed%20April%207,%2 2014. 8. World Health Organisation. 20 questions on genetically modified foods. http://www.who.int/ mediacentre/news/notes/np5/en/ 9. Atanasova I, Terziivanov D. Advanced molecular therapies of the 21st century I. Recombinant drug products – proteins and vaccines. Journal of Clinical Medicine 2010;3:9-18. 10. Groskreutz DJ, Sliwkowski MX, Gorman CM. Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin. Journal of Clinical Medicine 1994;269:6241–6245. 11. Ethical Futures: Bioscience and Food Horizons: EurSafe 2009. Nottingham, United Kingdom: European Society for Agricultural and Food Ethics. United States Congress 2009;445. 12. European Science Foundation. Marine Biotechnology: A New Vision and Strategy for Europe. http://www.marinebiotech.eu/sites/marinebiotech.eu/files/public/library/MBT%20publications/2010%20ESF%20Position%20Paper.pdf 13. Richmond RH. Environmental protection: applying the precautionary principle and proactive regulation to biotechnology. Trends in Biotechnology 2008;26:460– 467. 14. Leary D, Vierros M, Hamon G, et al,. Marine genetic resources: a review of scientific and commercial interest. Marine Policy 2009;33;183–194. 15. Danciu, A. The implications of the biotechnology for bioterrorism. UASVM Agriculture 2011;68:513- 517. 16. The Convention on Biological Diversity. What is the Convention?. (http://www.cbd.int/) 17. Gallant D. Biopiracy and bioprospection: a new terminology for an old problem. (http://www. scopemed.org/?mno=152460 ) 18. Caulfield T, Bubela T, Murdoch CJ. Myriad and the mass media: the covering of a gene patent contro-versy. Genetics in Medicine 2007; 9:850–855.

PRINCIPLES OF TISSUE ENGINEERING

CHAPTER 11 PRINCIPLES OF TISSUE ENGINEERING Lubos Danisovic


1. Stem Cells ..................................................................................................................... 175

1.1 Brief History of Stem Cell Research ............................................................................ 175

1.2 Definition and Basic Biological Properties of Stem Cells ............................................. 176

1.3 Types of Stem Cells ..................................................................................................... 177

1.3.1 Embryonic Stem Cells ........................................................................................ 177

1.3.2 Primordial Stem Cells ......................................................................................... 178

1.3.3 Induced Pluripotent Stem Cells .......................................................................... 178

1.3.4 Foetal Stem Cells ................................................................................................ 178

1.3.5 Adult Stem Cells ................................................................................................ 179

2. Bioactive Molecules ...................................................................................................... 180

2.1 Hormones ................................................................................................................... 180 2.2 Cytokines .................................................................................................................... 181

2.3 Growth Factors ........................................................................................................... 181

3. Scaffolds ....................................................................................................................... 182

3.1 Natural Polymers for Scaffold Fabrication ................................................................... 182

3.2 Synthetic Polymers for Scaffold Fabrication ................................................................ 183

4. Conclusions ................................................................................................................. 184

Acknowledgements ................................................................................................................ 184 Summary Box ........................................................................................................................ 184 Review Questions and Answers .............................................................................................. 184 Further Readings ................................................................................................................... 185 PRINCIPLES OF TISSUE ENGINEERING

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Summary

R

ecently, despite the current medical advances, there are still many pathological conditions due to diseases, injuries or congenital anomalies which cannot be healed by the conventional therapeutic approaches. In many cases, these conditions may lead into significant reduction in the quality of life and to social isolation of the affected individuals. Moreover, in the extreme cases it may cause a total collapse of the individual leading to death. For that reason clinicians and researchers are forced to find appropriate alternative methods to solve this problem. Promising approaches are provided by tissue engineering. Tissue engineering is multidisciplinary field which applies the basic principles of cell biology and materials technology to replace, repair, and/or regenerate the damaged or lost tissues and organs (Figure 1).

large scale of the subject matter, the present chapter is devoted only to the dissemination of fundamental principles of tissue engineering. Three main topics are considered and form the basic frame of the text; stem cells, bioactive signal molecules and scaffolds (substitutes for extracellular matrix).

Figure 2. Basic concept of tissue engineering.

Fabrication of artificial tissues for clinical application starts with the procurement of tissue samples necessary for cell isolation and expansion in vitro to obtain a sufficient quantity of cells. Obtained cells are usually cultured within various types of scaffolds in bioreactors with the addition of specific growth factors. Prepared artificial tissues have to be carefully tested for pathological events and potential microbiological contamination before application in human medicine.

1. Stem Cells Figure 1. The “triad” of the tissue engineering.

Tissue engineering belongs to the fastest developing fields of biomedical science. From its introduction in the 1990s, a plenty of artificial tissues and organs were produced, including urinary bladder, urethra, skin, cartilage, bones, vessels, heart valves, etc. Many of them are recently in different stages of preclinical and clinical testing. However, further investigations have to be carried out for safe utilization of tissue engineering products in practical clinical applications (Figure 2). Moreover, it will be necessary to resolve many ethical issues. Due to the PRINCIPLES OF TISSUE ENGINEERING

1.1 Brief History of Stem Cell Research Stem cell research has its roots in the 19th century, when Robert Remak claimed that new cell arises only from other already existing cell by cell division. This claim was later adopted by Rudolf Virchow who published it as a part of the cell theory (Omnis cellula e cellula). At the beginning of 20th century, in 1909, Alexander Alexandrowitsch Maximow developed and introduced a unitarian theory of hematopoiesis. CHAPTER 11

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He stated that all blood cells develop from a common precursor cell and therefore he is considered the discoverer of hematopoietic stem cell. Later, in 1917, Artur Pappenheim published his assumption about the existence of undifferentiated cells, which he called “gemeinsame Stammzelle”. Moreover, he postulated that haematopoietic stem cells have the potential for treatment of diseases. Other important experiments on stem cells were performed by Ernest A. McCulloch and James E. Till in the 1960s who studied the radiation sensitivity of bone marrow progenitor cells. They transplanted bone marrow cells into irradiated mouse. Afterwards, they observed macroscopic splenic colonies, and speculated that each nodule arose from a single stem cell. In later work, in collaboration with Lou Siminovitch, they provided evidence of stem cells self-renewal. In 1976, Owen and Friedenstein experimentally demonstrated that the cells isolated from the bone marrow have the ability to produce fibroblast colonies. Moreover, they supposed that stromal cells are capable of generating progenitors of adipocytes and osteocytes. It was later confirmed by Keating and colleagues in 1982. In 1978, haematopoietic stem cells were isolated from human umbilical cord blood. An important event was the isolation of embryonic stem cells from mouse embryoblast in 1981. Later, in 1998, James Thompson and co-workers established the first human embryonic stem cell line. In the same year, John Gearhart isolated stem cells from foetal tissue – primordial stem cells. Since the beginning of the 21st century, many research papers focused on the analysis of biological properties of stem cells were published and began their gradual transfer into clinical practice.In 2006, the first induced pluripotent stem cells were produced from somatic cells of mouse. One year later two independent groups generated the first human induced pluripotent stem cells by reprogramming mature fibroblasts.

1.2 Definition and Basic Biological Properties of Stem Cells Stem cells are generally characterized as basic, primitive undifferentiated cells which occur in all mul176


ticellular organisms during all stages of ontogenesis. They possess the unique potential of long term self-renewing (being able to reproduce itself ) and capability of differentiation into specialized cells of the body, so-called plasticity. Both of these characteristics are also referred to as “stemness”. Due to these properties, stem cells perform important roles during embryonic development and in the mature organism they provide a supply of cells necessary for regeneration, growth and repair of terminally differentiated cells. Long term self-renewing is attributed to two types of cell division. Stem cells can reproduce symmetrically and asymmetrically (Figure 3). The balance between both divisions determines appropriate the quantity of stem cells and differentiated daughter cells. During a symmetric division, both daughter cells gain the same fate – both of them are undifferentiated or differentiated. Asymmetric cell division produces one copy of a stem cell, which remains undifferentiated and a second cell that is intended to terminal differentiation.

Figure 3. Symmetric and asymmetric cell division of stem cells.

Stem cell division is controlled by intrinsic and extrinsic factors. Cell components, including cell polarity regulators, cell fate determinants and orientation of the mitotic spindle belong to intrinsic factors. The asymmetric position of two cells and specific growth factors within a stem cells niche belong to extrinsic factors. Genomic analysis showed that genes oct-4, sox-2 and rex-1 as well as transcription factors, including leukaemia inhibitory factor (LIF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) are essential for maintaining the undifferentiated state. Plasticity is another defining characteristic of stem cells. It means the ability of tissue-specific stem cell to differentiate into cells of other tissues (Figure 4). This potential is determined by its potency.


1.3 Types of Stem Cells 1.3.1 Embryonic Stem Cells

Figure 4. Plasticity of stem cells.

Totipotent stem cells (zygote and blastomeres) have the ability to form an entire organism. They give rise to all embryonic and extra-embryonic structures. Pluripotent stem cells (embryonic stem cells derived from the inner cell mass of the blastocyst) are able to generate cells of all three embryonic layers (endoderm, mesoderm and ectoderm). On the other hand, they are not capable of producing cells of extra-embryonic structures, which are essential for the development of the entire organism. Multipotent stem cells (adult/somatic stem cells) have the potential to form multiple cell types. They are capable of differentiation into specialized cells of cartilage, bone, fat etc. Oligopotent stem cells also termed as progenitor cells can differentiate into only a few cell types. Examples of oligopotent stem cells are the lymphoid or myeloid stem cells. Unipotent stem cells so called precursors are able to form cells from a single lineage, for example spermatogonial stem cells. The stimulus for differentiation is usually specific differentiation (growth) factor, for example transforming growth factor β (TGF-β), bone morphogenetic protein (BMP), insulin like growth factor (IGF) etc. The differentiation is also influenced by various biologically active molecules and chemical elements, such as insulin, valproic acid, beta mercaptoethanol, izobutylmetylxantine, ascorbic acid, transferrin, selenium etc. Behaviour of stem cells is also affected by contact with other cells and extracellular matrix copmonents. PRINCIPLES OF TISSUE ENGINEERING

Embryonic stem cells are isolated from the inner mass of cells of embryoblast (Figure 5). They are pluripotent and give rise to all fetal and adult cell types of all three germ layer. On the other hand, they are not capable of producing cells of the extra-embryonic structures, which are essential for proper development of the individual. Another important feature of the embryonic stem cells is increased telomerase activity. Human embryonic stem cells are characterized by the expression of some specific transcription factors and surface markers. The most important are Oct4, nanog and Sox2. They also express glycolipids SSEA3 SSEA4 as well as keratan sulfate antigens Tra-1-60, Tra-1-81, GCTM2 and GCT343. Moreover they are positive for CD9 and CD90; and produce tissue-nonspecific alkaline phosphatase. In addition, embryonic stem cells may produce teratomas and chimeras after application in vivo.

Figure 5. Isolation and in vitro culture of embryonic stem cells.

There are several methods of isolation and in vitro expansion of embryonic stem cells. The first one was developed by Martin Evans and Matthew Kaufman in 1981. They isolated embryonic stem cells from mice. This method includes progesterone treatment which leads into the embryo implantation delay and allowing the inner cell mass to increase. After 4–6 days, early embryos were harvested and grown under defined in vitro conditions until the CHAPTER 11

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inner cell mass forms “egg cylinder-like structures” which were dissociated into single cells. They were cultured on the feeder layer of mitomycin-c treated fibroblasts with the presence of LIF. In the same year, Gail Martin independently introduced different method of embryonic stem cells isolation. She obtained embryos approximately 76 hours after fertilization and cultured them overnight in a medium supplemented with serum. Next day, the inner cell mass was extracted and were cultured on mitomycin-c treated fibroblasts to form colonies. Embryonic stem cells obtained by these two methods were pluripotent and posses ability to form teratomas and embryoid bodies. In 1998, the first human embryonic stem cells were isolated and propagated in vitro by James A. Thomson and co-workers. They used donors’ fresh or frozen cleavage stage human embryos, produced by in vitro fertilization for clinical purposes. Embryos were cultured to the blastocyst stage. Cells from embryoblast were obtained and propagated in vitro on mouse embryonic fibroblast feeder layer in presence of FGF-2. Despite the broadest differentiation potential of ESCs, their experimental and therapeutic use is restricted in many countries due to ethical considerations.

1.3.2 Primordial Stem Cells Primordial (germinal) stem cells are undifferentiated cells which represent precursors of gametes. In humans, they arise from the basis of yolk sac during the third week of embryonic development and gradually migrate along dorsal mesogastrium of the primitive gut to the genital ridge. During this process primordial stem cells undergo extensive nuclear reprogramming to regain differentiation totipotency and reset genomic imprinting. Finally, they start to differentiate into oogonia and spermatogonia. Primordial stem cells are pluripotent and share expression of several markers with embryonic stem cells, including Oct-4, nanog, SSEA-1 and SSEA3. Moreover, they produce tissue-nonspecific alkaline phosphatase and possess increased telomerase activity. 178


1.3.3 Induced Pluripotent Stem Cells Induced pluripotent stem cells (IPSCs) are produced from differentiated somatic cells by reprogramming into pluripotent state by ectopically expressing a combination of several transcription factors, which are present in embryonic stem cells. They are capable of forming teratomas which can develop into all three germ layers. Induced pluripotent stem cells are familiar with embryonic stem cells. It was proven that they express similar surface antigens as well as they have the same cell cycle and kinetics of proliferation. The first induced pluripotent stem cells were produced from mouse in 2006 and from human cells in 2007. Until now, induced pluripotent stem cells were obtained by reprogramming of fibroblasts, hepatocytes, exfoliated renal epithelial cells etc. The pluripotency is often achieved by transfection of certain stem cell-associated genes into somatic cells. Viral vectors are used for transfer of selected genes, including Oct-3/4, Sox2, c-Myc and Klf4. Induced pluripotent stem cells have the potential to be used as a substitute of ethically controversial embryonic stem cells. A great advantage is also the possibility to produce patient-specific pluripotent stem cells for transplantation therapy without immune rejection. However, clinical utilization still has several restrictions, including retroviral vectors, possibility of activation of proto-oncogens and low induction efficiency. Therefore, the scientists are forced to find new reprogramming techniques, for example alternative vectors (adenoviruses, plasmids, transposons), usage of micro RNA or molecules able to mimic the effect of transcription factors. At the moment, once these limitations have been solved, induced pluripotent stem cells will become a universal tool for personalized regenerative medicine.

1.3.4 Foetal Stem Cells Foetal stem cells can be isolated from all foetal tissues. Their differentiation potential and other biological characteristics are similar to adult stem cells (see section “Adult stem cells”).


1.3.5 Adult Stem Cells Adult stem cells are undifferentiated cells that are found in the tissues of multicellular organisms throughout their lives. The first adult stem cells were discovered 52 years ago. They were isolated from bone marrow and gave rise to all blood cells. These stem cells were termed as haematopoietic stem cells. The second cell population was also discovered in bone marrow. It was a highly heterogenic population of stem cells capable of chondrogenic, osteogenic and adipogenic differentiation. They were named as stromal stem cells. After these discoveries adult stem cells were isolated from many different tissues including cord blood, adipose tissue, skin, dental pulp, pancreas, liver etc. They were termed as mesenchymal or somatic stem cells. Adult stem cells, like other stem cells are undifferentiated cells with ability of self-renewing and plasticity. The main task of adult stem cells is the participation in regeneration processes or in replacement of worn-out cells. In adult tissues they represent a very rare population of “dormant” cells (1 stem cell/10–15 000 differentiated cells). When the tissue is aged adult stem cells are activated; they undergo cell divisions and they actively migrate to damaged tissues to promote regenerative processess. Due to these properties adult stem cells represent a unique tool of modern medicine. Furthermore, adult stem cells have several advantages over other stem cells. The major advantage is a relatively simple method of isolation and subsequent cultivation in vitro. When compared with embryonic stem cells they are more acceptable from an ethical and religious point of view. Despite current knowledge, it is hard to determine the exact phenotype of adult stem cells. After isolation and adaptation to in vitro conditions they have typical morphology of fibroblast like cell (Figure 6), which may vary depending on the culture conditions. Therefore, it is necessary to combine more of their characteristics.


Figure 6. Fibroblast-like morphology of adult stem cells isolated from bone marrow stained by Giemsa (400x).

In pioneering work, characterization of stem cells isolated from the bone marrow of rodents was based on their ability to adhere to the bottom of culture vessel and to produce CFU-F (colony forming unit-fibroblast) after their seeding at high dilutions. They were named as mesenchymal progenitor cells or mesenchymal stem cells. Similar cells have been isolated from bone marrow of humans, monkeys, dogs, cats, sheep, and rabbits. Since then, their multilineage potential has been proven. Recently, stem cells have also been characterized according to expression of specific surface antigens. The most explored is Stro-1. The cells positive for Stro-1 are capable of producing CFU-F and have multilineage potential. On other hand, Stro-1 antigen is not expressed only by MSCs (mesenchymal stem cells). Moreover, its expression decreases with increasing passage. According to the International Society for Cytotherapy, MSCs must express the surface antigens CD73, CD90 and CD105, and they have to be negative for CD14, CD34 and CD45, which are typical for hematopoietic and endothelial stem cells. Moreover, MSCs have to be capable of differentiating into at least three lines – chondrogenic, osteogenic and adipogenic. In addition to the above mentioned markers, some spermatogonial stem cells (SSCs) express CD13, CD29, CD44, CD59, CD106, CD133, CD140b, CHAPTER 11

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CD166 and CD271. Most of the MSCs are also positive for HLA-A, B, C on the other hand they are negative for HLA-DR. Table 1 shows some phenotypic characteristics of adult stem cells isolated from different sources. Surface antigens Source +

-

Bone marrow

CD13, CD29, CD44, CD73, CD90, CD105, CD106, CD166, CD271

CD14, CD31, CD34, CD45

Mobilized peripheral blood

CD90, CD105, CD133

CD14, CD34, CD45

Cord blood

CD13, CD29, CD44, CD73, CD90, CD95, CD105, CD133, CD166

CD14, CD34, CD45

Wharton’s jelly

CD29, CD44, CD51, CD105

CD34, CD45

Placenta

CD9, CD13, CD44, CD63, CD73, CD90, CD105, CD349

CD31, CD34, CD45

CD44, CD73, CD90, CD105, CD140b

CD34, CD45

Endometrium

CD14, CD31, CD45

Skeletal muscle

CD29, CD44, CD59, CD73, CD90, CD105, CD106

CD14, CD34, CD45

Skin

CD29, CD44, CD71, CD73, CD90, CD105, CD166

CD14, CD31, CD34, CD45, CD133

Pancreas

CD29, CD44, CD51, CD58, CD81, CD90

CD7, CD34, CD45

Dental pulp

CD29, CD44, CD106

CD14, CD34, CD45

Periosteum

CD9, CD44, CD90, CD105, CD166

CD14, CD34, CD45

Table 1. Different tissue sources of adult stem cells and their phenotypic properties.

2. Bioactive Molecules Bioactive molecules are an important pillar of tissue engineering. They represent heterogenic group CHAPTER 11 / L. Danisovic

Figure 7. The most common way of the cell signalling.

CD13, CD29, CD44, Adipose tissue CD73, CD90, CD105, CD146, CD166

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of molecules with ability of controlling and regulating each event within organism to maintain its homeostasis and integrity. Their action is realized on basis of cascade reactions and feedback. The most common way of the cell signalling is demonstrated Figure 7. It involves several event, including releasing the signal molecule, transport, perception of the signal by receptor, transmission into the cell, cell signalling cascade, arriving of the signal to the final destination in the cell and response by the cell.

Cells may communicate with each other by different ways. Many authors have classified this according to the distance between the signalling cell and the target cell as autocrine, paracrine, endocrine and neurocrine communication. The transmission of signalling molecule is provided by extracellular fluids (e.g. plasma, blood) or by changes in the electrical potential across the plasma membrane of neurons. In the respect of the tissue engineering most interesting are extracellular signalling molecules, such as hormones, cytokines and growth factors. Each of them effects the proliferation, migration and differentiation of cell.

2.1 Hormones Hormones are generally characterized as molecules secreted by endocrine glands, cells and tissues directly to the blood stream. They are transported to target cell with specific receptor to provoke specific response. They are divided into several classes:


small water soluble molecules, peptide hormones and lipophilic molecules. Peptide hormones have the highest impact for tissue engineering. For example insulin, which is produced by β cells of the islets of Langerhans of pancreas, increases the cell proliferation, amino acid uptake and protein synthesis. Moreover, insulin in low concentration promotes chondrogenic differentiation of stem cells. More recently, it was shown that it has synergic effect with TGF-β1 and BMP-2. Another hormone in this class is follicle stimulating hormone (FSH) and it also influences the behaviour of various cells in vitro. FSH has the ability to induce proliferation of primordial stem cells after cryopreservation. Its anti-apoptotic effect in some cancer cells was also shown. Hydrocortisone belongs to lipophilic molecules which are also present in fetal bovine serum (cell culture supplement). Hydrocortisone promotes cell attachment, effects cell proliferation and differentiation. On the other hand, in higher concentration it has inhibitive effect.

2.2 Cytokines Cytokines represent a group of peptide molecules which have insightful action on cells. They are produced by many cell types (e.g. lymphocytes, monocytes) and affect other cells within short distance. Their main function is coordination of the immune responses. Some of them are pro-inflammatory; they are necessary for initiation of inflammatory response to recruit granulocytes, and later on, lymphocytes, to fight disease. Others are anti-inflammatory and serve to reduce inflammation and promote regeneration. They are generally divided into several groups: interleukins, interferons, tumour necrosis factors and chemokines. Interleukins represent a large family of bioactive molecules, which have 35 members. Except regulation of immune responses they have a variety of functions, including controlling cell growth and differentiation. Interferons are proteins which are released as a response to the presence of pathogens such as viruses, bacteria, parasites or tumour cells. PRINCIPLES OF TISSUE ENGINEERING

They allow communication between cells to trigger the protective effect of the immune system that eradicates pathogens or tumours. Moreover they affect cell proliferation, differentiation and promote apoptosis, both in vivo and in vitro. Tumour necrosis factors represent a family of bioactive peptides, which have 19 members. They are involved in process of cell death. Moreover, they stimulate cell proliferation and induce cell differentiation. Chemokines represent a family of small chemotactic cytokines. Some chemokines are considered pro-inflammatory and can be induced during an immune response to recruit immunocompetent cells to infected tissue. Others are involved in controlling cell migration, including stem cells during embryogenesis and regeneration.

2.3 Growth Factors Growth factors (GF) represent a group of biologically active polypeptides produced by the body, which can stimulate the cell proliferation, survival and differentiation. They are involved in the regulation of homeostasis and integrity, as well as in development of organism. They may act alone, but in many cases they have synergic and additive effects when operate together. Recently, over 50 known proteins which have growth-like factor effect have been reported. There are many families, but in respect to the tissue engineering, TGF-β, FGF (Fibroblast FG), EGF (Epidermal GF), IGF (Insulin-like GF) and PDGF (Platelet-derived GF) seem to be most important. Members of TGF -β superfamily secreted by many cell types play a pivotal role in cell proliferation, apoptosis and differentiation. Moreover, they are involved in the regulation of the cell cycle and immune system. BMP (BMP2 through BMP7) belong to this superfamily and they have a significant action in the chondrogenic and osteogenic differentiation. They are also involved in prenatal development and postanal growth of many tissues. Members of FGF family are heparin-binding polypeptides which are employed in the various cellular events, such as proliferation, differentiation and CHAPTER 11

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motility. They also play a very important role during embryogenesis, angiogenesis and wound healing. Moreover, some of them have been considered proto-oncogenes. The members of EGF family belong to potent growth factors that stimulate proliferation and migration of epidermal and epithelial cells. They are also necessary for appropriate wound healing. The members of IGF family are mitogenic polypeptides similar to insulin that stimulate the proliferation, differentiation and survival of various cell types. They have also a significant anabolic effect. The members of PDGF family represent proteins which are synthesized by platelets and are involved in the process of embryogenesis, cell proliferation and migration. They play a significant role in angiogenesis as well as in the growth of blood vessels from already-existing blood vessel tissue.

3. Scaffolds Substitutes of extracellular matrix (scaffolds) belong to key components for tissue engineering. They are produced from natural or synthetic biomaterials (Table 2) and may be in the form of hydrogels, sponges, fibrous meshes and nanofibers. Materials for scaffolding have to be non-toxic, sterile, biodegradable and biocompatible with cells. The structure of the surface, pore size, porosity and structural strength are also important characteristics, which influence their final utilization. These scaffolds do not have only a mechanical function, but also support cell attachment, migration, proliferation and differentiation for expression of desirable phenotypes. Recent technologies allow various modifications (e.g. cross linking, enzymatic changes, adjustment of biodegradability, etc.) of scaffolds into appropriate form in respect to potential utilization. Moreover, it is possible to prepare smart scaffolds with bioactive substances (e.g. antibiotics, growth factors) bind to their structure. These substances may release from the scaffolds to influence cultured cells. Biodegradable polymers seem to be the most appropriate materials for scaffolding, because their 182


interactions with cultured cells lead to incremental biological degradation. After their application, there is no need to undergo other surgery to remove foreign material from the patient’s body. Natural

Synthetic

Agarose

Poly (2-hydroxyethyl methacrylate)

Alginate

Poly (a-hydroxy esters)

Arabinogallactan

Poly (dimethylsiloxane)

Cellulose

Poly (ethylene glycol/oxide)

Collagen

Poly (glycolic acid)

Dextran

Poly (lactic acid)

Chitosan Chondroitin sulphate Elastin

Poly (lactic-co-glycolic acid)

Fibrin

Poly (propylene oxide) – Pluronics®

Gelatin

Poly (styrene)

Hyaluronic acid

Poly (tetrafluoroethylene) – Teflon®

Silk fibroin

Poly (urethane)

Starch

Poly (vinyl alcohol)

Poly (methyl methacrylate) Poly (propylene fumarate)

Table 2. Overview of natural and synthetic materials used in the preparation of extracellular matrix substitutes.

3.1 Natural Polymers for Scaffold Fabrication Natural polymers are obtained from different sources, including micro-organisms, fungi, plants and animals. These materials possess a large variety of structures and diverse physiological conditions. Moreover, they contain bioactive molecules which increase cell attachment and effect their proliferation and differentiation. Therefore they have significant potential for tissue engineering and regenerative medicine. There are two main groups of natural polymers used for the scaffold fabrication – polypeptides and polysaccharides. Both of them are biocompatible and biodegradable. Moreover, it is possible to set up and control their characteristics by relatively simple chemical methods. The most important polypeptides utilized in tissue engineering are collagen, elastin and silk. Collagen


is major physiological component of mammalian tissues and is main component of extracellular matrix. Currently, 28 different types of collagen have been identified. Collagen is composed of three polypeptide chains that form structure of triple helix. The chains are connected each other through hydrogen bonds. Moreover, collagen contains adhesion domains which are necessary for cell attachment. Collagen is enzymatically biodegradable and has tendency to degrade quickly. This problem can be overlapped by chemical cross-linking. The problem of collagen antigenicity was also solved by enzymatic removal of telopeptides from its molecule or by chemical cross-linking, so it is safe for producing various scaffolds (Figure 8) to prepare artificial skin, cartilage and bone.

Figure 8. Example of collagen-based scaffold.

Elastin is important structural component of extracellular matrix of connective tissues and is essential for stretching and relaxing. Elastin consists of water soluble tropoelastins which are cross-linked with lysine to make a massive insoluble, durable cross-linked array. Recently, we are able to produce well-defined elastin by recombinant technologies. These elastin-like proteins have unique properties that make them attractive for tissue engineering of vascular tissues and cartilage. Silks are fibers produced by spiders and silkworms. They are composed of repetitive sequences of glycine-rich proteins. The secondary structure of silks is β-sheet that results in enormous mechanical strength. Therefore it is suitable for tissue engineering of skin and tendon.


The important polysaccharides utilized in tissue engineering are agarose, chitosan and hyaluronic acid. Agarose is polysaccharide extracted from algae. It is water soluble polymer which forms thermally reversible gel. The basic properties of agarose gel, including strength and permeability can be adjusted by changing the concentration of agarose. Agarose gels are studied mainly in respect to cartilage tissue engineering. Chitosan is derivate of chitin that occurs in the exoskeleton of crustaceans and insects. It is linear biodegradable polysaccharide. The properties of chitosan may be adjusted with changing the pH of solutes to form insoluble scaffolds. Chitosan was used as a scaffold (Figure 9) for cultivation of many types of cells, including fibroblast, hepatocytes, chondrocytes, etc.

Figure 9 Example of chitosan-based scaffold.

Hyaluronic acid is abundant in the extracellular matrix of many tissues. It is a biodegradable polysaccharide with proper characteristics, including biocompatibility and low immunogenity. Hyaluronic acid has been used for osteochondral repair in form of gels and sponges.

3.2 Synthetic Polymers for Scaffold Fabrication Besides the above-mentioned natural biopolymers, a variety of synthetic polymers may be utilized with respect to tissue engineering. They can be modified in many ways such as mechanical properties, degradation rate and chemical modification. Moreover, they can be manufactured in unlimited scale. CHAPTER 11

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The most widely used synthetic polymers are polyglycolic acid (PGA), polylactic acid (PLA) (and their co-polymer), polyethylene glycol (PEG) and polypropylene fumarate (PPF). PGA is linear aliphatic polyester which is formed by polymerization of cyclic diesters of glycolide. PGA is characterized by a high melting point and low solubility in organic solvents. PGA have been intensively investigated as a scaffold to support various cells, including fibroblasts, chondrocytes and smooth muscle cells. PLA is a biodegradable polymer that can be produced from lactic acid, which can be fermented from crops. PLA is structurally similar to PGA, with addition of pendant methyl group. This decreases its melting point and increases hydrophobicity. The scaffold produced from PLA was used in regeneration of nerves, muscles and cartilage. PEG is linear-chained polymer which contains ethylene oxide repeat unit. It is hydrophilic prepared in process of anionic/cationic polymerization. They have best characteristics in form of hydrogels. PEG was extensively studied in respect to cartilage tissue engineering. PPF is linear polyester with repeating unit of two ester groups. It was shown that by cross-linking it is possible to produce scaffold with characteristics suitable for bone tissue engineering.

4. Conclusions Recently, tissue engineering has begun to provide alternative possibilities in clinical practice for healing patients with damaged or lost tissues. It combines cells, different bioactive signal molecules and appropriate scaffolds to prepare a biological substitute of tissues and organs. Further investigations will be focused on producing and testing of new bioactive and biocompatible polymers with well-defined conditions, such as controlled incremental biodegradation, non-immunogenity. Considerable progress can be expected on the field of stem-cell research. Special attention will be paid to comprehensive characterization and output control of in vitro expanded cells. It will be also essential to find proper methods for their administration and to determine the effective dose. Moreover, new approaches will be probably introduced, especially 184


in combination with gene therapy. These examples will generate new opportunities and new therapies for personalized regenerative medicine.

Acknowledgements This work was supported by the grant of Ministry of Health of the Slovak Republic no. 2012/4UKBA-4, by the grant VEGA no. 1/0706/11 and by the grant of the Slovak Research and Development Agency no. APVV-0434-12.

Summary Box Tissue engineering is multidisciplinary field which applies the basic principles of cell biology and material technology to replace, repair, and/or regenerate the damaged or lost tissues and organs. Currently, a variety of cells (including stem cells), bioactive signalling molecules and scaffolding materials of natural and synthetic origin have been proposed for the production of artificial tissues and organs. Since 90s of last century, a plenty of artificial tissues and organs were produced, including urinary bladder, urethra, skin, cartilage, bones, vessels, heart valves, etc. Many of them are recently in different stages of preclinical and clinical testing. However, further investigations have to be carried out for safe utilization of tissue engineering products in practical clinical application

Review Questions and Answers Q1. What are the main components of tissue engineering? A1. The main components of tissue engineering are cells, bioactive molecules and scaffolds. Q2. What are the main characteristics of stem cells? A2. The main characteristics of stem cells are long term self-renewing and plasticity. Both of them are termed as “stemness”. Q3. What are main effects of bioactive molecules? A3. Bioactive molecules influence cell proliferation, survival and differentiation.


Q4. What are the main prerequisites for fabrication of substitutes of extracellular matrix suitable for regenerative medicine?

acteristics, and applications in maxillofacial surgery. Journal of Oral and Maxillofacial Surgery 2007;65:1640-7.

A4. Synthetic extracellular matrix has to be non-toxic, sterile and biocompatible. Moreover, possibility of controlling of their biodegradability also affects their utilization.

6. Kolf CM, Cho E, Tuan RS. Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation. Arthritis Research & Therapy 2007;9:204.

Q5. Which naturally occurred polypeptides are most commonly used in fabrication of substitutes of extracellular matrix? A5. Collagen, elastin and silk.

Further Readings 1. Langer R, Vacanti JP. Tissue engineering. Science 1993;206:920-6. 2. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al,. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145-7. 3. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663-76. 4. Takahashi K, Tanabe K, Ohnuki M, et al,. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:86172. 5. Shanti RM, Li WJ, Nesti LJ, et al,. Adult mesenchymal stem cells: biological properties, char-


7. Graf T, Enver T. Forcing cells to change lineages. Nature 2009;462:587-94. 8. Jackson L, Jones DR, Scotting P, et al,. Adult mesenchymal stem cells: differentiation potential and therapeutic applications. Journal of Postgraduate Medicine 2007;53:121-7. 9. Spradling A, Fuller MT, Braun RE, et al,. Germline stem cells. Cold Spring Harbor Perspectives in Biology 2011;3:a002642. 10. Bellin M, Marchetto MC, Gage FH, et al,. Induced pluripotent stem cells: the new patient? Nature Reviews Molecular Cell Biology 2012;13:713-26. 11. Dominici M, Le Blanc K, Mueller I, et al,. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8315-7. 12. Hancock JT. Cell signalling. Third edition. New York: Oxford University Press 2010;341. 13. Fisher JP, Mikos AG, Bronzino JD. Tissue engineering. Boca Raton: CRC Press 2007; 600.

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BACTERIOPHAGES AND THEIR APPLICATIONS

CHAPTER 12 BACTERIOPHAGES AND THEIR APPLICATIONS Ľubomíra Tóthová


1. Bacteriophages ............................................................................................................. 189

1.1 Lytic and Temperate Phages ....................................................................................... 190

1.2 Classification of Bacteriophages ................................................................................. 191

2. Phage Typing ................................................................................................................ 192

3. Phage-Based Detection Systems of Bacterial Targets .................................................... 193

3.1 Reporter Phages ......................................................................................................... 194

3.2 Quantum Dots .......................................................................................................... 194

3.3 Phage Amplification ................................................................................................... 194

3.4 Phage-Based Biosensors .............................................................................................. 195

4. Phage-Based Controlling of Bacteria in Agriculture and Food Processing .................... 195

5. Phage Therapy ............................................................................................................. 198

5.1 Animal Experiments .................................................................................................. 199

5.2 Phages in Medicine .................................................................................................... 202

6. Conclusions .................................................................................................................. 203

Review Questions and Answers ............................................................................................. 204 Further Readings ................................................................................................................... 204


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188

CHAPTER 12 / Ľ. Tóthová


Summary

B

acteriophage are viruses that recognize, infect and replicate within bacteria. They are fascinating organisms that play a key role in bacterial genetics, physiology and ecology. As model organisms, they allow elucidation of fundamental biological processes at the molecular level. Advances in techniques of molecular biology, especially the possibility to acquire complete genomic sequences (of phage and their hosts), has renewed interest in research on bacteriophage. Scientists focus not only on characterization of the phages themselves, but especially on the phage-host interactions, their influence on the evolution of microbial populations, their ecological role, as well as the possibilities for their application. Recently, a variety of rapid, sensitive and easy to perform phage-based detection approaches have been established for identifying their host bacteria. These improved detection systems have quickly found their place in the monitoring of bacterial pathogens in food, biotechnological, medical or even agricultural industries. Bacteriophage can be produced in large-scale at minimal costs, phage preparations are relatively stable and can be easily stored. Several other advantages make them a very useful tool for humans. As natural antimicrobial agents, bacteriophage are widely used in the food industry and agriculture. Some phage preparations are already approved by the Food and Drug Administration (FDA) for controlling bacterial pathogens. These products are mostly utilized in animal products or in agricultural production. Using appropriate phage can prevent the spread of bacterial infections and thus improve and ensure the general safety of food or plant processing, production and handling. Another potential application of phages is their use in medicine – as phage therapy. Phage therapy is the use of bacteriophage to treat bacterial infections in humans. This idea was first proposed in the early 20th century, but the discovery and production of antibiotics in the 1940s suppressed further development in this field. Nowadays, in the era of increasing number of bacterial strains multiresistant to most available antibiotics, the idea of phage therapy has emerged BACTERIOPHAGES AND THEIR APPLICATIONS

again. The advantage of phage therapy is that bacteriophage specifically kill pathogenic bacteria leaving the accompanying microflora untouched, which is not the case with conventional antibiotic therapy. Phage therapy should also be an option for patients who are allergic to certain antibiotics; no case of allergic reaction has been previously reported with phage therapy. Bacteriophage have a great evolutionary potential, which provides them with a quick response to the emergence of phage-resistant bacteria. Potential problems of bacterial resistance can also be avoided by the use of various phage in a therapeutic cocktail. Moreover, the use of purified phage components with antimicrobial properties is an alternative to classical phage therapy with complete phage particles. Many studies have confirmed the high efficiency of phage in this form, but they lack formal clinical tests carried out in accordance with the current requirements. In general, interest in phages and phage gene products as potential therapeutic targets has rapidly increased and is likely to have a profound impact on biotechnology and pharmaceutical industries in the upcoming years. Bacteriophages can be exploited in biotechnology, medical research and agriculture. In this chapter, we focus in detail on several topics, including phage for rapid identification of bacteria, therapeutic use of phage in medicine and phage as biocontrol agents in food industry. However, for a long time phage have been used in several other research applications that are not covered by this chapter (e.g. phage display, vaccine delivery and targeted gene delivery).

1. Bacteriophages Bacterial viruses - bacteriophage - form a separate group of viruses that are able to infect only bacterial cells. Bacteriophage are ten times more abundant than bacteria, and it is estimated that about 1031 phage are present in biosphere. Although they are among the most abundant organisms on the planet, phage have not been studied extensively in terms of biodiversity, biogeography and phylogeny. Bacteriophage can be detected from different CHAPTER 12

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environments (water, soil, surface of plants and animals, and others). They were isolated wherever their hosts were present, from fresh and marine waters, marine sediments, soil, from human and animal organisms, and various organs and body fluids. Without realizing it, we consume phage every day in yogurt, sauerkrauts, sausages, pickles etc. Their occurrence in the nature is directly related to the presence of a suitable host. Recent studies suggested that some phage are characterized by global distribution, while others are rather endemic to particular environments.

1.1 Lytic and Temperate Phages Based on the life cycle of bacteriophages, they are divided into two basic groups: lytic and temperate (Figure 1). Lytic (or virulent) phage infects a host cell by injection of phage nucleic acid, which independently of the chromosome of the host cell replicates and encodes the synthesis of phage proteins necessary for the formation of new phage particles. Afterwards, the cell lyses as a result of the release of newly-formed phage particles. This replication mechanism passes the following stages: Binding- when phage particle interacts with susceptible host cell by binding to specific receptors on the cell surface; Penetration- when the genetic material is injected into the bacterial plasma membrane; Replication- when viral mRNA is synthesized by the use of host cell transcription apparatus (DNA viruses) or by viral enzymes (in case of RNA viruses). Subsequently, mRNA is translated into proteins (with help of host cell protein translation mechanisms). Multiple copies of phage genomes are produced; Assembling- phage components associate together (viral proteins and replicated genomes) – making phage progeny virions; Releasing- newly formed phage particles are released all at once, which cause the lysis of the host cell. Temperate phages cause either, similarly to virulent phages, lysis of the host cells or the phage DNA integrates into the host chromosome (in the form of so-called prophage), so the cell survives the infection. The entire process leading to the integration of phage nucleic acid into the host cell chromo190


some is called lysogeny. Under certain circumstances, the prophage can be spontaneously released from the bacterial chromosome and thus can start a lytic cycle. Such spontaneous release occurs with a very low frequency. However, several different factors can induce the release of prophage including UV-radiation, X-rays, mitomycin C, novobiocin or high temperatures. Lysogenic cells (bacterial cells containing prophage) are immune to superinfection by phage of the same species/genera. The nature of this immunity rests in production of a “repressor” encoded by the prophage, which specifically terminates the synthesis of phage virions homologous or closely related to itself. Lysogenic conversion is a process that is characterized as a gain or loss of phenotypic characteristics by integrating the phage genome into the host cell chromosome. In lysogenic conversion, frequent changes in virulence of infected bacterial cells take place, mainly due to the newly acquired ability to produce toxin genes encoded by the phage. The production of toxins after bacteriophage lysogenic conversion has been demonstrated in several bacteria, e.g. in Corynebacterium diphtheriae, Clostridium botulinum, Vibrio cholerae and even Escherichia coli. In recent years, large scale sequencing of bacteria has revealed that most of them contain prophage in their genomes, which highlighted the importance of bacteriophage in the evolution of new bacterial strains.

Figure 1. Lytic and lysogenic life cycle of bacteriophage. After recognition of the host cell receptors, phage nucleic acid may either enter the lytic or lysogenic cycle. During the lytic cycle, stages A-E take place. After adsorption on the surface of the bacterial cell phage injects its nucleic acid


into the host, which leads to replication of its genome. After gathering all virion components, nucleic acid is packaged into the virus head, and new phage is completed. Then the lysis of host cells occurs and bacteriophage are released into the environment. If the lysogenic cycle is triggered, the phage incorporates its nucleic acid into the bacterial chromosome as a prophage (H-F). At this stage, phages do not directly lyse the host cell, nevertheless, as prophages they replicate along with the bacterial chromosome and are transmitted to the hosts next generation (adapted from http://www.nicerweb.com/bio1903/Locked/media/ ch18/18_07LamdaLyticLysoCycle.jpg).

Filamentous bacteriophage encompass a special group of phages, which is represented by the M13 phage. After the infection by M13 phage, host cells do not lyse immediately, however they continuously produce the virions, which are afterwards released into the environment (several hundred particles in one bacterial cell cycle). Bacterial cells infected with M13 are able to grow, but at a slower rate compared to uninfected cells.

1.2 Classification of Bacteriophages In 1915, a British pathologist Frederick William Twort described the transformation of micrococcus colonies by unknown agents. Independently, Félix Hubert d’Herelle, a French-Canadian scientist working at the Pasteur Institute in Paris, observed decay of bacteria of the genus Shigella. D’Herelle found that the essence of this phenomenon was the viruses named bacteriophage. He also classified them into separate species Bacteriophagum intestinalis with multiple genera. Currently, the International Committee on Taxonomy of Viruses (ICTV) divides phages into 17 families. Salterprovirus is still not included in the specific family (http://www. ictvdb.org/Ictv/index.htm). Other two recently isolated phages, namely “Serpentine-Lake-Hispanica” and “Sulfolobus turreted icosahedral-Virus” are also considered as two separate families by some authors. In addition, some other families are waiting to be classified. Bacteriophage taxonomy is based mostly on their morphological and biochemical

Table 1. Basic characteristic of bacteriophage families, adapted by Ackermann. BACTERIOPHAGES AND THEIR APPLICATIONS

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Table 2. Basic characteristic of bacteriophage families, adapted by Ackermann.

characteristics and thus has more or less predictive value. Particular families and their basic characteristics are summarized in Tables 1 and 2.

this,but because of unknown phage morphology it is not possible to include these prophage in taxonomic classes.

Bacteriophage are composed of two basic components: proteins and nucleic acid. Some phages are coated with an additional lipid layer. Virions are polyhedral or tailed, filamentous or pleiomorphic. Genetic information can be stored in the form of either DNA or RNA. Most bacteriophages contain double-stranded (ds) DNA, but there are phages with single-stranded (ss) DNA, ssRNA or ds RNA with different topologies (linear or circular). Bacteriohage with binary symmetry (phage with tails) represent about 96% of all bacteriophage. Together, they form a separate order named Caudovirales with three large and phylogenetically related families: Podoviridae, Myoviridaeand Siphoviridae. Cubic, filamentous and pleiomorphic phage include only about 4% of the viruses, but they are classified into 13 small families (see Tables 1 and 2). They differ significantly in their basic properties and seem to come from different evolutionary lines. Classification of bacteriophage on the basis of the morphology and biochemistry of phage particles is not sufficient in terms of molecular phylogeny. The discovery of different prophage in bacterial genomes from sequencing projects demonstrates

2. Phage Typing

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Although many sophisticated techniques for characterization of bacterial species are available (ribotyping, polymerase chain reaction with random primers - RAPD, amplified fragment length polymorphism - AFLP, etc.), nucleic acid amplification methods are expensive and time-consuming. Therefore, phage typing remains a traditional phenotyping assay in many laboratories due to high sensitivity and simplicity. Different sets of bacteriophage are used to distinguish or discriminate between specific strains within the bacterial species. Drops of selected bacteriophage are spotted onto specific bacterial lawns grown on agar plates. Lysis results for each phage are recorded and a characteristic pattern is obtained, which enables the strain characterization. The sensitivity and reproducibility of this method is affected by the number of phages used. Phage typing, however requires skilled and experienced personnel and the upkeep of many bacteriophage stocks, as well as reference bacterial strains. On the other hand, this technique is cheap, rapid and reliable. Different phage typing schemes


are available worldwide for various clinically relevant bacteria such as Salmonella, Campylobacter, Escherichia, Enterococci, Listeria and many other species. For example, the phage typing of Salmonella serovars uses different typing schemes containing different number and types of bacteriophage. At the present time, more than 200 phage types of Salmonella typhimurium alone exist. The phage for these typing schemes are lysogenic or lytic, and may be isolated from various environments (soil, water, faeces, sewage etc.).Their selection could be helpful with regard to the development of effective mixtures of bacteriophages against specific (sensitive) bacterial pathogen. This technique has also implications in phage therapy or theoretical medicine. However, for global clinical and epidemiological relevance, sufficient standardization and use of the same typing schemes in national reference centres around the world is crucial.

3. Phage-Based Detection Systems of Bacterial Targets Methods for identification of bacteria by phages provide several advantages over the commonly utilized methods. For example, the production of antibodies for bacterial identification is expensive and time consuming compared to phage preparation. Another advantage is that phages detect only viable bacteria, thus eliminating possible false positive signals, which are a common problem in bacterial molecular detection methods. Applications of bacteriophage as a tool to identify bacteria include food technology, agriculture, medicine, etc.

3.1 Reporter Phages Specific and rapid detection of bacterial strains by measuring the activity of reporter genes, located within the phage genome and expressed only after the onset of infection, was first described in 1987. The expression of bacterial luciferase gene (lux) of Vibrio fisherii cloned into the phage lambda vector was able to detect a minimum amount of ten E. coli cells in milk. Vectors derived from lambda phage BACTERIOPHAGES AND THEIR APPLICATIONS

have a large cloning capacity, but since it is a temperate phage with a narrow host range, their use is limited. Unfortunately, in the case of most phage, direct cloning is ineffective. However, reporter genes can be inserted into bacteriophage through homologous recombination with plasmids. The amount of DNA inserted into to the phage genome is limited, so most of the phages use lux reporter gene, which requires the addition of an external substrate. Typically, the addition of an aldehyde substrate and the phage infection of specific hosts will thereby generate a bioluminiscent light. Later, in 1997, Loessner et al. constructed a recombinant luciferase reporter phage (LRP-phage) for the detection of Listeria strains. Virulent and host-specific bacteriophage A511 was able to infect up to 95% of clinically important serovars of Listeria monocytogenes. The luxAB gene was inserted into the phage by homologous recombination; gene transcription was under the control of the major capsid promoter of the phage. Easy and rapid methods for the detection of Listeria contamination in food samples (cabbage, lettuce, cheese, and others) was thus achieved. When no bacterial host is present, no expression of luxAB occurs. But once a specific host is present, phage infection results in a light signal, detectable as a certain level of bioluminescence. An innovative lux reporter assay was established by Ripp et al., in which self-generated bioluminescent light production was achieved. They constructed a bacterial bioluminescent bioreporter, where all luxCDABE genes were inserted into the bacterial genome. Quorum sensing chemical N-3-(oxohexanoyl)-1-homoserine lactone (OHHL) is produced by the luxI gene, which was inserted into the phage lambda genome. As soon as this modified phage infects E.coli cells, luxI expression is achieved. In addition, in medium with luxCDABE-based bioreporters the interaction triggers autonomous bioluminescence. This assay is rapid and easy to perform, while no personal manipulation is needed. Many different reporter genes have now been incorporated into bacteriophage genomes including eukaryotic luciferase, lacZ gene encoding the β-galactosidase enzyme, for detection of various clinical or CHAPTER 12

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food-borne pathogens. For detailed information several comprehensive reviews are available. Another approach for detection of bacteria in their natural environment employs fluorescently labelled phages. Detection of E. coli cells using green fluorescent protein (GFP) was demonstrated by Funatsu et al.. Distinct differentiation of E.coli cells (sensitive to phage infection) from Mycobacterium smegmatis (resistant to phage infection) were observed using epifluorescent microscopy. Although the use of these phage is beneficial, methods using fluorescent dyes are relatively expensive. This disadvantage was overcome by the construction of auto-fluorescent bacteriophages. Mutant T4 phage was prepared, which is unable to produce lysozyme, the enzyme responsible for host cell wall degradation, and subsequent lysis. The small outer capsid protein of this mutant phage also presented GFP to the viral capsid. Extended incubation times of bacterial cells with these newly constructed phage increased fluorescence intensity, allowing efficient fluorescent quantification to be achieved within a one hour assay duration. Fluorescence microscopy, flow cytometry or microplate fluorometry are suitable methods for the detection of fluorescent signals.

3.2 Quantum Dots Fluorophores used as reporters such as luciferase or GFP, have two main drawbacks: low ratio of specific signal-to-noise in bacterial cells or clinical samples and low photostability. Therefore Edgar et al. in their work chose a very innovative approach. Their unique detection system combines in vivo biotinylation of recombinant (host-specific) phage and conjugation of this phage to streptavidin-coated quantum dots (QD- fluorescent probes, colloidal semiconductor nanocrystals with a diameter of a few nanometres). The major capsid protein of bacteriophage T7 was fused with a small peptide, capable of subsequent biotinylation. Thus, if the sample contains bacteria sensitive to a particular phage type, thevirions produced become biotinylated. Biotin present in living cells is attached post-translationally to lysine residues on the fusion peptide, and the biotinylation process is highly 194


conserved among different bacterial species. The biotinylated phage particles in the lysate (evidencing the presence of sensitive bacteria) are detected by conjugation to the fluorescent streptavidin QD. This very fast system allows for specific detection of less than 10 bacterial cells per ml, and is useful for the identification of slow-growing bacterial cultures such as Mycobacterium or highly infectious bacterial strains such as Bacillus anthracis.

3.3 Phage Amplification Phage amplification assays represent another phage detection system, based on amplification of large amounts of phage particles after infection of specific bacterial hosts. Once released, this burst of amplified phage particles is detected by another non-pathogenic bacterial strain, known asso called helper cells. Prior to detection, remaining free phage are eradicated by using a virucide. After neutralizing the virucide, fresh helper cells are added and amplified phages from infected cells are measured by a basic plaque test. The major advantage of phage amplification is that virtually any lytic phage can be used without the need for any phage genome manipulations. This system is being successfully applied for detection of Mycobacterium tuberculosis. Comprehensive results from up to thirteen mycobacterial studies confirmed the specificity of these tests, and these assays also showed better accuracy when compared to culture methods or sputum microscopy. Classical microbiological identification of mycobacteria can take up to several weeks, whereas the phage-based technologies are easier, cheaper and faster. Since discovery of this method for rapid Salmonella detection in 1983, several modifications of phage amplification assays have been developed. Immunomagnetic separation of target cells was proposed in order to avoid the addition of virucide. Salmonella cells were locked onto the paramagnetic beads, while free phage were removed by washing. Another improvement utilised the measurement of optical density, instead of plaque assays for assessment of infection. The Endpoint measurements by quantitative real-time (RT)-PCR were reported as novel adaptations of phage amplification for de-


tection of Yersinia pestis by Sergueev et al.. Yersinia pestis specific bacteriophages QA1122 and L-413C were tested for identification of specific bacteria via RT PCR with primers specifically designed against their genomes. Phage QA1122 possesses higher (maximum) sensitivity for this assay when compared to phage L-413C; on the other hand, phage L-413C provided higher specificity. Using both phages in RT-PCR, phage amplification assay provide rapid , simple and definitive identification of Yersinia pestis within four hours,without the need for template DNA extraction.

3.4 Phage-Based Biosensors With better understanding of phage-hosts interactions and more complex knowledge about bacteriophage generally, bacterial detection methods have moved forward to sophisticated phage-based biodetection approaches. Knowing that numbers different bacteriophage are available, specific detection of all kinds of bacterial pathogens, foodborne, clinical or even waterborne, is possible. More recently, biosensors have emerged as an attractive tool for bacterial detection. They overcome some disadvantages of common microbiological, molecular biological or immunological detection methods, through lower detection limits and requiring minimal sample preparation. Biosensor detection of bacterial pathogens has two major components. The first is a probing element, which is a biological agent with specificity to the analyte and its recognition; the second is a transducing platform, which alters the interaction between the analyte and the probe into a measurable signal. As a biorecognition probe DNA, RNA, enzymes, phages, phage display peptides or antibodies can be exploited. Also, a number of transducers have been reported, such as surface plasmon resonance, electrical, piezoelectric or optical. Herein we report especially on immobilized phage virions. In 2007, Lakshmanan et al. reported a phage-based magnetoelastic sensor for detection of Salmonella typhimurium. Filamentous bacteriophage were immobilized by physical adsorption onto the sensor surface. Since the pathogen interacts with the immobilized bacteriophage, the BACTERIOPHAGES AND THEIR APPLICATIONS

response of the magnetoelastic sensor is changed, but proportional to the number of salmonella cells in different liquid media. Using this phage-based technology, the detection limit for salmonella cells was 5x103 colony forming units (CFU/ml) in water or fat free milk. The disadvantage of this system is that physical phage adsorption may result in lower sensitivity of the sensor due to non-complex surface functionalization procedures. This problem may be bypassed either by the use of chemically anchored bacteriophages onto the sensor platform or by genetic manipulation of bacteriophages (in order to achieve more effective immobilization). For further reading see also the extensive review published by Singh et al. All mentioned assays possess several advantages, as well as disadvantages, but many proof-of principle experiments confirmed that phage-based techniques for bacterial detection are sensitive, specific, rapid, reliable and cheap. In addition, there are many ongoing projects searching for new potentially therapeutic phages, sequencing new phage genomes, and extensive development of new molecular methods for engineering of desired phage. Moreover, the use of some phage components such as lysins, holins or phage receptor binding proteins offer another opportunity and challenge toward wide assay formats to fulfil different defined needs. Altogether, phage assays definitely have their place in the agricultural, food, medical and biotechnological industries.

4. Phage-Based Controlling of Bacteria in Agriculture and Food Processing Recently, the number of known diseases caused by pathogenic bacteria has increased. The evolution and frequency of multidrug resistant pathogens has become a worldwide problem. They are widespread in our environment, but especially among livestock, where they may also cause disease, but livestock often remain asymptomatic. Consuming food prepared from raw materials of animal oriCHAPTER 12

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gin that have not undergone sufficient heat treatment or secondarily contaminated serves as a direct threat to humans. Currently, many research teams focus on these issues and try to find appropriate methods to improve food safety in the production process. Bacteriophages, thanks to their host specificity and ability to eliminate bacteria, have become ideal candidates for research in this area. There are four potential phage application types that could be beneficial in the decontamination process: 1. Animal feeding - bacteriophage therapy administered to living animals should be appropriate to reduce or eliminate colonization by intestinal bacteria that pose a potential risk to humans. Alternatively, phage could be added to feed for animals just prior to slaughter. This approach is probably preferrable in terms of obtained resistance, because phage administered to animals in the slaughterhouse are not able to be recycled back to the farm. 2. Application to raw food – a direct application of phage onto already processed but not yet cooked, food. 3. Application to fresh and processed food intended for immediate consumption. 4. Increasing the shelf life of foods - some bacteria have an effect on product quality by reducing the shelf life of products or causing odour issues, therefore use of bacteriophages could also be useful in this area. Monitoring various phases of the food production process, so-called “farm to fork” concept, is a major requirement in the food industry, as stated in the European Commission White Paper on Food Safety (2000; http://ec.europa.eu/food). This term refers to critical steps in the production process, which may contaminate food and where the application of bacteriophage could be of benefit. Here, we focus on the frequently occurring bacterial food pathogens such as the genera Salmonella, Escherichia, Campylobacter, Listeria, Clostridium or Staphylococcus. Nowadays, acute enteric infections caused by Salmonella are a global health problem. The emergence and rapid spread of multidrug re196


sistant strains represents a significant risk, along with resistance to commonly used therapeutic agents. Despite strict control of this pathogen in food production, the main source of Salmonella infections is usually primarily infected animals,mostly poultry. Several studies have examined the possibility of applyingbacteriophage to eliminate Salmonella in broiler chickens. Various phage were applied to chickens artificially infected with Salmonella enteritidis, S. typhimurium or S. hadar. The authors observed pathogen eradication in the broiler intestine. Similar results were also obtained when applying a mixture of 3 lytic phages directly onto the skin of animals. Chicken legs were immersed in a suspension containing 106 CFU of S. enteritidis PT4 per ml on the day of the killing at the slaughterhouse. A phage cocktail of 109 plaque forming units (PFU/ml) was administered the day after. Numbers of Salmonella colonies were significantly lower 3, 6 and 9 days after administration. Although all studies clearly confirmed the reduction of pathogenic bacteria in chickens, Salmonella was not definitively cleared completely in either case. Another study published by Bigwood et al. applied bacteriophages to Salmonella typhimurium PT160 and Campylobacter jejuni infected raw and cooked meat to reduce pathogen numbers. The authors simulated different conditions (high/low amount of pathogen and phage, miscellaneous food storage temperatures), while the obtained results confirm that the phages are useful in biocontrol of food pathogens. These assumptions were also confirmed by another study, where the authors applied bacteriophage to sprout seeds. The eefficiency of using phage lysates in experimentally contaminated seeds was lower compared to in vitro, which may be due to the less stringent nature of in vitro conditions compared to the complexity of a system including seeds. Carlton et al. tested the effectiveness and safety of a commercial mixture of bacteriophages to effectively lyse different Listeria strains (Listex TM P-100; http://www.micreosfoodsafety.com). Toxicity studies used 10 rats, which received high doses of oral phage (5x1011 PFU/ml) for 5 days. No changes in


the behaviour of rats and no short-term side effects of therapy were recorded. The efficacy of the product has been evaluated in experimentally infected Muenster cheese by L. monocytogenes at a dose of 2x101 CFU/cm2. According to the results of this study the effects of ListexTM P-100 no the bacterial host are dose dependent. Similar results were observed using raw salmon and catfish flesh. ListexTM P-100 has already received the designation of GRAS (generally recognized as safe) for use in the food industry. A similar preparation named LMP-102TM contains a mixture of six bacteriophages targeting L. monocytogenes (http://www.intralytix.com). This product was applied on ready-to-eat products without effecting the taste, colour or smell of the food products. This preparation effectively reduces the amount of L. monocytogenes 100 to 1000-fold in the case of food contamination by this pathogen. The company has also licensed the rights to other phage preparations (for example: SPLX-1 Timothy, 1 Timothy PLSV-against Salmonella and INT-401TM against Clostridium perfringens) for veterinary use in animals just prior to slaughter. In the last phase of clinical trials is another product ECP-100TM effective against E. coli O157: H7. In addition, Biotech Company, which deals with bacteriophage technology, provides AgriPhageTM (http://www. omnilytics.com). This product is used in agriculture against the pathogenic bacteria Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. Tomato and has been approved by the U.S. Environmental Protection Agency –(EPA). Two other products are aimed at eliminating bacteria from animals prior to slaughter. Both are designated as BacWashTM and are effective against Salmonella and E. coli. They can be applied directly to live animals. Other available commercial phage preparations are described in more detail by Monk et al. or for further information you can visit the respective companies websites. Rozema et al. have investigated the effects of bacteriophage administration into infected cattle. Experimental animals were infected with E. coli O157: H7 (resistant to nalidixic acid) and subsequently specific phage cocktails were administered orally, BACTERIOPHAGES AND THEIR APPLICATIONS

rectally or both in multiple doses to the animals. The lowest number of bacteria was detected in a group of bulls. In another study, nine different bacteriophages targeting various enterotoxigenic E. coli (ETEC) have been isolated in Ontario from more than 30 pig farms. All phage were extensively characterized and identified as suitable candidates for the prophylaxis or treatment of diarrhoea in young pigs. Several other bacteriophages able to lyse shiga toxin-producing E. coli (STEC O157: H7) were also recently described. The relatively quick and easy isolation of bacteriophage from different environments suggests their enormous potential for biocontrol of various food pathogens. On the other hand, recently published papers reported unsuccessful effects of orally administered phage therapy in different animals, and suggested the low pH of gastrointestinal tract (GIT) or insufficient amount of phage reaching the GIT, may be responsible factors. In 2005 two experimental studies first described the possible application of specific bacteriophages against Campylobacter infection in chicken. Preventive, as well as therapeutic treatment was tested on artificially contaminated broilers. Successful elimination of bacteria was observed in the therapeutic group; on the other hand, in the preventive group only delayed onset of bacteria colonization was observed compared with a control group. None of the treated animals showed any side effects after phage administration. Loc Carrillo et al. selected highly lytic phages for their in vivo experiment, in which they resuspended phage in antacid solution in order to protect them from harsh intestinal conditions such asadverse pH or proteolytic enzymes. Afterwards, different doses of phage vs. host were tested. Dose and time dependent reductions of campylobacter were observed, with an optimal dose of 7 log10PFU. Similar results were also concluded from study of Carvallo et al., regardless of the elimination of Campylobacter bacteria, by the use of novel phage cocktails. Besides, they presented a new approach of phage application- in feed, which was also proved to be effective in reducing Campylobacter colonies in faecal samples. A unique CHAPTER 12

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study was recently performed by Siringan et al., who were the first to describe postharvest application of bacteriophage on Campylobacter jejuni biofilms formed on glass. The use of two previously described lytic bacteriophages was able to reduce viable bacterial cell counts by 1 to 3 log10 CFU/cm2. There is a great potential for use of lytic phages in controlling bacterial biofilm formation, although some resistant bacterial cells survived after treatment. Nevertheless, Campylobacters, that survived the phage therapy in broilers, were found to be physiologically compromised. Further experiments will be necessary for better understanding of phagehost interactions under biofilm conditions. Increasing numbers of new studies from academia as well as biotech companies confirm that bacteriophages play an important role in biocontrol of pathogenic bacteria in food and agriculture. Application of these bacterial viruses in food processing is a very straightforward approach. Optimising the composition of the phage preparations, dosage and method of transport to ensure efficient entry of phage particles into the bacteria are important in improving the effectiveness of reducing pathogens in food. For safety reasons, it is important to use only lytic viruses that do not contain virulence genes or genes capable of transduction. Also from a practical point of view, it is necessary to apply sufficient amounts of infectious phage particles in the most appropriate part of the production process.

5. Phage Therapy Before using bacteriophage in vivo it is essential to understand in detail the phage-host relationship and to assess phage activity and amplification ability in vitro. It is apparent that in well-defined in vitro conditions (enough air, culture medium, dose response, etc.), the phage-host interactions are likely to be different when compared to in vivo situations. Clinical environments that include other nutritional components, physiological conditions (pH, ionic strength) may also affect the bacterial gene expression and phenotype. In addition, upon infections, the interactions between the immune system and 198


the bacteria, as well as the therapeutic bacteriophages occur. Before using the selected phage as a therapeutic agent, phage should be clearly examined in terms of their genomic sequences, host specificity, virulence, stability, etc. Bacterial eestriction-modification systems can represent a limiting factor in the ability of phage to target particular bacterial species. This problem may be overcome by modification of the bacteriophage genome, which would remove restriction sites recognized by the host. Another possibility is the production of phages in a bacterial strain that allows DNAmodification, thus escaping further restriction in the target bacteria. Knowing the sequence of the bacteriophage and its subsequent comparison with sequences available in databases may also help to detect toxic genes, resistance genes, pathogenicity islands and genes that are subject to integration into the bacterial genome. Phage preparations may contain contaminants from bacterial cells (including exo/endo toxins), if not sufficiently purified. These contaminants may cause increased morbidity or mortality, and thus lower the efficiency of treatment. For clinical practice, a careful purification by cesium chloride gradient as used in most publications, or ultrafiltration through a membrane polysulfone-chromatography on Sepharose and cellulofinesulfate is likely to be necessary. Advantages (A) and disadvantages (D) of phage therapy: A • Bacteriophage infect only bacterial cells and areharmless to eukaryotes. For each bacterial species occurring in nature, there is at least one complementary phage. Thus, phage therapy could be appliied to any bacterial infection. A • Bacteriophages are not only capable of (self ) replication, but also (self ) elimination because their reproduction is limited by the presence of sensitive bacteria. A • Bacterial imbalance or “dysbiosis” caused by antibiotic therapy can lead to secondary infection by other resistant pathogens, which prolongs hospitalization and the total cost of patient treatment.


This problem can be avoided by the application of bacteriophage with narrow host range which do not affect the natural microflora. A • During phage therapy trials (experimental or clinical), no serious side effects or allergic reactions to the phage therapy have been reported. Phage therapy is therefore suitable also for patients allergic to certain antibiotics. A • Nowadays, manifold applications of phage therapy are available, including oral drugs, injections, nasal sprays, ointments, etc.. A • Bacteriophage in combination with antibiotic therapy may reduce the occurrence of bacterial resistance. A • Since phages grow exponentially in the presence of sensitive bacteria, a single dose may be sufficient to cure the patient as opposed to multiple doses of antibiotics. Besides, large scale phage production is relatively quick and inexpensive.

5.1 Animal Experiments In recent years, interest in phage therapy has markedly increased (Figure 2). This revaluation is likely due to the expansion of multiresistant bacterial strains and the inefficiency of antibiotics to combat them. In the light of previously published information about phage biology and the results of clinical testing of bacteriophages as therapeutic agents, the present status of research is highly encouraging. Some scientists, however, point to the lack of definitive information in the field of phage biology. Skurnik and Strauch pointed out the need for a safe and controllable phage therapy, as well as a need for more information on the properties and behaviour of specific phage-bacterial host systems both in vitro, but primarily under in vivo conditions.

D • Since some bacteriophage are highly species-specific, it is necessary to precisely characterize the infectious bacterial agent prior to their application. D • One cannot guarantee that the phage, which has a strictly lytic life cycle in precisely defined in vitro environments a strictly lytic life cycle, will remain in this cycle unchanged under the physiological conditions of the human body. D • A frequently mentioned disadvantage of phage therapy is the possibility of toxic shock after bacterial lysis and subsequent amplification of neutralizing antibodies during the re-treatment of the patient. This can be avoided by passaging bacteriophages in murine cell lines. However, this approach is not effective when using phage cocktails consisting of different phages. Another way to solve this problem is to use phage deficient in the lytic system. Such lysis-deficient phage are capable of infecting, replicating and killing the host bacteria, however, without releasing the phages or endotoxins into the environment. Macrophages will subsequently detect and kill the pathogenic bacteria. BACTERIOPHAGES AND THEIR APPLICATIONS

Figure 2. Increase in number of phage therapy-related publications by year. Based on the keywords “phage therapy” searched in Pubmed database.

Treatment of Staphylococcal infections by phages in animal model has been described by Soothill et al. in 1992. Unpurified phage ф-131 that is already in use in Poland, was not successful in eliminating the bacterial infection in mice. Phage ф-131 has been less active when compared with results of in vitro testing. Among others who have described the successful phage therapy of bacterial infections is Matsuzaki et al.. The authors applied Staphylococcus aureus either sensitive to phage фMR11 or a lysogenic strain intraperitoneally, which was phage resistant. Then a polyvalent bacteriophage фMR11 CHAPTER 12

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was applied to both groups, what led to elimination of the pathogen in case of the sensitive strain. The results obtained in this study also showed that phage particles themselves are not capable of inducing an antibacterial response. Indeed, if the therapeutic effect of phage was mediated by phage-induced immune response, phage preparation would be effective in both treated groups. Presented experiment also confirmed that the mechanical lysate of S. aureus without a phage had no protective effect. Capparelli et al. confirmed again the protective effect of phage therapy in mice with Staphylococcal infection. Bacteriophage Msa was effective in the systemic, local and intracellular infections caused by MSSA (methyciline sensitive Staphylococcus aureus) strains, as well as MRSA (methyciline resistant Staphylococcus aureus). The authors observed no adverse effect of treatment due to rapid bacterial lysisin mice. Also noteworthy is that they were able to prepare bacteriophage able to survive in the reticulo-endothelial system of mice for much longer period. While Wsa phage (wild-type) persisted in the circulation for 2 days, obtained selected phage Msa (mutant) was present in circulation for more than 20 days. A similar selection system in mice was used in 1996 when two variants of E. coli phage lambda and phage P22 of Salmonella Typhimurium were determined. The method of multiple passages (serial-passage selection method) acquired lambda phage variants amounting to 13,000 to 16,000-fold greater ability to survive in the mouse bloodstream (to overcome the immune system) compared to the wild type phage. Very similar results were achieved in the case of phage P22. The longer circulating mutants also showed more successful treatment of bacterial infection compared with wild type ones and morbidity and mortality of infected animals was lower. The results of the above-mentioned papers suggest a relatively simple way of selection of longer circulating phages, enabling their use in the prophylaxis of bacterial infections. Capparelli et al. examined the efficacy of phage therapy in already infected animals using a low, non-lethal dose of 5x106 CFU/ml. Even after 10 days, the Msa phage was 200


able complete eliminate the infection. These results demonstrated that the ability of phages to kill bacteria is not limited by time, and thus it would be possible to use bacteriophages also for the treatment of long-term infections. A study of Polish authors focused on the detection of prophylactic effects of phage in immunosuppressed mice. Animals were first injected with a dose of the immunosuppressive drug cyclophosphamide, 4 days later they were intraperitoneally administered a specific bacteriophage 30 minutes prior to the application of S. aureus. The results confirmed a significant reduction in the number of bacteria in infected mice (without functional phagocytes), but also revealed a beneficial effect of S. aureus specific phage A5/L on the immune system. However, in this field of research quite a few contrasting results have been published, so further studies are more than necessary. The ideal tools for phage therapy are lytic bacteriophage, which upon contact with bacteria-sensitive strains immediately induce their lysis. However, the ability of temperate phage to mutate allows changing their life cycle to an exclusively lytic mode. Two types of mutations come into play - “clear or vir plaque.” The loss of phage receptors, which are responsible for maintaining the bacteriophage in a prophage stage, results in clear plaque mutants. In contrast, vir plaque mutants have lost the area to which the repressor binds. An important functional difference between vir and clear mutants is that the vir mutants are able to infect a lysogenic strain (for the particular phage) from which the mutation arose. Clear mutants do not have this ability. Garcia et al. isolated lytic phage mutants of temperate phage targeting S. aureus, by use of 100 mM sodium pyrophosphate, causing random DNA deletions. In these experiments it was not possible to determine precisely the mutant type (clear or vir). However, it is clear that the dynamics of temperate phage, including recombination, intragenomic translocation, duplication and other processes contribute to the evolution of S. aureus strains and their adaptation to the host.


Biswas et al. isolated bacteriophage effective against vancomycin-resistant Enterococcus faecium (VRE) from sewage samples. Afterwards, they artificially induced bacteremia in mice by intraperitoneal administration of VRE. Phage therapy applied within 45 minutes of an outbreak of infection had a 100% protective effect. This monophage preparation, administered to mice with a delay of more than 24 hours after the bacterial inoculation, showed a therapeutic effect in up to 50% of the mice. Uchiyama et al. isolated 30 different bacteriophages specific for Enterococcus faecalis. Virulent phage фEF24C with the widest host range has been sequenced and subjected for detailed analysis. In the murine model of bacteraemia induced by two strains of E. faecalis (one of them is among the vancomycin-resistant) they have shown that a single dose of фEF24C is sufficient to successfully overcome infection. Intraperitoneal application of phage did not cause any side effects in experimental, or control animals. Additionally, no adverse effects or changes in behaviour of mice were observed even after repeated administration of phage (7 times every 4 days). Valuable results have been obtained from studying the therapeutic effects of bacteriophage in mouse models of Pseudomonas aeruginosa infections. Experiments involved the inoculation of laboratory animals with bacterial strains P. aeruginosa (resistant to imipenem) and subsequent application of specific phage фA329. Treatment was 100% successful when phage were administered intraperitoneally within 60 minutes after bacterial inoculation. Survival rates of animals significantly decreased with later administration of purified phage preparations (50% and 20% when administered after 180 and 360 minutes, respectively). Even in this case, phage therapy did not cause any side effects, even in mice treated with high doses of phage фA329. Five newly isolated bacteriophages from different environments, tested in vitro for specificity to lyse various P. aeruginosa strains, were later used in a study to eliminate this pathogen in burn wound infection mice models. In vivo testing of phage survival after intraperitoneal administration (3x108 PFU/ ml) demonstrated phage particles were present in BACTERIOPHAGES AND THEIR APPLICATIONS

the blood, the peritoneal fluid, the lung, and in the skin. The highest titre was detected 3 hours after administration; however, phage numbers dropped to zero after 36 hours. Mice infected with P. aeruginosa (subcutaneously onto the burn) were administered also with a dose of the respective phage. This, however, had no protective effect. Similarly, disappointing results were obtained with repeated administrations of the same virus. Interestingly, successful curing of infections caused by Klebsiella pneumoniae was observed in the same animal model and in the same laboratory. These results demonstrated once again that the phage-host relationship does not behave identically in in vivo systems and under in vitro conditions. Chibana-Chennoufi et al. found that orally administered bacteriophages (T4-like coliphages) were able to pass through the mouse digestive tract without loss of viability. Phage were absorbed in the intestine and no virions were detected in the mesenteric lymph nodes or liver. Oral application of bacteriophages did not cause any histopathological changes in the mucous membranes of the intestine. Another important result of this study was the finding that only recently introduced bacteria in the gut were sensitive to theintroduced phage. Accompanying microflora remained intact despite containing bacteria sensitive to this bacteriophage in vitro. In other experiments, a therapeutic cocktail of three highly virulent E. coli phages has been used successfully to eliminate strain E. coli O157:H7 in the gastrointestinal tract of mice. However, it should be noted that a single dose of phage cocktail was not sufficient to eradicate E. coli O157: H7 from the gastrointestinal tract. Testing the stability of the phage cocktail in acidic environments in vitro revealed that phages significantly lose viability at pH 2. Therefore, the authors neutralize the phage mixture by adding 0.25% CaCO3 prior to in vivo experiment. Wang et al. addressed the potential use of phage to fight strains of E. coli producing β-lactamase with a broad spectrum of activity (ESBLs-extended-spectrum beta-lactamase producing E. coli). They used newly isolated bacteriophage Ø9882, which showed lytic activity against severCHAPTER 12

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al resistant clinical isolates of E. coli. Intraperitoneal injection of bacteria (3x107 CFU/ml) caused bacteremia and mice died within 24 hours. After a rapid phage application up to 40 minutes after administration of bacteria, 100% of the animals survived. In another group of animals treated with heat-inactivated virus particles, the survival rate decreased to 0%. A similar “therapeutic effect” from heat-inactivated bacteriophage has been described by Biswas et al. and Wang et al.. Taken together, these results confirm that the therapeutically effective mechanism by which phages kill susceptible bacteria is direct lysis. The aforementioned experiments demonstrate the importance of animal models to study the effectiveness of phage therapy, although no universal model has been proposed. These studies indicated that for successful phage therapy several factors are especially important, including the right timing of application of therapeutic phages, selecting the right application mode and knowledge about phage-host interactions in order to choose the right MOI (multiplicity of infection).

5.2 Phages in Medicine Nowadays, with the steadily growing number of bacterial strains resistant to available antibiotics, pharmaceutical companies are struggling to develop new effective agents. Their development and testing is a time- and money-consuming process. During the period from 1998 to 2003, the FDA has approved only nine new antibiotics, from which only two belong to a new class. The actual trend should be directed towards other ways to eliminate pathogenic bacteria. One such options could be the use of bacteriophages. These viruses can be used in medicine as a substitute for conventional antibiotics, which are often ineffective owing to the rise of resistant strains. Recent clinical studies have shown some important properties of phages including their antimicrobial activity against Gram+ve and Gram-ve bacteria, their safe use, good systemic distribution, including the nervous system, and the relatively low chance of resistance in bacteria.

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The idea of phage therapy historically preceded the concept ofantibiotics. However, the lack of knowledge about the biological properties of phages, highly controversial results of phage therapy and the extensive onset of antibiotic use in Western countries completely stopped their use. Only in Eastern Europe, especially in Poland and Georgia, has intensive research on phage and their therapeutic potential continued to this day. The Eliava Institute in Tbilisi (Georgia), a specialized institute for phage therapy, has been the sole provider of bacteriophage products against various pathogenic bacteria for many years. Phage preparations are used in different areas of medicine: in surgery, gynaecology, urology, ophthalmology, etc. The Institute produces monophage preparations (phages against pathogenic genera including Staphylococcus, Streptococcus, Escherichia coli), but also different types of phage cocktails (namely Pyophage, Intestiphage) etc. Results from clinical trials using these phage preparations are published in several articles, although mostly written in the Russian or Georgian languages. The Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Warsaw has been devoted to studying the biological properties and possible applications of bacteriophage for several decades. In 2005, the Institute opened its own Center for Phage Therapy, which receives patients internationally. Operation is fully consistent with domestic legal and ethical requirements related to the experimental treatments performed. Phage products manufactured at this facility may be used for patients with infectious diseases caused by strains of Staphylococcus, Enterococcus, Escherichia, Citrobacter, Enterobacter, Klebsiella, Shigella, Salmonella, Serratia, Proteus, and Pseudomonas. The vast majority of the publications on phage therapy in medicine originate from Poland and Georgia. One of the first human phage safety tests was carried out according to standard protocols by Swiss scientists Bruttin and Brüssow in 2005. Fifteen healthy volunteers received either low (103 PFU/ml) or high (105 PFU/ml) dose of Escherichia coli phage T4 and


placebo. The presence of bacteriophages in faeces was detected in volunteers who received a high phage dose, immediately after the first day of the application. This detection was 50% lower among those who received the low phage dose. One week after a two-day test on safety of oral phage application, these phages were not present in the stools. In this context, the administered phages did not cause loss of population in the total amount of E. coli or of the accompanying stool intestinal microforms. Also, the volunteers did not show any side effects of the “therapy”. Neither serum transaminase levels, nor T4 phage particles and the specific antibodies were present in the serum. The promising use of bacteriophages in the prevention and treatment of bacterial infections (chronic as well as infections caused by resistant bacteria) has been proposed by a large number of authors. Leszczynski et al. suggested the successful elimination of infection by MRSA using phage therapy for medical workers. Gastrointestinal tract MRSA colonisation was followed by urogenital infection with the same pathogen. Phage preparations consisted of three highly lytic, polyvalent orally administered bacteriophages. Resulting swab cultures were negative even after 6 months post-decolonization. Recently, Letkiewicz et al. described successful uses of phage preparations in the treatment of chronic bacterial prostatitis. In all 3 patients, Enterococcus faecalis was culture-confirmed (the presence of other unusual bacteria was excluded), and all underwent several previous unsuccessful attempts of antibiotic treatment, autovaccination or transrectal laser biostimulation as adjunctive therapy. Ten millilitres of bacteriophage preparation (107-109 PFU/ml) was administered rectally for 28-33 days. In all cases, there was a significant eradication of the bacteria, reduction in the size of the prostate, as well as the disappearance or alleviation of pain. In a clinical study, Rhoads et al. aimed to determine the safety of the use of a new bacteriophage preparation effective in the treatment of wounds in 39 patients with chronic venous leg ulcers. Cocktails containing 8 lytic phages specific to Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia BACTERIOPHAGES AND THEIR APPLICATIONS

coli were administered topically to the site of the ulcers over 12 weeks without serious side effects. Another product that is under clinical investigation is PhagoBioDerm (http://www.intralytix.com). PhagoBioDerm is a biodegradable polymer matrix, composed of amino acids and impregnated with bacteriophage and ciprofloxacin. It is effective in the treatment of chronic bacterial infections of burns and wounds caused by various injuries. For many years now, it has been used to treat these conditions in Georgia, after which the cases were described in several publications. Examples are two loggers from Georgia who were exposed to radioactive strontium-90 and with subsequent extensive infection by S. aureus. One month after conventional therapy failed, PhagoBioDerm was applied and after another 7 days there was a significant decrease in numbers of S. aureus. Although, to date no large clinical study has been performed, this case highlights the possibility of using phage therapy in humans if conservative treatment fails. Biophage-PA is currently undergoing phase II clinical trials (I/II). It is a product of Biocontrol Limited (http://www. ampliphibio.com), which is used to treat infections caused by P. aeruginosa in chronic otitis. It contains 6 bacteriophages (each 105 PFU/ml) as a cocktail, dissolved in 10% glycerolin phosphate buffer, which is directly applied to the patient’s external auditory canal. In these clinical tests, 24 patients were enrolled and in 50% of them, there was a significant decrease in symptoms (only 20% in the control group). Additionally, after 3 weeks, there was an 80% reduction in numbers of bacteria. No side effects were observed during this therapy.

6. Conclusion The acceptance and prevalence of phage-based methods of detection, diagnostics and therapy of bacterial pathogens will expand along with related knowledge and technical progress. In order to survive, and propagate for millions of years, phage have evolved mechanisms to withstand environmental conditions and be able to infect hosts in complex conditions. They are logical candidates for future antibacterial strategies. The extensive research on CHAPTER 12

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phage will likely result in their general application in pathogen biocontrol and their commercial applications will soon become more widely available. One can assume that even the most virulent phage are safe for humans and will eventually become a part of health policy in order to preserve food safety and public health. However, several issues still need to be resolved. Firstly, the amount of phage in nature is vast, and therefore more robust and stable cost-effective and specific phage based probes should be identified. Secondly, when applying phages in practice, regardless of the field of usage, phage administration and routes of delivery, concentrations and timing of application are just some of the many parameters that must be set carefully according to results found in literature, since the efficacy of each phage or phage cocktail depends on the phage-host systems. As research efforts help to optimize transition from culture based to more complex systems, it is likely that phage based diagnostics or detection, therapy, and food safety approaches start to fulfil their potential role. Lastly, the future of phage exploitation is remains under governmental regulatory agencies that still show some incredulity regarding phage usage, mainly due to the lack of scientific evidence generated in fully controlled clinical trials under the control of ethical committees and in compliance with the highest regulatory standards. There is also a great need to educate all involved professionals and the general public about the advantages of phage use, in order to fully take the benefits of bacteriophage use into account.

Review Questions and Answers Q1. Please briefly characterize the life cycle strategies of bacteriophages. A1. Lytic cycle of phage – when the phage enters the cell, it starts to reproduce itself rapidly and independently on chromosome, using the cell`s enzymes. After gathering all virion components, nucleic acid is packaged into the virus head. Newly-formed phage particles are released into the environment, while causing the host lysis. In lysogen204


ic cycle of phage – when the phage enters the cells, its genetic material is incorporated into the bacterial chromosome (prophage). At this stage, phages do not directly lyse the host cell, nevertheless, as prophages they replicate along with the bacterial chromosome and are transmitted to the hosts next generation. When some harsh conditions occur, phage switches to lytic cycle. Q2. Could you name 3 different phage-based detection systems for targeting bacteria? A2. Lux reporter phage assay, phage amplification and phage-based quantum dots Q3. What are the main pros and cons of using the phage therapy as an alternative to antibiotic treatment of bacterial infections? A3. Pros: Bacteriophages infect strictly bacteria without having negative effects on humans. Phages are eliminated from environment, when no specific bacteria are present. Preparation of large amount of phages is fast and cheap, etc. Cons: Prior to bacteriophage application, their precise characterization, as well as the infectious bacterial agent is necessary . Although, the phage would be precisely defined in in vitro environment (as strictly lytic), we don’t know whether this cycle remain unchanged also under the physiological conditions of the human body, etc.

Further Readings 1. Ackermann HW, Prangishvili D. Prokaryote viruses studied by electron microscopy. Archives of Virology 2012;157:1843-9. 2. Arya SK, Singh A, Naidoo R, Wu P, McDermott MT, Evoy S. Chemically immobilized T4- bacteriophage for specific Escherichia coli detection using surface plasmon resonance. Analyst 2011;136:486-92. 3. Baggesen DL, Sorensen G, Nielsen EM, Wegener HC. Phage typing of Salmonella Typhimurium is it still a useful tool for surveillance and outbreak investigation? Euro Surveill 2010;15:19471. 4. Balogh B, Jones JB, Iriarte FB, Momol MT. Phage therapy for plant disease control. Current Pharmaceutical Biotechnology 2010;11:48-57.


5. Bigwood T, Hudson JA, Billington C, CareySmith GV, Heinemann JA. Phage inactivation of foodborne pathogens on cooked and raw meat. Food Microbiology 2008;25:400-6. 6. Boratynski J, Syper D, Weber-Dabrowska B, Lusiak-Szelachowska M, Pozniak G, Gorski A. Preparation of endotoxin-free bacteriophages. Cellular and Molecular Biology Letters 2004;9:253-9. 7. Brussow H, Fremont M, Bruttin A, Sidoti J, Constable A, Fryder V. Detection and classification of Streptococcus thermophilus bacteriophages isolated from industrial milk fermentation. Applied and Environmental Microbiology 1994;60:4537-43. 8. Bruttin A, Brussow H. Human volunteers receiving Escherichia coli phage T4 orally: a safety test of phage therapy. Antimicrobial Agents and Chemotherapy 2005;49:2874-78. 9. Calendar R. The Bacteriophages 2006. 10. Capparelli R, Parlato M, Borriello G, Salvatore P, Iannelli D. Experimental phage therapy against Staphylococcus aureus in mice. Antimicrobial Agents and Chemotherapy 2007;51:2765-73. 11. Carvalho CM, Gannon BW, Halfhide DE, et al,. The in vivo efficacy of two administration routes of a phage cocktail to reduce numbers of Campylobacter coli and Campylobacter jejuni in chickens. BMC Microbiology 2010;10:232. 12. Clokie MR, Millard AD, Letarov AV, Heaphy S. Phages in nature. Bacteriophage 2011;1:31-45. 13. Edgar R, McKinstry M, Hwang J, et al,. Highsensitivity bacterial detection using biotin-tagged phage and quantum-dot nanocomplexes. Proceedings of the National Academy of Sciences 2006;103:4841-5. 14. Esteban JI, Oporto B, Aduriz G, Juste RA, Hurtado A. A survey of food-borne pathogens in free-range poultry farms. International Journal of Food Microbiology 2008;123:177-82. 15. Favrin SJ, Jassim SA, Griffiths MW. Development and optimization of a novel immunomagnetic separation- bacteriophage assay for detection of Salmonella enterica serovar enteritidis in broth. Applied Environmental Microbiologyl 2001;67:217-24. BACTERIOPHAGES AND THEIR APPLICATIONS

16. Favrin SJ, Jassim SA, Griffiths MW. Application of a novel immunomagnetic separation-bacteriophage assay for the detection of Salmonella enteritidis and Escherichia coli O157:H7 in food. International Journal of Food Microbiology 2003;85:63-71. 17. Foley SL, Nayak R, Hanning IB, Johnson TJ, Han J, Ricke SC. Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production. Applied Environmental Microbiologyl 2011;77:4273-9. 18. Funatsu T, Taniyama T, Tajima T, Tadakuma H, Namiki H. Rapid and sensitive detection method of a bacterium by using a GFP reporter phage. Microbiology and Immunology 2002;46:365-9. 19. Gasanov U, Hughes D, Hansbro PM. Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: a review. FEMS Microbiology Reviews 2005;29:851-75. 20. Gorski A, Borysowski J, Miedzybrodzki R, Weber-Dabrowska B. Bacteriophages in medicine. Caister Academic Press 2007;126–58. 21. Gorski A, Miedzybrodzki R, Borysowski J, et al,. Bacteriophage therapy for the treatment of infections. Current Opinion in Investigational Drugs 2009;10:766-74. 22. Hagens S, Loessner MJ. Bacteriophage for biocontrol of foodborne pathogens: calculations and considerations. Current Pharmaceutical Biotechnology 2010;11:58-68. 23. Handa H, Gurczynski S, Jackson MP, Mao G. Immobilization and molecular interactions between bacteriophage and lipopolysaccharide bilayers. Langmuir 2010;26:12095-103. 24. Hanlon GW. Bacteriophages: An appraisal of their role in the treatment of bacterial infections. International Journal of Antimicrobial Agents 2007;30:118-28. 25. Hopkins KL, Desai M, Frost JA, Stanley J, Logan JM. Fluorescent amplified fragment length polymorphism genotyping of Campylobacter jejuni and Campylobacter coli strains and its relationship with host specificity, serotyping, and phage typing. Journal of Clinical Microbiology 2004;42:229-35. CHAPTER 12

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26. Chibani-Chennoufi S, Sidoti J, Bruttin A, Kut¬ter E, Sarker S, Brussow H. In vitro and in vivo bacteriolytic activities of Escherichia coli phages: implications for phage therapy. Antimicrobial Agents and Chemotherapy 2004;48:2558-69. 27. Chirakadze I, Perets A, Ahmed R. Phage typing. Methods in Molecular Biology 2009;502:293-305. 28. Jikia D, Chkhaidze N, Imedashvili E, et al,. The use of a novel biodegradable preparation capable of the sustained release of bacteriophages and ciprofloxacin, in the complex treatment of multidrug-resistant Staphylococcus aureus infected local radiation injuries caused by exposure to Sr90. Clinical and Experimental Dermatology 2005;30:23-6. 29. Kalantri S, Pai M, Pascopella L, Riley L, Reingold A. Bacteriophage- based tests for the detection of Mycobacterium tuberculosis in clinical specimens: a systematic review and meta- analysis. BMC Infectious Diseases 2005;5:59. 30. Kumari S, Harjai K, Chhibber S. Bacteriophage Treatment of Burn Wound Infection Caused by Pseudomonas aeruginosa PAO in BALB/c Mice. American Journal of Biomedical Sciences 2009;1:385-94. 31. Kumari S, Harjai K, Chhibber S. Efficacy of bacteriophage treatment in murine burn wound infection induced by klebsiella pneumoniae. Journal of Microbiology and Biotechnology 2009;19:622-8. 32. Kumari S, Harjai K, Chhibber S. Characterization of Pseudomonas aeruginosa PAO Specific Bacteriophages Isolated from Sewage Samples. American Journal of Biomedical Sciences 2009;1:91- 102. 33. Kutateladze M, Adamia R. Bacteriophages as potential new therapeutics to replace or supplement antibiotics. Trends in Biotechnology 2010;28:591-5. 34. Lakshmanan RS, Guntupalli R, Hu J, et al,. Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium. Journal of Microbiological Methods 2007;71:55-60. 35. Leszczynski P, Weber-Dabrowska B, Kohutnicka M, Luczak M, Gorecki A, Gorski A. Successful 206


eradication of methicillin-resistant Staphylococcus aureus (MRSA) intestinal carrier status in a healthcare worker-case report. Folia Microbiologica (Praha) 2006;51:236-8. 36. Letkiewicz S, Miedzybrodzki R, Fortuna W, Weber-Dabrowska B, Gorski A. Eradication of Enterococcus faecalis by phage therapy in chronic bacterial prostatitis - case report. Folia Microbio¬logica 2009;54:457-61. 37. Li J, McClane BA. A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates. PLoS Pathogens 2008;4:e1000056. 38. Loc Carrillo C, Atterbury RJ, el-Shibiny A, et al,. Bacteriophage therapy to reduce Campylobacter jejuni colonization of broiler chickens. Applied and Environmental Microbiology 2005;71:6554-63. 39. Loessner MJ, Rudolf M, Scherer S. Evaluation of luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods. Applied and Environmental Microbiology 1997;63:2961-5. 40. Mahony J, McAuliffe O, Ross RP, van Sinderen D. Bacteriophages as biocontrol agents of food pathogens. Current Opinion in Biotechnology 2011;22:157-63. 41. Matsuda T, Freeman TA, Hilbert DW, et al,. Lysis-deficient bacteriophage therapy decreases endotoxin and inflammatory mediator release and improves survival in a murine peritonitis model. Surgery 2005;137:639-46. 42. Matsuzaki S, Yasuda M, Nishikawa H, et al,. Experimental protection of mice against lethal Staphylococcus aureus infection by novel bacteriophage phi MR11. The Journal of Infectious Diseases 2003;187:613-24. 43. McGrath S, van Sindern D. Bacteriophage: Genetics and Molecular Biology. Caister Academic Press 2007. 44. McNerney R, Kambashi BS, Kinkese J, Tembwe R, Godfrey-Faussett P. Development of a bacteriophage phage replication assay for diagnosis of pulmonary tuberculosis. Journal of Clinical Microbiology 2004;42:2115-20.


45. Merril CR, Biswas B, Carlton R, et al,. Longcirculating bacteriophage as antibacterial agents. Proceedings of the National Academy of Sciences 1996;93:3188-92. 46. Monk AB, Rees CD, Barrow P, Hagens S, Harper DR. Bacteriophage applications: where are we now? Letters in Applied Microbiology 2010;51:363-9. 47. Newell DG, Koopmans M, Verhoef L, et al,. Food-borne diseases - the challenges of 20 years ago still persist while new ones continue to emerge. International Journal of Food Microbiology 2010;139 Suppl 1:S3-15. 48. O’Flaherty S, Ross RP, Coffey A. Bacteriophage and their lysins for elimination of infectious bacteria. International Journal of Food Microbiology 2009;33:801-19. 49. Petty NK, Evans TJ, Fineran PC, Salmond GP. Biotechnological exploitation of bacteriophage research. Trends in Biotechnology 2007;25:7-15. 50. Rhoads DD, Wolcott RD, Kuskowski MA, Wolcott BM, Ward LS, Sulakvelidze A. Bacteriophage therapy of venous leg ulcers in humans: results of a phase I safety trial. Journal of Wound Care 2009;18:237-8, 40-3. 51. Ripp S. Bacteriophage-based pathogen detection. Advances in Biochemical Engineering 2010;118:65-83. 52. Scott AE, Timms AR, Connerton PL, Loc Carrillo C, Adzfa Radzum K, Connerton IF. Genome dynamics of Campylobacter jejuni in response to bacteriophage predation. PLoS Pathogens 2007;3:e119. 53. Sergueev KV, He Y, Borschel RH, Nikolich MP, Filippov AA. Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR. PLoS One 2010;5:e11337. 54. Sillankorva SM, Oliveira H, Azeredo J. Bacteriophages and their role in food safety. International Journal of Microbiology 2012;2012:863945. 55. Silva J, Leite D, Fernandes M, Mena C, Gibbs PA, Teixeira P. Campylobacter spp. as a Foodborne Pathogen: A Review. Frontiers in Microbiology 2011;2:200. BACTERIOPHAGES AND THEIR APPLICATIONS

56. Singh A, Poshtiban S, Evoy S. Recent advances in bacteriophage based biosensors for food-borne pathogen detection. Sensors 2013;13:1763- 86. 57. Skurnik M, Strauch E. Phage therapy: facts and fiction. International Journal of Medical Microbiology 2006;296:5-14. 58. Smartt AE, Xu T, Jegier P, et al,. Pathogen detection using engineered bacteriophages. Analytical and Bioanalytical Chemistry 2012;402:3127-46. 59. Soni KA, Nannapaneni R, Hagens S. Reduction of Listeria monocytogenes on the surface of fresh channel catfish fillets by bacteriophage Listex P100. Foodborne Pathogens and Disease 2010;7:427-34. 60. Tanji Y, Furukawa C, Na SH, Hijikata T, Miyanaga K, Unno H. Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage. Journal of Biotechnology 2004;114:11-20. 61. Tanji Y, Shimada T, Fukudomi H, Miyanaga K, Nakai Y, Unno H. Therapeutic use of phage cocktail for controlling Escherichia coli O157:H7 in gastrointestinal tract of mice. Journal of Bioscience and Bioengineering 2005;100:280-7. 62. Tolba M, Minikh O, Brovko LY, Evoy S, Griffiths MW. Oriented immobilization of bacteriophages for biosensor applications. Applied and Environmental Microbiology 2010;76:528-35. 63. Uchiyama J, Rashel M, Takemura I, Wakiguchi H, Matsuzaki S. In silico and in vivo evaluation of bacteriophage phiEF24C, a candidate for treatment of Enterococcus faecalis infections. Applied and Environmental Microbiology 2008;74:4149-63. 64. Wang J, Hu B, Xu M, et al,. Therapeutic effectiveness of bacteriophages in the rescue of mice with extended spectrum beta-lactamase-producing Escherichia coli bacteremia. International Journal of Molecular Medicine 2006;17:347-55. 65. Wendlandt S, Schwarz S, Silley P. MethicillinResistant Staphylococcus aureus: A Food-Borne Pathogen? Annual review of food science and technology 2013;4:117- 39. 66. Wright A, Hawkins CH, Anggard EE, Harper DR. A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic resistant Pseudomonas aeruginosa; a preliminary report of efficacy. Clinical Otolaryngology 2009;34:349-57. CHAPTER 12

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OMICS SCIENCE

CHAPTER 13 OMICS SCIENCES Daniele Vergara, Pasquale Simeone, Claudia Toto, Michele Maffia


1. Genomics, Metabolomics, Lipidomics, Epigenomics .................................................... 211

2. The Post-Genome Era: Proteomics ............................................................................... 212

2.1 Methods for Protein Separation .................................................................................. 213

2.1.1 Gel-Based Proteomics: 2-DE .............................................................................. 213

2.1.2 Gel Free-Based Approaches ................................................................................ 214

3. Limitations of Current Proteomics Approach ............................................................... 215

3.1 Protein Abundance ..................................................................................................... 215

4. Clinical Applications of Proteomics ............................................................................. 217

5. Omics Science in 2013: Toward Omic Personalized Medicine ...................................... 219

Acknowledgement ................................................................................................................. 219 Summary Box ........................................................................................................................ 219 Review Questions and Answers .............................................................................................. 220 Further Readings ................................................................................................................... 220 OMICS SCIENCE

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CHAPTER 13 / D.Vergara, P. Simeone, C. Toto, M. Maffia

OMICS SCIENCE

Summary

T

he omic sciences of systems biology including genomics, transcriptomics, lipidomics, metabolomics, and proteomics, aim at understanding the biological mechanisms that give rise to the phenotype of an organism by using highthroughput technologies with the promise of great medical advances. The term “omics” represents the study of biological processes as systems. It deciphers the dynamic interactions between the numerous components of a biological system to analyze networks, pathways, and interactive relations that exist among them, such as genes, transcripts, proteins, metabolites, and cells. This new scientific vision has opened the way to new research strategies and experimental technologies that have transformed the study of virtually all life processes. Expansion of the “–ome” concept was incessant and has created a host of new terms, including bacteriome, cardiome, epigenome, erythrome, immunome, microbiome, neurome, connectome, osteome, physiome, proteinome, transportome, degradome, psychome, transcriptome, and many others. In this book chapter, different omics technologies are briefly introduced with a major focus towards proteomics.

1. Genomics, Metabolomics, Lipidomics, Epigenomics Genomics is the study of the genomes of organisms. For several years genomics was at the forefront of omic sciences, we were in the “Genomic Era”. Because many diseases are so intimately associated with genetic mutations, the idea that the solutions for human pathologies lie on genes has catalysed the interest of scientists for years, making genomebased analysis methods a central approach in omics science and setting the scene for the completion of the Human Genome Project (HGP), undoubtedly a major landmark event in the field of genomics after the discovery of the double-helical structure of DNA. Since the completion of the human genome project, our ability to explore genome function is OMICS SCIENCE

increased in specificity. In fact, substantial changes have occurred in the study of genome owing to the introduction of several approaches to DNA sequencing and expression. The massive quantification of messenger RNA (mRNA), genome copy number, and single nucleotide polymorphisms (SNPs) by microarray technology has enabled to assess the expression of tens of thousands of genes shedding light on the mechanisms underlying human pathologies, providing the basis for stratifying patients and predicting outcomes in a variety of diseases. Together with microarrays, recent advances in DNA sequencing with the introduction of next-generation sequencing (NGS) technologies have made possible an unprecedented extensive analysis of genome of individuals. Presently, there are three main NGS systems: the Roche/454 FLX, the Illumina/ Solexa Genome Analyzer, and the Applied Biosystems SOLiDTM System. Each one, by a different approach, seeks to amplify single strands of a fragment library and perform sequencing reactions on the amplified strands. Together with these technologies, a new generation of single-molecule sequencing technologies is now emerging offering advantages over current sequencing methods including small amounts of starting material (theoretically only a single molecule may be required for sequencing), and low cost. An important consequence of this new emerging scenario was the creation of multi-disciplinary teams and the formation of large-scale collaborative networks to handle and integrate these large amounts of data. The HGP was the first example of a large collaborative project; others include the Cancer Genome Atlas (TCGA) and the 1000 genome project. TCGA has achieved comprehensive sequencing, characterization, and analysis of the genomic changes of major types of human cancers providing also a platform for researchers to search, download, and analyse data sets generated by TCGA (http://cancergenome.nih.gov). The 1000 genome project aims to establish an extensive catalogue of human variations from 25 populations (www.1000genomes.org). The project provides an international open access resource that serves as CHAPTER 13

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a basis for subsequent phenotype related studies (www.1000genomes.org). Metabolomics is devoted to the study of global metabolite profiles in cells, tissues, and organisms. Most commonly used techniques for metabolomics are nuclear magnetic resonance (NMR), gas chromatography coupled to mass spectrometer (GC–MS) and mass spectroscopy (MS). Recently published papers describe the application of metabolomics in the study of heart disease, cancer, and other human pathologies. Another potential application of metabolomics involves the definition of biochemical pathways that contribute to drug response. Pharmaco-metabolomic signatures have also been identified for several drugs to predict individual responses to broader medical, dietary, microbiological or physiological challenges. Data generated experimentally by metabolomics are available in metabolite databases such as the Human Metabolome Database (HMDB). HMDB (www. hmdb.ca) is a resource dedicated to providing scientists with the most current and comprehensive coverage of the human metabolome. It contains information on biological properties, ontology, spectra and physical properties of human metabolites as well as data regarding metabolite concentrations, disease associations and tissue locations. Lipidomics, a sub-discipline of metabolomics, aims to define all of the lipid molecular species in a cell and understand how lipids function in a biological system. In detail, lipidomics involves the identification of individual cellular lipid species, including the type and number of individual atoms in each lipid species, and their stereoelectronic interactions with other lipids and proteins. Cells use 5% of their genes to synthesize their lipids that fulfil three main functions. Lipids not only forms the bilayer matrix, not only are used as energy storage, but can also act as second messengers and participate in signalling via specialized microdomains, lipid rafts, that have large amounts of lipids. The field of lipidomics is rapidly growing as demonstrated by the great utility of this approach to improve diagnostic–prognostic capabilities for human disorders, and for the identification of new classes of lipids. Early separation and 212

identification of lipids started with GC and HPLC (High Performance Liquid Chromatography), but other technologies coupled to chromatographic methods, such as MS, Matrix-assisted laser desorption-ionization/time of flight (MALDI/TOF), NMR, and quadrupole–linear ion trap (QTRAP), provide now a powerful approach to the global analysis of complex lipid mixtures. Given the enormous complexity of cellular lipidomics, it has been estimated to encompass around 180000 – 200000 different lipid species, high-throughput technologies are needed to approach the entire lipidome of cells. MALDI can also be used to reveal the distribution of lipids in tissues with the technique of imaging mass spectrometry (IMS) obtaining information relevant to the local distribution of lipids as they occur in tissues. The term ‘‘epigenetics’’ was originally coined by Conrad Waddington to describe heritable changes in a cellular phenotype that were independent of alterations in the DNA sequence. DNA methylation, the transfer of a methyl moiety from S-adenosylmethionine (SAM) to the 5-position of cytosines in certain CpG dinucleotides, represents the most studied of epigenetic processes with a great impact on gene expression. Evidence is mounting for a direct link between DNA methylation and human diseases. Chromatin changes are another central epigenetic process with a role in transcription, repair, replication, and condensation. Overall, there are now at least four different DNA modifications and 16 classes of histone modifications. Gene-specific techniques for determining DNA methylation include bisulfite sequencing, methylation-specific PCR (MSP) and quantitative MSP. The coupling of NGS platforms with established chromatin techniques such as chromatin immunoprecipitation (ChIP-Seq) represents the standard for identifying binding site locations for individual proteins and histone modifications.

2. The Post-Genome Era: Proteomics The complete characterization of all proteins has been the goal of proteomics since its inception


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more than 20 years ago. Proteins are the real-time executors of many biological functions and proteomics is the large-scale study of proteins, including their structures, localizations, post-translational modifications (PTMs), and functions. Proteomics experiments also provide information on protein interactions and complex formation. For example, proteins interact with each other as part of large complexes that serve to execute most biological processes including signal transduction, transcription, and translation. A literature search at the start of 2013 showed there were 38031 articles published on proteomics encompassing several research strategies. In both bacteria and eukaryotes, the cellular concentrations of proteins do not completely correlate with the abundances of their corresponding mRNAs. They often show a squared Pearson correlation coefficient of about 0.40, this means that6about 40 % of the variation in protein concentration can be explained by knowing mRNA abundances. This demonstrates that proteomics represents a complementary to genomics approaches. The classic proteomics screening methodology combine two different approaches. The first one, called expression-based proteomics, has the aim to define the expression of all proteins present in biological samples. Traditionally, it is performed through the combination of several sequential steps including protein extraction, separation and identification. The general starting point is the protein separation by an electrophoresis system, one- or two-dimensional electrophoresis (1-DE or 2-DE), and the subsequent identification of digested proteins by MS. Alternatively, proteins can also be digested using a specific protease and the resulting peptides separated and analysed immediately by MS. Such approach, namely as shotgun, is considered the method of choice for the large-scale analysis of proteins. The strength of this approach is that it is unbiased; a drawback is that the outcome relies on analysis and interpretation of experimental data. By contrast, targeted proteomic using multiple-reaction monitoring mass spectrometry (MRM-MS) allows the selective detection and quantification of OMICS SCIENCE

selected peptide ions. Such approach uses the capability of triple quadrupole mass spectrometers to act as ion filters. In a MRM-MS experiment, the precursor ion is isolated in the first quadrupole (Q1), fragmented within Q2 producing fragment ions that are monitored using Q3. The second approach, functional proteomics, aims to define the biological role of proteins and to identify protein–protein interactions, or interactomes. Protein complexes can be purified in several ways, one very common approach is to use an affinity tag to the protein of interest and purify the interacting partners. Proteomics has emerged more than two decades ago as a post-genomic technology with the promise to unravel the cellular mechanisms of diseases and to develop reliable markers of diagnosis or treatment. However, such studies remain challenging owing to the high degree of complexity of cellular proteomes, in particular the serum/plasma proteome, and the low abundance of regulatory proteins hidden by abundant proteins. Due to the enormous variation in protein diversity, there is currently no single methodological platform that can be used for a full characterization of the proteome.

2.1 Methods for Protein Separation The separation of all the proteins contained within cells, tissues, and biofluids remains a challenging analytical problem. Existing methodologies are not adequate to completely isolate and resolve the large number of proteins present at such different levels of concentration. Proteomic approaches can be classified as either gel-based or gel-free methods that can be further subdivided in “label-free” or “label-based”.

2.1.1 Gel-Based Proteomics: 2-DE Since its introduction by Kolin in 1954, protein separation by 2-DE has broadly affected life science research and successfully used applied to the study of biological or clinical samples for the purposes of identifying novel disease-specific protein biomarkers or gaining better understandings novel protein CHAPTER 13

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targets for therapeutic interventions and drug developments. During these years the technique has undergone several advances that have enhanced resolution, detection, quantitation, and reproducibility. One of the most notable improvements was the introduction of immobilized pH gradient (IPG) gels that led to standardized procedures for 2-DE permitting higher resolution and improved reproducibility for inter laboratory comparisons. More recently, the development of 2-D differential in-gel electrophoresis (DIGE) in 1997 overcame problems of reproducibility and quantitation because allowed running test and control sample in the same gel. Although there has been a significant progress towards liquid chromatography and MS methods to separate and analyse proteins, 2-DE still remain a popular technique for conducting proteomic studies. In a classical 2-DE experiment, proteins are separated according to their isoelectric point (pI) in the first dimension and by their molecular weight in second dimension. Proteins of interest are excised from the gel, proteolytically digested, and identified using MS (Figure 1). In a single run, up to 1,000 – 2,000 protein species from one complex sample can be separated. In specialised laboratories, using large-gel 2-DE method, the number of protein spots detected were drastically increased up to 10, 000.

Figure 1. A schematic workflow of two-dimensional gel electrophoresis (2-DE). 2-D gel electrophoresis is an experimental technique that combines two separation methods.

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Biological samples are grown under different conditions and total proteins are extracted and subjected to isoelectric focusing (IEF) (first-dimension electrophoresis), where proteins are separated according to their isoelectric point (pI). After first dimension, IPG-strips are re-equilibrated to the second dimension buffer conditions, and transferred to the SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels. Proteins on gels are visualized by MS-compatible stains, including Coomassie or silver staining. Software-based image analysis is then crucial for the biological interpretation of experiments. After statistical validation, differentially expressed spots from 2-DE gels are excised, and a tryptic digestion is performed to generate tryptic peptide mixtures of the proteins that are applied to MALDI- or LC-MS/MS for identification of the excised proteins. The peptide data then are compared with the entire protein database (Swiss-Prot, NCBI).

2.1.2 Gel Free-Based Approaches A high resolution liquid chromatography (LC) separation coupled on line with a mass spectrometer is the central component of a gel free-approach. Complex protein mixtures are digested by trypsin into polypeptides, which are then separated by LC and analysed by MS via an electro spray ionization (ESI) interface. For this purpose, chromatographic separations are performed using flow rates in the range of low nanoliter per minute (nano-flow liquid chromatography or nanoLC). The relative quantification of peptides usually involves either label-free or stable isotope labelling techniques to identify differences in protein abundances. The labelling methods can be classified into two main groups: chemical isotope tags and metabolic labelling. A variety of labelling approaches including, Isotope-Coded Affinity Tags (ICATs), Isobaric Tags for Relative and Absolute Quantification (iTRAQ), and Stable Isotope Labelling by Amino Acids in Cell Culture (SILAC) are valuable techniques in quantitative proteomic analysis. The rationale behind each labelling strategy is to create a mass shift that distinguishes identical peptides that exhibit the same chromatographic and ionisation properties, from different samples within a single MS analysis.


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3. Limitations of Current Proteomics Approach In recent decades gel based proteomics techniques became robust and reproducible, however two distinctive issues remains a challenge: the problems to detect low abundance and hydrophobic proteins. The question of under-representation of hydrophobic protein and in particular membrane proteins is well known and to explain this problem very different possible mechanisms have been proposed: (i) aggregation caused by the low solubility of these protein species in the aqueous media; (ii) protein loss over the sequential steps of the 2-DE processes; (iii) precipitation at the protein corresponding pI during isoelectrofocusing phase; (iv) expression in low copy numbers; (v) difficulties to identify them by MS than hydrophilic proteins. Several fruitful strategies were considered to solve the problem. Usually, the best strategy in 2-DE experiments is to solubilize proteins from the lipid layers by detergent and chaotropic salt. This can be performed by applying a solution of 2M thiourea, 8M urea, and 4% chaps. In the most recent years, other solutions were proposed: use of different zwitterionic detergents, nonionic n-dodecyl β-D-maltoside and zwitterionic amidosulfobetaine ASB-14, and 1,2-diheptanoyl-sn-glycero-3-phosphatdiyl choline (DHPC). Two organic solvents have been recommended for miscible extraction of red blood cells membrane proteins using methanol (MeOH), 2,2,2-tri- fluoroethanol (TFE) and urea. Despite the many solutions proposed in the recent years the problems with hydrophobic proteins, on 2D gels are widely unsolved. In fact, membrane protein solubility is low at their pI, and therefore membrane proteins tend to precipitate at their pI range. It is evident that the issue represents a builtin problem for all 2D electrophoresis systems IEFbased, for this reason IEF-free separation systems represent a natural alternative in the analysis of membrane proteins.

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3.1 Protein Abundance The large number of gene and gene splice-variants that encode proteins, as well as the extensive posttranslational modifications of eukaryotic proteins renders proteomic studies extremely difficult. The detection of specific, disease-related protein markers, notoriously difficult to identify, because expressed at low concentration, can be extremely challenging on a classical proteomic experiments where highly abundant proteins could obscure the rare ones. Consequently, there has been an extensive investment into developing techniques and methods capable of revealing the so named “hidden proteome”. This cannot be achieved by one single approach. In fact, several methods are used for the enrichment and visualization of the lowabundance proteins and also for the depletion of the high-abundance proteins. It was demonstrated that the 10% most-expressed gene products represented the 75% of the total protein content, and the 2/3 of less-expressed only 10% of the protein content. In this situation is simple to argue that the signal of high-abundance proteins tends to hide the signal of rare species. Some of these technical difficulties can be bypassed loading more sample, exploiting the great capacity of 2D gels, allowing many of low abundant proteins to be detected because above the detection limit. But high-loading approach is restricted by gel crowding and is related to the strong presence of normal and modified forms of high abundance protein species. This strategy gives gel with completely saturated zones with no increased performance in the visualization of low abundant species. Possible solutions proposed the use of giant gels with greater resolution and capacity. However, this technology is inadequate and difficult to use due to the extreme fragility of the gels employed in the analysis. To address these issues, analytical chemists have attempted to develop pre-fractionation methods to separate large numbers of proteins. Fractionation based-methods that take advantage of proteins function or structure are extensively used, allowing the isolation of specific proteomes: glyco-proteome CHAPTER 13

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Figure 2. Figure shows the 2-DE maps (pH 3-10) obtained from initial Jurkat cell line extract (top) as compared with those obtained from: (A) 2.5 mg/ml of proteins treated with library and eluted with RS solution (2 M thiourea, 7 M urea, 4% CHAPS); (B) 5 mg/ml of proteins treated with library and eluted with RS solution (2 M thiourea, 7 M urea, 4% CHAPS); (C) and (D) 14 mg/ml of proteins treated with library and eluted with RS solution (2 M thiourea, 7 M urea, 4% CHAPS) and RSA solution (2 M thiourea, 7 M urea, 4% CHAPS, acetic acid to pH 3.3), respectively.

by lectin columns, phospho-proteome by antiphospho-aminoacid antibodies or metal-chelating resins. However, these protocols do not resolve the challenge of signal suppression due to high-abundance species present. Immunodepletion columns, containing immobilized antibodies addressing the highest abundance proteins, were proposed as a possible solution. Though, these approaches cause the dilution of the initial sample, rendering it even more difficult to detect low-abundance proteins. In this scenario, combinatorial hexapeptide ligand libraries have arisen as a powerful method for sample handling and are recently used to better elucidate and obtain extensive information on the protein composition of complex samples like 216

serum, bile fluid, human urine, platelet extracts, and red blood cell lysate. The ligand libraries, designed as batch of chromatographic beads, are synthesized by modified Merrifield approach described by Lam and collaborators. The library consists of millions of affinity baits (hexapeptides) so that each bead comprises multiple copies of the same bait. The beads represent the affinity solid phase of chromatographic column. This hexapeptide ligand library is assembled in order to be able to bind each single protein species present in a given biological extract. On the basis of the saturation-overloading chromatographic principle the loaded proteins are captured by their respective specific ligand until saturation whereas the excess, unbound proteins are washed away. The proteins are captured from


OMICS SCIENCE

the peptide library by several and different combination of interacting forced: Van der Walls interactions, hydrogen bonding, structural docking, hydrophobic associations etc. Thus, high represented proteins species rapidly saturate their specific bead ligands while the excess of the same protein remains unbound. On the contrary, low-abundance proteins were concentrated by their ligand up to saturation. Washing steps eliminate the protein excess not bound to the library and are removed from the chromatographic column. The protein species bound are eluted by a single appropriate elution buffer able of destroy proteins–hexapeptides interaction or by a sequence of desorbing agents, each of them addressing a selected type of binding. These fractions can then be analysed using well-known methods, such as SDS-PAGE, 2D electrophoresis and MS. An application of hexapeptide libraries to cellular lysates is reported in Figure 2. Proteins obtained from the human T cell lymphoblast-like cell line Jurkat were used as starting material to show the feasibility of this approach. Jurkat cell line was maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics. Cells were harvested by centrifugation followed by lysis via sonification in Tris buffer containing protease inhibitors. Varying amounts of cell lysate (2.5 mg/ml, 5 mg/ml, 14 mg/ml) were subjected to column chromatography over a solidphase combinatorial ligand library (ProteoMiner, Biorad). Following washing, each individual column was subjected to two distinct elutions using a RS solution (2 M thiourea, 7 M urea, 4% CHAPS) and a RSA solution (2 M thiourea, 7 M urea, 4% CHAPS, acetic acid to pH 3.3) respectively. RSA eluted sample was precipitated with 2D Clean-Up (GE Healthcare) and resuspended in RS. For 2-DE studies, 80 μg of proteins were dissolved in sample buffer and isoelectric focusing of protein samples was carried out by using commercial 13 cm IPG polyacrylamide strips (pH 3 to 10 NL). Separation in the second dimension was carried out in 12% SDS-PAGE gels. Silver stained gels were scanned and analyzed by the software Image-Master 2-D Platinum. OMICS SCIENCE

4. Clinical Applications of Proteomics With the advent of proteomics several large-scale studies were launched to investigate the protein profile in different biological systems with the aim of discovering potential diagnostic and prognostic biomarkers. Specifically, this technology offers the possibility of identifying and quantifying proteins associated with a particular disease by means of their altered levels of expression and/or PTMs between the control and disease states. This type of comparative analysis enables correlations to be drawn between the range of proteins, their variations and modifications produced by a cell, tissue and bio-fluids and the initiation, progression, therapeutic monitoring or remission of a disease state. Then, clinical proteomics should be defined as the application of proteomic analysis with the aim of solving a specific clinical problem within the context of a clinical study. As clinical proteomics consists of a variety of experimental procedures, pre-analytical variability, as well as analytical and post-analytical procedures can markedly affect a proteomic experiment. Collection of appropriate clinical specimens (e.g. urine, blood, and tissue), duration of storage, number of freeze-thaw cycles, analysis of proteins and peptides of interest, data interpretation, data validation of protein dataset in a specific clinical context should effectively be standardized to reduce bias. As a result, recommendations concerning minimal information about a proteomic experiment (MIAPE) were released from the Human Proteome Organisation (HUPO). Reference materials are also expected to support both qualitative and quantitative proteomic measurements. Despite substantial progress in the field, clinical proteomic approaches have not matured into routine diagnostic applications. As described above, proteomic analysis of blood and other body fluids and tissues is extremely difficult due to the complexity of samples and the dynamic range of concentrations of proteins in biological fluids. Major CHAPTER 13

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challenges exist for plasma biomarkers discovery, where the large dynamic concentration range of up to ten orders of magnitude for plasma proteins and the presence of very high abundance proteins such as serum albumin and immunoglobulins mask the lower abundance plasma biomarkers. Moreover, to validate biomarkers in a clinical setting it is required the analysis of hundreds (and perhaps more) of high-quality clinical samples. In fact, a huge amount of data and samples is necessary to ensure a bio-statistical significance. This large set of samples is in contrast with the time consuming and intensive proteomic approach that are distant from the routine of a clinical laboratory. One of the major factors for successful proteomic analysis of clinical samples is the selection of an appropriate workflow. For instance, in studies that use biological fluids, samples should be pre-treated to remove high-abundance proteins (running the risk also to eliminate proteins of interest because of protein-protein interactions) or concentrated to enrich the protein fraction (in the case of urine). In biomarkers discovery, caution must be exercised in preserving samples for protein degradation, a problem that can lead to misinterpretation of data. Working with proximal fluids (synovial fluid, pleural fluid, peritoneal fluid, ascites) it should be necessary to eliminate the contamination of mucosa and salts before sample separation by 2DE-MS, LC-MS), or capillary electrophoresis coupled to mass spectrometry (CE-MS). With regard to clinical proteomics, among the strategies that have the highest potential to reduce the gap between proteomics and its clinical application there is the possibility to conduct a differential proteome analysis on tissue samples with the advantage to investigate the disease directly at the origin. Moreover, biomarkers present in tissue are more concentrated than those released in the blood making this biological sample suitable for specific isolation or fractionation schemas. However, researchers who work with this type of sample are well-aware that the great heterogeneity of human tissues represents a well-known limit in the investigation of 218

biomarkers. Several approaches were developed to overcome this problem with the potential to be clinically useful. 2D electrophoresis or SELDI (surfaceenhanced laser desorption/ionization) have been coupled to laser capture microdissection (LCM), allowing the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization, or laser microdissection and pressure catapulting techniques (LMPC). In this last procedure, after microdissection, the sample is directly catapulted into an appropriate collection device. As the entire process works without any mechanical contact, it enables pure sample retrieval from morphologically defined origin without cross contamination. Among the strategies that have the highest potential to reduce the gap between proteomics and its clinical application there is the possibility to conduct a differential proteome analysis directly on tissue samples with the advantage to investigate the disease directly at the origin. In these years, MALDI Imaging has emerged has another promising technique for the combined morphologic and molecular tissue analyses. In detail, MALDI Imaging allows to image/profile intact tissue sections placed onto a conductive glass side obtaining information about protein expression and localization. Because of its practical simplicity and ability to obtain reliable information from tissue section, MALDI imaging might have the potential to complement histopathologic evaluation for assisting in diagnostics, patient stratification, or predicting drug response. Together with technological challenges, other issues that could affect proteomic results should not be underestimated including harvesting, handling and storage of samples. Considering that the proteome is dynamic over time and expression of a myriad of factors, researchers should also consider the clinical history of the patient such as age, sex, and race. To minimize these systemic problems is desirable to establish a specimen bank (biorepository). A sample, to become eligible for a biorepository, must be collected and analysed immediately because cells and proteins degradation and/or modification may affect the analysis. Moreover, sample should be subject to


OMICS SCIENCE

an accurate quality control and catalogued according to trusted, safe and standardized clinical data. Based on these observations we conclude that proteomics can be considered as a main strategy for biomarker discovery. However, special attention has to be paid to reduce pre-analytical variables, analytical variability, and biological variation. This will require a close interdisciplinary collaboration involving clinicians, statisticians/bioinformatics, epidemiologists, chemists, biochemists and biologists.

5. Omics Science in 2013: Toward Omic Personalized Medicine The end of the 20th century was marked by the genomics revolution. However, over the past decades it has become clear that common diseases develop as a result of multiple defects at different levels including proteins, lipids and metabolites, defects can that cannot be completely predicted by the simple analysis of genes. A systems-level approach, that integrate the results of genomics with those obtained by the analysis of metabolomes and proteomes, has enabled researchers to utilize novel strategies to tackle unexplored research questions in human diseases. The ultimate goal is to evolve an integrated omics picture of the genes, transcripts, proteins, and metabolites to fully describe cellular functioning. This will be important for diagnosing and treating of human diseases which developed as a consequence of the dysfunction of multiple systems, including DNA mutations, epigenetic mechanisms, and altered signalling pathways, but also for predicting disease onset. In fact, the greatest benefits for patients are likely to be realized from the monitoring and management of early stage disease rather than from treatment of late stage disease. In this field, a comprehensive integrative omic profiles was applied with success to perform an integrated Personal Omics Profiling (iPOP) on a single healthy individual. Authors of this study combined genomic, transcriptomic, proteomic, metabolomic, and autoantibody profiles, in conjunction with routine laboratory testing, from a single OMICS SCIENCE

individual over a 14 months period, generating an individual iPOP over the course of healthy states and two viral infections that occurred during the study interval. Peripheral blood mononuclear cells, plasma and serum were collected and results from whole-genome sequencing predicted an increased disease risks for various diseases, including hypertriglyceridemia, and type 2 diabetes (T2D). Markers associated with T2D became elevated during the course of study, in particular, following a respiratory infection. Moreover, data from omics experiments before and after viral infections allowed for the creation of a dynamic picture of this process. Omics-based approaches will identify at risk groups supporting the implementation of risk-stratified health screening. This may lead to significant costsavings at the societal level.

Acknowledgement This work was supported by the PS105 ARTI strategic project “Development and realization of biochip for molecular diagnostic and typization of human pathogenic viruses (HPV, HCV)” of Apulia Region. We also acknowledge support by the ‘‘ANGELA SERRA’’ Foundation for Cancer Research (Parabita - Lecce, Italy), the PONa3_00334 “Research Center for Environment and Human health”, the PON02_00563_34847 “RINOVATIS”, and the PRIN 2010FPTBSH “NANO Molecular Technologies for Drug delivery - NANOMED”.

Summary Box Given the complexity of cellular systems, several techniques have been developed over the years for the comprehensive analysis of molecular components. In particular, the advent of omic sciences has changed the way in which human diseases are studied making possible the simultaneous interrogation of thousands of molecular species at the system level. It was a technological revolution that modified the way in which experiments are designed, moving from mostly hypothesis- based approaches to studies that are largely hypothesis free. In this book CHAPTER 13

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chapter, we briefly discuss classical omics approaches including next-generation sequencing and metabolomics. More in detail, we have opted to focus our attention on proteomics, a complementary approach to genomics that over these years has led to important insights in the comprehension of cellular biological processes and human diseases. Current experimental limitations including the enormous complexity and the dynamic nature of proteomes are also discussed. When integrated among them, omics approaches have the great potential to provide insight into the molecular alterations that drive disease pathogenesis.

Review Questions and Answers Q1. What do we intend for unbiased experimental approaches? A1. There are no specific assumptions about which molecules are likely to be involved in a particular process. Biomarkers discovery experiments are generally performed on an omics-platform for a global unbiased analysis of the samples. Q2. What are the main steps of proteomic analysis of human samples? A2. A typical proteomic workflow starts by extracting proteins from tissues or biological fluids. Proteins are then separated of fractionated before MS analysis. MS is used to identify and quantify the proteins present in the sample.

Q5. Which are the principal methods to reduce protein complexity? A5. Proteins can be fractionated by several methods including SDS-Page or 2-DE, chromatography, immunoprecipitation, affinity depletion, ligand libraries, phospho- or glycocapture.

Further readings 1. Keusch GT. What do omics mean for the science and policy of the nutritional sciences? The American Journal of Clinical Nutrition 2006;83:520S522S. 2. Venter JC, Adams MD, Myers EW, et al,. The sequence of the human genome. Science 2001;291:1304-51. 3. Lander ES, Linton LM, Birren B, et al,. Initial sequencing and analysis of the human genome. Nature 2001;409:860-921. 4. Xuan J, Yu Y, Qing T, et al,. Next-generation se¬quencing in the clinic: Promises and challenges. Cancer Letters 2012;pii: S0304-3835(12)006726. 5. 1000 Genomes Project Consortium, et al,. An integrated map of genetic variation from 1,092 human genomes. Nature 2012;491:56-65. 6. Jennifer LS, Natalie JS, S. Gail Eckhardt, Clinical Applications of Metabolomics in Oncology: A Review. Clinical Cancer Research 2009;15:431– 440.

Q3. How do I prepare my sample for 2-DE analysis?

7. Loizides-Mangold U. On the future of mass spectrometry based lipidomics. FEBS J 2013;280:2817-29.

A3. Proteins must be solubilized in a buffer containing chaotropic agents, and zwitterionic detergents such as CHAPS.

8. Dawson MA, Kouzarides T. Cancer epigenetics: from mechanism to therapy. The Cell 2012;150:12-27.

Q4. What is a mass spectrometer?

9. Vogel C, Marcotte EM. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nature Reviews Genetics 2012;13:227-32.

A4. Mass spectrometer is an instrument that ionizes molecules and separates the generated ions according to their mass-to-charge (m/z) ratio. Mass spectrometers consist of an ion source, a mass analyzer, and a detector.

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10. Kolin, A. Separation and concentration of proteins in a pH field combined with an electric field. The Journal of Chemical Physics 1954;22:16281629.


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11. Vergara D, Martignago R, Bonsegna S, et al,. IFN-beta reverses the lipopolysaccharide-induced proteome modifications in treated astrocytes. Journal of Neuroimmunology 2010;221:115-20. 12. Bjellqvist B, Ek K, Righetti PG, et al,. Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. Journal of Biochemical and Biophysical Methods 1982;6:317-39. 13. Unlü M, Morgan ME, Minden JS. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 1997;18:2071-2077. 14. Klose J, Kobalz U. Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis 1995;16:1034-59. 15. Wilkins MR, Gasteiger E, Sanchez JC, et al,. Two-dimensional gel electrophoresis for proteome projects: the effects of protein hydrophobicity and copy number. Electrophoresis 1998;19:1501-5. 16. Corthals GL, Wasinger VC, Hochstrasser DF, et al,. The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis 2000;21:1104-15. 17. Rabilloud T, Adessi C, Giraudel A, et al,. Improvement of the solubilization of proteins in two di-mensional electrophoresis with immobilized pH gradients. Electrophoresis 1997;18:307-16. 18. Chevallet M, Santoni V, Poinas A, et al,. New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 1998;19:1901-9. 19. Duncan R, McConkey EH. How many proteins are there in a typical mammalian cell? Clinical Chemistry 1982;28:749–55. 20. Wilkins MR, Sanchez JC, Williams KL, Hochstrasser DF. Current challenges and future appli-

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cations for protein maps and post-translational vector maps in proteome projects. Electrophoresis 1996;17:830–8. 21. Herbert BR, Righetti PG, Citterio A, et al,. Sample preparation and prefractionation techniques for electrophoresis-based proteomics. In Proteome Research 2007;15–40. 22. Echan LA, Tang HY, Ali-Khan N, et al,. Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma. Proteomics 2005;5:3292- 303. 23. Sennels L, Salek M, Lomas L, Boschetti E, Righetti PG, Rappsilber J. Proteomic analysis of human blood serum using peptide library beads. Journal of Pro¬teome Research 2007;6:4055-62. 24. Mischak H, Apweiler R, Banks RE, et al,. Clinical proteomics: A need to define the field and to begin to set adequate standards. Proteomics Clin Appl 2007;1:148- 56. 25. Emmert-Buck MR, Bonner RF, Smith PD, et al,. Laser capture microdissection. Science 1996;274:998-1001. 26. Caprioli RM, Farmer TB, Gile J. Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS. Analytically Chemistry 1997;69:4751-60. 27. Chaurand P, Caprioli RM. Direct profiling and imaging of peptides and proteins from mammalian cells and tissue sections by mass spectrometry. Electrophoresis 2002;23:3125-35 28. Franck J, Arafah K, Elayed M, et al,. MALDI imaging mass spectrometry: state of the art technology in clinical proteomics. Molecularl Cell Proteomics 2009;8:2023-33. 29. Chen R, Mias GI, Li-Pook-Than J, et al,. Personal omics profiling reveals dynamic molecular and medical phenotypes. The Cell 2012;148:1293307.

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ENZYMES AND PROTEINS - A BIOTECHNOLOGY TAILORING POINT OF VIEW

CHAPTER 14 ENZYMES AND PROTEINS - A BIOTECHNOLOGY TAILORING POINT OF VIEW Yusuf Deeni, Nuruddeen Sojimade CONTENTS Summary ............................................................................................................................... 225

1. Enzymes ....................................................................................................................... 225

2. Enzyme Engineering .................................................................................................... 225

2.1 Directed Evolution .................................................................................................... 226

2.2 Semi-Rational Design ................................................................................................ 226

2.3 Rational Design ......................................................................................................... 226

2.4 De Novo Design ........................................................................................................ 226

3. Drug Metabolising Enzymes ........................................................................................ 227

3.1 Cytochrome P450 Enzymes: Structure, Evolution And Function ............................... 228

3.1.1 Engineering of Cytochrome P450s ..................................................................... 229

4. P450 Enzyme Engineering and Methodology Tools ..................................................... 229

4.1 Polymerase Chain Reaction ........................................................................................ 230

4.2 Transformation of Cells .............................................................................................. 230

4.3 Purification of Protein ................................................................................................ 230

4.4 SDS Polyacrylamide Electrophoresis and Western Blot .............................................. 231

4.5 Lysate Assay ............................................................................................................... 231

5. Case Study .................................................................................................................... 232

5.1 Cytochrome P450 CYP2D6-NADPH Reductase Fusion Protein ................................ 232

5.2 Change of Selectivity (Regioselectivity and Enantioselectivity) .................................... 233

5.3 Change of Protein Stability (Thermostability) ............................................................. 234

5.4 Designing and Tailoring (Redesigning) Enzyme for New Substrates/Reactions ............ 234

5.5 Sequence-Based Enzyme Redesign .............................................................................. 235

5.6 Structure-Based Enzyme Redesign ............................................................................... 235

5.7 Computational Enzyme Redesign ............................................................................... 236

5.8 De Novo Enzyme Redesign (Rosetta) ........................................................................... 236

6. Conclusions .................................................................................................................. 236

Review Questions and Answers .............................................................................................. 237 Further Readings ................................................................................................................... 237 ENZYMES AND PROTEINS - A BIOTECHNOLOGY TAILORING POINT OF VIEW

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Summary

E

nzymes are biological catalyst essential to life, which have highly evolved to carry out specialised functions in cells, tissues and organs. Most enzymes are proteins built from a set of 20 naturally occurring amino acids and structurally ascribed to function under certain environmental conditions within certain space and time. Thus protein enzymes are naturally or artificially engineered to degrees of suitability, sensitivity, specificity, selectivity, flexibility, malleability, efficiency and stability. These properties typify enzymes, but are however not perfect for catalysts in bioprocessing and in large-scale manufacturing. Protein enzymes can therefore be engineered or designed industrially to enhance or alter them and their properties into suitable and stable catalysts. This chapter introduces concepts of protein enzyme engineering or tailoring from biotechnological perspectives and applications. It used some case studies to highlight, support and re-enforce the concepts. Different concepts, like direct evolution approach, rational approach, semi-rational approach and de novo approach, have been applied to engineer or make enzymes. These approaches have led to the evolution of a logical basis of forecasting experimental findings, and given hypothesisdriven enzyme engineering a boost over discovery based methods. In the discourse some practical knowledge and novel applications of biotechnology regarding certain aspects of protein enzymes and their engineering, especially the cytochrome P450 monooxygenases (P450 or CYP) as drug metabolising enzymes (DMEs), are embedded in the chapter. The emphasis on P450s is informed by (1) P450s constitute a superfamily of structurally diverse and functionally versatile enzymes with over 15 000 known genes distributed across biological species, genera, families, orders, classes, phyla and kingdoms; (2) P450s can catalyse a wide spectrum of complex biotransformations; (3) the academic and industrial drive and motivation for using P450s to develop and produce new medicines and agrochemicals, as well as the use of their enzymatic ENZYMES AND PROTEINS - A BIOTECHNOLOGY TAILORING POINT OF VIEW

properties in other biotechnological applications and innovations.

1. Enzymes Enzymes are biological reagents that stimulate essential life activities continually under the most benevolent experimental and environmental conditions. They are proteins that have highly evolved to carry out specialised functions in cells, tissues and organs. Most enzymes are built from a set of 20 naturally occurring amino acids except for few that are made from RNA e.g. ribozymes. These assembled amino acids carry functional groups that structurally support the enzyme molecules and also provide the reactive centres and species necessary for their catalytic functions. Characteristically, enzymes are sensitive, extremely active, very specific, highly selective, and unstable at certain temperature, and operate well in an aqueous environment. Although these properties typify enzymes, they are however not perfect for as catalysts in large-scale manufacturing and unfavourable in bioprocessing. They can therefore be engineered industrially to enhance or alter them into more suitable and stable catalysts. Enzymes are very diverse in nature and function some representatives of which are the drug metabolising enzymes (DMEs). This chapter will present a general and simplistic overview on enzyme engineering concepts. Then use DMEs to introduce and exemplify the concept of enzymes, their structures and functions, as well as their evolution. A general overview regarding the activity, selectivity, stability, substrate specificity of DMEs will be highlighted. The application of myriad biotechnological tools to adjust some of the structural or functional parameters for a tailored DME property will also be addressed. Finally, other complementary approaches like in silico and de novo design or redesign of enzymes will be highlighted.

2. Enzyme Engineering Enzyme engineering has so far proved to be a formidable biotechnological instrument for creating a more robust enzyme that can function as bioCHAPTER 14

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catalyst to stimulate biochemical transformations. Engineered enzyme can be employed for industrial purposes as well as in evaluating protein structure and function relationships. Enzyme engineering involves the alteration of protein sequence leading to a modified structure and to an enzyme with enhance functionality such as thermo-stability, specificity and selectivity towards non-natural substrates. Thus, the limitations posed by natural enzymes as a biocatalyst on industrial scale are being surmounted. Different approaches have been applied to engineer enzymes which include; direct evolution approach, rational approach, semi-rational approach and de novo approach (Figure 1). These approaches have thus far led to the evolution of a logical basis of forecasting experimental findings, giving hypothesis-driven enzyme engineering a boost over discovery based methods.

2.1 Directed Evolution Targeted enhancement of enzymes characteristics by directed evolution approach has revolutionised enzyme engineering and it involves the creation of modified libraries of genes and the use of sophisticated screens that select for specific features of an enzyme (Figure 4). This approach begins with pinpointing a target enzyme to be optimized and subsequently cloning of its gene which is then used as template for the next round of directed evolution. In contrast to rational design, directed evolution can be used to modify and generate a functional naturally occurring enzyme that possesses either one or all of the following features; activity, stability, selectivity and solubility without comprehensive information on the structure, function or mechanism of the enzyme. Directed evolution has utilised advancement in molecular biology techniques to achieve success in the creation of enhanced enzymes that are highly sort after in industries.

2.2 Semi-Rational Design This approach employs site-saturated mutagenesis to alter multiple residues of enzymes whose structural or functional properties are already known 226


leading to the creation of a smart library that will produce beneficial result. Semi-rational approach uses a combination of protein sequence, properties and computational predictive algorithms to identify potential target sites with reduced sequence diversity. Semi-rational approach is more desirable in circumstances without advanced screening technique because it makes the generation of small libraries quite easy and encourages development and use of ‘less-throughput’ techniques for the evaluation of library members, unlike in directed evolution approach where high-throughput screening method is required.

2.3 Rational Design Rational approach is the earliest method used in enzyme engineering and it has shown to be quite valuable in modifying an enzyme’s reaction process to catalyse new reactions, altering, expanding and improving on substrate specificity. In rational approach, the emphasis is on mutations that are close to the active site of the protein that are created through site-specific mutagenesis. Rational approach is however much compounded and requires comprehensive knowledge of the target enzyme’s structure and function. This requirement is difficult to guarantee due to the structural dynamism of enzymes. This makes the approach limited in its application. However, the approach has the potential of improving the chance of functional mutations and the library size required is reduced, which equally reduces the time needed for screening the library.

2.4 De Novo Design Enzymes are said to be created de novo if they are created from the scratch independent of any parent enzyme’s substrate or reaction machinery and so far very few enzymes has been engineered de novo. The task of generating an enzyme de novo begins with in silico (computational) rational design and dependent on dependable structure estimation, provisos for protein stability and good knowledge of the inter-molecular interactions of enzymes.


Figure 1. Schematic overview of protein engineering approaches and requirements for successful enzyme engineering.

De novo design is driven by the desire to generate enzymes with enhanced functions, wider substrate flexibility and novel properties needed to develop improved industrial products. The main limitation to encouraging the industrial adoption of this method is that since de novo designs are not dependent on natural sequences, products of this engineering approach are less functional compared to natural enzymes. However, this limitation would be overcome in due course with advancement in the understanding enzymes’ structure-function as well as in the field of synthetic biology. The potential of de novo enzyme engineering cannot be overemphasised considering the fact that it does not require prior knowledge of enzyme properties and functions. ENZYMES AND PROTEINS - A BIOTECHNOLOGY TAILORING POINT OF VIEW

3. Drug Metabolising Enzymes Drug metabolising enzymes (DMEs) are known to metabolise several exogenous and endogenous compounds or xenobiotics ranging from drugs, environmental pollutants, steroids to prostaglandins and are particularly noted for exhibiting a degree of specificity for their substrates. Theoretically, DMEs are categorised into either oxidative or conjugative DMEs. The oxidative DMEs hydroxylate or demethylate a compound by attaching an oxygen atom into its substrate, while the conjugative DMEs are central to the metabolic deactivation of pharmacologically functional substances via conjugation reactions such as glucuronidation. Metabolism carried out by DMEs consists of three phases (I, II & III). Phase I DMEs introduce a polar group CHAPTER 14

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into the substrate molecule, increase its water solubility and most importantly render the xenobiotic to a suitable substrate for Phase II reactions. Phase II DMEs altered substrates and complexes to produce a water-soluble conjugation product that is readily excreted. Phase III DMEs transport (uptake/entry and export /exit) both exogenous and endogenous xenobiotics, including the Phase I and phase II altered complexes and soluble conjugation products that are readily excreted. Important phase I metabolising enzymes are the Cytochrome P450 (CYP450) enzymes. CYP450s usually modify substrates molecules by the attachment of an oxygen atom to the molecules leading to the hydroxylation or de-methylation of such molecules (Evans and McLeod, 2003). They are present in all organisms in the biological kingdom and their activities are determined by a diverse super family of genes which if altered can have major impact on enzyme substrate products, activity and specificity.

3.1 Cytochrome P450 Enzymes: Structure, Evolution and Function Cytochrome P450s are highly versatile enzymes that were discovered in 1958 to be pigmented molecules with a distinctive UV absorption soret maximum at 450 nm (Figure 2) following their binding by carbon monoxide. They have been widely studied by scientists for reasons ranging from their multifaceted chemical mechanism to their part in primary and secondary metabolism, as well as their significant role in the biotransformation of drugs. The CYP450 enzyme biocatalyst system is made up of a P450 and a NAD(P)H-associated P450 reductase which it employs in catalysing monooxygenation reactions and the atomic structures (crystals) for several P450 variants have been established. The numbers of available structures since the identification of the first CYP450 structure are adequate enough to suggest that CYP450s are structurally conservative with similar fold that is exclusive to this group of enzyme (http://drnelson.uthsc.edu/ CytochromeP450.html) and in particular tailored 228


for redox partners binding, oxygen activation and substrate recognition. This unique fold has two major subdomains consisting of larger a-helix-rich (alpha helices) and smaller b-sheet-rich (beta sheets). CYP450s can be grouped into four classes on the basis of the partners they required for redox reaction. Class I require flavin adenine dinucleotide (FAD) containing NADPH P450 reductase. Class II require a permeating FAD/flavin mononucleotide (FMN)-containing NADPH P450 reductase). Class III however have endogenous substrates (endoperoxide or hydroperoxide) that they use in reaction and do not need external supply of electrons and are found in mammals and plants. Class IV do not need external supply of electron because they receive supply directly from reduced pyridine nucleotides. CYP450s in prokaryotes and eukaryotes are evolutionarily divergent with former being soluble cytoplasmic enzymes while the latter are membrane bound proteins. Eukaryotic CYP450s are usually within 400 to 600 primary amino acids chain in length and are divided into three main groups depending upon their subcellular localisation. Many of them are either found in endoplasmic reticulum (microsomal type), in the mitochondria (mitochondrial-type) or are cytosolic type. There may possibly be some CYP450s in the nucleus (nucleolar), however, the existing evidence is weak, quite debatable and somewhat controversial. CYP450s seem to have evolved as a result of the need for them to be able to manage specific substrates. Most of the variation in CYP450 enzymes appears to stem from gene duplication leading to their attainment of new roles. Despite the characteristics substrate-dependent specificity of most CYP450s, a host of them demonstrate significant level of substrate promiscuity. Majority of CYP450s enzymes are utilised endogenously for growth regulation, biosynthesis of toxins and pigments formation as in plants. In metazoans (human included), CYP450s undertake many functions ranging from biosynthesis, biotransformation and bioconversion of both edogenous and


exogenous xenobiotics, including such physiologically vital compounds like steroids, fatty acids, fatsoluble vitamins and degradation of herbicides and insecticides. In spite of their versatility, fewer mammalian CYP450s are being utilised industrially because they are membrane-associated and naturally insoluble, coupled with low level of expression and low activity level that is not sufficient to act as biocatalysis. It will therefore be of significant value if the activity level of CYP450 enzymes can be enhanced by adjusting their properties such as substrate selection, selectivity (regio- and stereo-), inhibition, thermostability, solvent tolerance, oxidative stability, and substrate and product tolerance to give desired results, as well as solubility. The tasks of adjusting CYP450 enzymes in new products are enormous and emphases are recently being place on it through protein engineering.

3.1.1 Engineering of Cytochrome P450s CYP450s has been engineering for decades with the aim of making recombinant P450s that are soluble in order to be able to crystallise them before ascertaining their structure, which precedes the engineering of unique function for industrial applications. This aim is so far elusive to fully actualise due to the inadequate understanding of basic structure– function relationships in CYPP450s. Equally, the inability to identify, clone and co-express the redox partner required for effective P450 activity has also been an obstacle to CYP450 engineering, since P450s need redox reducing partners to bind NAD(P)H reductase to execute their catalytic functions. Redox partner fusion was proposed as a formidable approach to surmounting the problem of identifying and cloning natural redox partners. The CYPP450 fusion species to be identified was the flavocytochrome P450 BM3 from Bacillus megaterium. It fuses to a mammalian-like diflavin reductase as its redox partner to create a model soluble enzyme known as CYP102A1. Following the initial generation of this recombinant P450 (CYP102A1), many research has been carried out using enzyme engineering approaches such as ENZYMES AND PROTEINS - A BIOTECHNOLOGY TAILORING POINT OF VIEW

rational or random design to engineered the wildtype CYP102A1 to produce new enzymes with tiny structure-activity relationship to the native substrates of CYP102A1. P450 BM3 is a catalytically independent enzyme (self-sufficient), a character which when fuse with obtainable crystal structures and proficient purification techniques makes the enzyme a model candidate for protein engineering. Mutant BM3 that can competently metabolise both natural and artificial substrates with enhanced properties and altered regio-and stereo-selectivity has been generated. P450 BM3 is a highly adaptable, desirable and standard enzyme with the highest activity level ever recorded for P450s; a kcat value of up to 17 000 min1.

4. P450 Enzyme Engineering and Methodology Tools The evolution and rapid development of new technologies particularly that of recombinant DNA technology has greatly influence and enhance protein engineering thus making new enzymes that were not naturally available a possibility. These new enzymes with improved structural flexibility and superior properties are create from genetic manipulation using an array of molecular and biochemical techniques. The goal of enzyme engineering is to induce the transcription/translation of enzyme gene into protein at the cellular level and this is achieved by the construction of an artificial expression system that consists of gene isolation, cloning onto a plasmid vector and transformation into a host cell. The two most important expression system used in enzyme engineering is that of the bacterium Escherichia coli (has no endogenous P450 or P450 reductase genes) and the yeast Saccharomyces cerevisiae (has three P450s and one associated NAD(P)H P450 reductase gene). Mammalian cell expression systems could also be developed including transgenic models. Generally, the focus of enzyme engineering is to exploit a particular property of an enzyme to improve on such property within the enzyme or introduce CHAPTER 14

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a desired property into another enzyme where such is lacking by the fusion of two enzymes (fusion enzyme). These are accomplished by the alteration of enzyme properties through the substitution of amino acid residue(s) known as site-specific mutagenesis, replacement of active sites between related enzymes through DNA shuffling, and/ or the introduction of an enzyme’s active site into protein fragment scaffold of the enzyme that is without its original active site. The capability to successfully create new enzymes is dependent on proper accomplishment of some of the following techniques; polymerase chain reaction (PCR), restriction enzyme digestion, gel electrophoresis, ligation, cloning and transformation. These techniques and how they are applied in enzyme engineering will be briefly discussed below using the cytochrome P450 enzymes as an example.

4.1 Polymerase Chain Reaction PCR is essentially used to replicate nucleic acids through the use of primers designed to specifically target a particular area of interest. There are several types of PCR and in the case of enzyme engineering, error-prone PCR is usually used to create a sort of hybrid DNA/gene often referred to as a library by bringing together similar DNA sequences from different parent genes through a method called DNA Shuffling. We have used the technique while constructing a human fusion enzyme, cytochrome P450 CYP2D6 NADPH-P450 reductase. Similar approach was also employed to create and express an artificial human gene, cytochrome P450 3A4, on the surface of bacteria (Escherichia coli; E. coli). Expression plasmid is used as a vehicle for holding each part of the fused enzymes above following the amplification by PCR of the newly created gene fragments, digestion, ligation, gel purification and then authentication by sequencing.

4.2 Transformation of Cells Transferring the plasmid encoded genetic material of the hybrid enzyme into a host cell is called transformation and it is achieved with the help of competent cells. Transformation is a two-step process 230


that requires the introduction of the cloned enzyme (cDNA) carried in a plasmid vector into the host cell and the antibiotic selection of cells that were able to take up the introduced plasmid encoded cDNA. The bacteria (usually E. coli) normally used in transformation is not naturally competent they therefore need to be made competent before transformation. DNA can be introduced DNA into E. coli through chemical treatment or electroporation. One of the aims of transformation is to exploit the bacterial host genome and machinery to produce more of the introduced DNA.

4.3 Purification of Protein This is a routine procedure following the successful transformation into the host bacteria in order to ascertain whether the intended cloned DNA is enclosed in the plasmid and it requires picking a single colony from the transformants and culturing it overnight. Other process such as cell harvesting, lysis and plasmid and nucleic acid recovery and column affinity purification follow the culturing. These procedures are what make up the miniprep or midiprep plasmid isolation/purification, and simple kits are commercially and readily available. The cloned enzyme of interest can also be expressed and isolated for further usage and characterisation. In the case of cloned P450s the membrane preparations or microsomes can be made from the P450 expressing E. coli or eukaryotic cells. In purifying our recombinant CYP2D6 fusion, solubilised membranes prepared from E. coli transformed to express the CYP2D6 fusion enzyme, then ultracentrifuged them before using a combination of ion exchange (Mono-Q), affinity (ADP-Sepharose 4B) and gel filtration (Sephacryl S-200 HR) chromatography to purify the recombinant CYP2D6 fusion protein. The eluted CYP2D6 fusion protein was concentrated, quantified and characterised in further studies. Some of the membrane preparations were homogenised in Tris sucrose EDTA buffer and used assess the catalytic functionality of the engineered CYP2D6 fusion protein (Figure 4). Spectrophotometry is used to quantify and determine the potential physical and enzymatic integrity


(spectral) of P450s. This is based on a method introduced by Omura and Sato in 1962. Reduced P450 can form complex with carbon monoxide (CO) to produce a characteristic absorbance soret maxima or peak difference at about 450 nm against CO-free reduced P450 (Figure 2). This has been traditionally the basis of naming these forms of cytochromes and DMEs as cytochrome P450s.

Figure 2. Characteristic absorption spectra of purified recombinant CYP2D6 fusion protein expressed in E. coli. (A) Typical CO difference spectrum of recombinant CYP2D6F membrane preparation showing a maximum absorbance at closely 445 nm following its reduction with Na2S2O4 and CO complex (Fe2+-CO) formation in 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol (v/v), 0.5 mM EDTA, and 0.1 mM DTT. (B) UV-Visible spectrum of purified recombinant YP2D6 fusion protein showing a shoulder peak at closely 418 nm and 445 nm for the oxidised feric form (Fe3+) and CO complex reduced ferous form (Fe2+-CO), respectively. (adapted from Deeni et al 2001).

4.4 SDS Polyacrylamide Electrophoresis and Western Blot The separation of proteins on the basis of their sizes can be done with a polyacrylamide gel (PAGE) (Figure 3A). The addition of SDS into the gel is to ensure that already denatured protein through heating is kept in a denatured state and also to create a net charged field. Separation is followed by the transfer of the gel components to a membrane which is then ENZYMES AND PROTEINS - A BIOTECHNOLOGY TAILORING POINT OF VIEW

probed with antibodies. The membrane (nitrocellulose) serves as platform for the protein to re-fold which is essential because it will enable the antibody to recognise and bind successfully to the protein. This is called western blotting and once the eluted proteins are applied to Western blot analysis, the band showing the molecular weight of the revealed protein should be approximately the size expected for the protein being screened for (Figure 3B).

Figure 3. SDS-PAGE (A) and Western blot analysis (B) of CYP2D6Fexpressed in E. coli. Membrane preparations from E. coli expressing control plasmid pCW (lane 1) or expressing recombinant CYP2D6F (lane 2) were solubilized in sample loading buffer (31 mM Tris-HCl,pH 6.8, 2% SDS, 10% glycerol, 100 mM 2-mercaptoethanol, 0.002% bromphenol blue). Approximately 10 mg of total proteins were resolved by 8% SDS-PAGE and stained with Coomassie brilliant blueR-250. Proteins were transferred to nitrocellulose and probed with human CPR antibodies. The bands indicated by arrows correspond to the recombinant fusion polypeptide. Protein molecular size markers are shown on the left.

4.5 Lysate Assay Protein screening can easily be done through functional assay on the protein itself. Lysate from the protein being studied can be screened using microplates reader to check the absorbance of the substrate or product of a newly formed enzyme. This technique is highly adaptable and can be easily applied. The product of this assay could also be easily identified using gas chromatography, high-performance liquid chromatography or mass spectromCHAPTER 14

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etry. The traditional liquid-based assays employed in screening are being reconsidered and efforts are being jeered towards miniaturised high throughput (HTS) screening assay techniques which will have the capacity to fast-track the screening process, shrink assay cost and conserve reagents.

5. Case Study 5.1 Cytochrome P450 CYP2D6NADPH Reductase Fusion Protein The extent with which Cytochrome P450 enzymes catalyse diverse xenobiotic compounds is enormous. This enduring function of CYP450 enzymes is mostly shadowed by low level of activity which hinders their application industrially. Deeni et al isolated and optimised the catalytic activity of the human debrisoquine-4-hydroxylase (CYP2D6) gene using the rational design method to generate a

fusion of the enzyme with its ancillary redox partner that is catalytically more efficient. Deeni et al cloned, expressed, purified and characterised a recombinant fusion protein that consists of a human CYP2D6 and human CPR (cytochrome P450 reductase). By cloning of CYP2D6 and CPR in a plasmid to express as a fused protein, the group was able to surmount the need of an ancillary redox partner and generate a catalytically self-sufficient active P450 protein (CYP2D6F). This was confirmed by the incubation of membrane fraction of CYP2D6F with substrate for CYP2D6 such as bufuralol, methoprolol and dextromethorphan compared with individually coexpressed CYP2D6 and human CPR (CYP2D6/R) in E. coli membranes (Figure 4). The result of the comparison showed that CYP2D6F has elevated substrate-dependent turnovers of about 2-fold in the presence of antioxidant GSH and was catalytically active.

Figure 4. Catalytic activity of engineered recombinant CYP2D6F Protein. Assays were performed with prototype CYP2D6 substrates to final concentrations of 20, 40 and 100 mM for bufuralol, metoprolol and dextromethorphan, respectively, using 20 pmol of CYP2D6F membranes. Each value represents the mean ± SD of triplicate determinations. Activities are expressed nmol/min/nmol P450.

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Specifically dextromethorphan O-demethylation, metoprolol α-hydroxylation and O-demethylation by CYP2D6F were higher as shown in Figure 4. Under these circumstances, however, bufuralol 1’-hydroxylation from CYP2D6F when compared to the one from CYP2D6/R was marginally lower, which suggest a substrate-dependent effect on catalysis. Deeni et al went on further to investigate the kinetic properties of CYP2D6F enzyme for (±)-bufuralol hydroxylation. Then established that CYP2D6F protein has very close kinetic properties to the holo CYP2D6 enzyme based on substrate turnover (kcat ; 4 and 6 min-1 ) and affinity (Km; 10 and 11 μM) values, respectively, and with equal catalytic efficiency. The findings were similar to that others have reported in the literatures regarding CYP2D6-dependent hydroxylation of bufuralol. Deeni et al showed also the capacity of purified recombinant CYP2D6F to metabolise bufuralol in the absence of phospholipids. Furthermore this CYP2D6F could additionally couple with another P450, including P450 reductase, in addition to dilution effect on its catalytic activity (Figure 5). Suggesting the recombinant CYP2D6F enzyme has both intramolecular and intermolecular coupling and electron transfer capacity. This is consistent with the demonstrated behaviour of other artificial or naturally occurring P450 fused enzyme chimera.

Figure 5. Coupling and catalytic activity of purified recombinant CYP2D6 fusion protein: (A) Effect of dilution on catalysis and (B) Demonstration of coupling capacity. Sample reactions (in A) containing 50 pmol of purified CYP2D6F were assayed with 20 mM buffuralol as substrate following serial dilutions of the purified recombinant protein. Then (in B) equimolar amounts (50 pmol) of purified ENZYMES AND PROTEINS - A BIOTECHNOLOGY TAILORING POINT OF VIEW

CYP3A4, CYP2D6F, and CPR were combined as shown. Bufuralol 19-hydroxylase (black column) and testosterone 6b-hydroxylase (white column) activities were assayed using 20 and 200 mM of bufuralol and testosterone, respectively in the presence of NADPH regenerating system. Results show the mean ± SD of triplicate determinations.

Deeni et al showed for the first time the ability to engineer a functional self-sufficient and completely human P450 fused enzyme chimera. Failed attempt reported by Parikh and Guengerich in 1997 has led to the assumption that the human P450s may only form functional fusion with other ancillatory redox partner reductases and not with the human form. The possibility for a self-sufficient and completely human CYP2D6F will create avenues to specifically investigate the physicochemical mechanisms underlying redox protein interactions and electron flow between human CPR and P450s. It will also provide opportunities of exploring the biotechnological application of CYP2D6F, by extension other self-sufficient and completely human P450 chimeras, toward the development of systems for high throughput screening of drugs metabolized by P450s. This has the potential to discover new ligands and the possibility to predict drugdrug interactions, as well as the development and emergence of novel engineered P450-dependent drug targeted therapies.

5.2 Change of Selectivity (Regioselectivity and Enantioselectivity) The reaction mechanism of P450s frequently generates two products; coupled products which are as a result of the pairing of the electron supplied by NADPH to substrate oxidation and uncoupled products which stems from the loss of electrons to reduced oxygen species. Conditions surrounding reaction are known to have radical effect on the rate of P450 catalysis but not on regio-selectivity and coupling. In order to find of the effect of environmental conditions on regio-selectivity and coupling, Traylor et al in 2011 investigated the effects of conditions CHAPTER 14

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such as buffer type, buffer concentration, pH and temperature on the oxidation of 7-ethoxymethoxy3-cyanocoumarin (EOMCC) by CYP1A2 by measuring the rate of substrate depletion, NADPH depletion and generation of O-dealkylated product simultaneously using a high-throughput optical technique. They found out that each rate increases with temperature peaking at temperature of 45oC. Phosphate buffer between 250 and 375 mM concentration was also noticed to increase each rate. Strong relationship was noticed between NADPH and EOMCC when subjected to variation in buffer concentration and temperature thereby suggesting that coupling was not significantly altered by these conditions. The relationship between EOMCC and 7-hydroxy-3-cyanocoumarin (7HCC), its product from oxidation by CYP1A2 does not also modify regioselectivity. Significant change of about 6-fold was however observed when temperature and buffer concentration were varied at pH 6.7. On the other hand, the correlation for both coupling and regionselectivity were weaker as a function of the performance of EOMCC and NADPH when subjected to conditions such as variation in buffer type and pH. The Coupling and regio-selectivity varied by 14.4% and 21.6%, respectively.

5.3 Change of Protein Stability (Thermostability) Cytochrome P450s are quite limited in-terms of their stability and robust method for the identification of thermophilic P450s may greatly resolve this problem. There are only two characterised thermophilic P450s to date; CYP119 (from Sulfolobus solfataricus and hydroxylates lauric acid) and CYP175A1 (from Thermus thermophilus and hydroxylates b-carotene at the 3- and 30-positions). The CYP119 has an exclusive electron transport system that is made up of ferredoxin (Fdx) and 2-oxoacid:ferredoxin oxidoreductase (OFOR) and uses pyruvate rather than NAD(P)H as its electron donor, while the CYP175A1 with innate electron 234


transport system uses NADPH rather than NADH and is made up of Fdx and a different form of ferredoxin–NADP+ reductase (FNR). Having realised the huge need for a thermostable CYP 450 enzyme, Mandai et al in 2010 constructed two fusion proteins made up of CYP175A1, Fdx, and FNR (H2N-CYP175A1- Fdx - FNRCOOH (175FR) and H2NCYP175A1 - FNR-Fdx - COOH (175RF)) and characterized them. This was followed by the engineering of the CYP175A1 domain of 175RF for the oxidation of testosterone, which is usually not a CYP175A1 substrate. Mandai et al discovered that on subjecting the two fusion protein to b-carotene hydroxylation the activity in 175RF was notably higher when compared to that of 175FR and it was suggested that the imbalance in this activity could be a resultant effect of intramolecular electron transfer and nearly optimal interaction between the three components. Equally, 175RF maintained 50% residual activity even at 800C suggesting an exceptionally thermostable self-sufficient P450. Analysis of the engineered CYP175A1 domain of 175RF for the oxidation of testosterone shows that the fusion protein 175RF did not hydroxylate testosterone whereas significant hydroxylation activity was observed when mutants hydroxylate testosterone was subjected to 175RF.

5.4 Designing and Tailoring (Redesigning) Enzyme for New Substrates/Reactions Undoubtedly one of the greatest gift of nature to man is a notable array of enzymes that has been of enormous use in the transformation of chemicals in (bio)chemical and pharmaceutical industries to create economically and environmentally friendlier synthetic chemicals. In spite of their diverse uses, the immense potentials of enzymes have not been fully tapped into due to constraint resulting from their instability, substrates peculiarity and necessity for expensive cofactors. To circumvent these constraints, enzymes are being designed to adjust to new substrates and reactions.


Designing enzyme for new substrates/reactions often referred to as “Enzyme Redesigning” requires information on protein structure, sequence and computational algorithms to identify potential focus sites with reduced amino acid diversity. Enzyme redesigning is achieved without interfering with the overall protein fold, but yet taking advantage of the conserved residues in the active site of the protein. Enzyme redesigning can therefore be sequencebased, structure-based and computational and they could be used for altering protein characters like substrate specificity, stereo-selectivity, and stability and for the fabrication of new function by de novo design (Lutz, 2010). They can also be tailored to manipulate product formation, enhance trace activities and new reactivity as well as reaction mechanisms.

5.5 Sequence-Based Enzyme Redesign Redesigning enzymes by exploiting their protein sequence functionality to produce new enzymes with required features is a popular method that basically uses the evolutionary information of the protein. Such information are generated using formidable tools such as multiple sequence alignments (MSAs) and phylogenetic analyses to dig deep into the conservational and ancestral connections between homologous protein sequences and structures with the aim of detecting functionally important area and evaluating amino acid alterability. Sequence based method utilises newly sequenced proteins in it redesigning process by introducing substitutions into sequences after ascertaining the sequence and structure alignments of an enzyme. The essence of carrying out an alignment is to be able to determine the conserved amino acids region of a protein considering the assumption that conserved amino acids influences the property of a protein. Substituting an enzyme’s hot-spots to redesign a needed one with enhanced property is undoubtedly more credible than random mutagenesis. In accordant with the assumption above, the number of enzyme super-families whose members have homologous structures and which use associated ENZYMES AND PROTEINS - A BIOTECHNOLOGY TAILORING POINT OF VIEW

methods to catalyse reactions is soaring and an amino acid substitution of a hotspot is most likely responsible for their varying actions which corroborates the assumption wide-ranging enzymatic activities stems conserved enzymes. Despite its proven credibility, several efforts at redesigning enzymes by substituting hotspot area have failed partly due to the inadequate knowledge of the essential mechanisms needed to create desired enzyme features and largely due to the rigid stance that amino acid replacement is only possible by primary amino acid sequence homologies. Sequence based enzyme redesign requires the verification of mutation by sequencing followed by the purification of the mutant enzymes to aid the determination of the kinetic and functional features of an enzyme.

5.6 Structure-Based Enzyme Redesign Effective functioning of a protein is mostly associated with its structure, thereby suggesting that the manipulation of protein sequences is not necessarily the action that affects the functions of a protein. In order to determine how structural modification affect enzyme functions, enzyme engineers focus on uncovering vital residues close to enzyme hotspot sites and at domain interfaces or hinge areas. Structure-based redesign of enzymes finds mutations by analysing active sites of enzymes with the aim of enhancing its binding and catalytic ability in a reaction. The identification of mutation is usually carried out through the bioinformatics analysis of protein sequences and the characterisation of the mutation impact on enzyme properties leading to the prediction of the mutation effect upon enzyme structure. New products created through this method are experimentally to understand the link between the enzymes’ structure and its biochemical features. Investigating newly created enzymes experimentally confines the structural revelation to proteins that can only be crystallized. Structurebased enzyme redesign method has been applied successfully to enhance the thermal stability of cellulosomal endoglucanase (Cel8A) from Clostridium thermocellum by 14-fold at 85°C without the loss CHAPTER 14

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of catalytic activity. Also the method has similarly been applied with penicillin G acylase (PGA) and glucose dehydrogenase (GDH) from Bacillus subtilis without loss of the enzymes’ activities.

5.7 Computational Enzyme Redesign Computational redesign, unlike other methods of enzyme redesign, is not restricted by homology with biological enzymes. It rather involves the computer aided creation of active sites designed around a particular reaction and inserting them into biologically known platforms to design proteins with new functions. Although computational enzyme engineering is still a budding discipline, many progresses have been made lately, in particular the de novo design of enzymes. The result of these advances has made it possible to simulate the laboratory evolution of enzyme in silico. In addition, in silico (computational) redesign has broaden the terrain of enzyme redesigning, making it possible to qualitatively and quantitatively probe and adjust enzyme activity, as well as increase the speed with which the detection of likely mutations that can influence enzyme activity and stability can be made. There is no doubt in going forward and that the potentials of creating desired enzymes by adoption and exploration of the tremendous opportunities in computational redesign of enzymes. However, current computational approaches lack provision for chemical awareness and the facility to be able to fit chemistry with platform unlike other previously discussed methods. Factoring these fundamental processes into in silico designing will indisputably hasten the creation of enzymes.

5.8 De novo Enzyme Redesign (Rosetta) ROSETTA is a de novo enzyme design software that has been used to create enzyme for the catalysis of several chemical reactions. It involves the selection of catalytic tool and model active site, detection of sites on protein scaffolds where this design activities can be achieved, enhancement of surrounding 236


residue’s features such that the interactions with the transition state and primary catalytic residues is stable and the assessment and classification of the ensuing designed sequences. ROSETTA was initially created for the de novo prediction of protein fold, however, it has over the years been broadened to incorporate methods for design, docking, proteinprotein interaction design and prediction, experimental verification of structure from datasets, RNA structure estimation, protein-DNA interaction prediction and design and enzyme design. ROSETTA has been used recently to create artificial enzymes that catalysed retro-aldol reaction and eliminate Kemp. In spite of its noteworthy accomplishment, ROSETTA application has experience some limitations however, but what differentiates ROSETTA is its proficiency for creating catalytic activity from an passive scaffold, unlike other experimental approaches, such as directed evolution methods where there is a huge dependence on present catalytic activity as an initiation point.

6. Conclusion Enzyme protein engineering will be pivotal to aim the increase in production processes of many industries ranging from development of new xenobiotics to detergent production. The expansion of this nascent field depends on several factors that include the reaction and enzyme of interest, existing biochemical and structural data and bioinformatics tools for example. Most importantly, the in-depth understanding of enzymatic activities will be crucial to the expansion of enzyme engineering. Moreover, to continue to make progress in this field, the further development of our deep knowledge of enzymology and refinement of enzyme structure at 2D and 3D, coupled with improvement in methods of designing artificial enzyme must increase and evolve in pari passu.


Review Questions and Answers Q1. What are cytochrome P450s and do you assay for their activity? A1. General features, distribution, classification, catalysis and function; Assays – membrane based; reconstitution based and whole cell based with the details and specifics therein. Q2. How would you engineer the “ideal” cytochrome P450 monooxygenase system? A2. This is open ended, flexible and to be informed by most of the sections and elements covered in this entire chapter, as well as some further readings. This is in addition to the use of independent thoughts and creativity.

Further Readings 1. Alderson RG, De Ferrari L, Mavridis L, et al,. Enzyme Informatics. Current Topics in Medicinal Chemistry 2012;12: 1911–1923. 2. Barrozo A, Borstnar R, Marloie G, et al,. Computational Protein Engineering: Bridging the Gap between Rational Design and Laboratory Evolution. International Journal of Molecular Sciences 2012;13:12428-12460 3. Chen R. Enzyme Engineering:Rational RedesignVersus Directed Evolution. Trends in Biotechnology 2001;19:13-14 4. Chica RA, Doucet N, Pelletier JN. Semi-Rational Approaches to Engineering Enzyme Activity: Combining the Benefits of Directed Evolution and Rational Design. Current Opinion in Biotechnology 2005;16:378–384 5. Deeni YY, Paine MJI, Ayrton AD, et al,. Expression, Purification, and Biochemical Characterization of A Human Cytochrome P450 CYP2D6NADPH: Cytochrome P450 Reductase Fusion Protein. Archives of Biochemistry and Biophysics 2001; 396:16–24 6. Di Nardo G, Gilardi G. Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites. International Journal of Molecular Science 2012;13:15901-15924 ENZYMES AND PROTEINS - A BIOTECHNOLOGY TAILORING POINT OF VIEW

7. Ding S, Yao D, Deeni YY, Burchell B, Wolf CR, Friedberg T. Human NADPH-P450 oxidoreductase modulates the level of cytochrome P450 CYP2D6 holoprotein via haem oxygenase-dependent and independent pathways. Biochemical Journal 2001;356:613-9 8. Eichelbaum M, Ingelman-Sundberg, M, Evans, W. E. Pharmacogenomics and Individualized Drug Therapy. Annual Review of Medicine 2006;57:119–137. 9. Evans WE, McLeod HL. Pharmacogenomics Drug Disposition, Drug Targets, and Side Effects. The New England Journal of Medicine 2003;348:538-549. 10. Gerlt JA, Babbitt PC. Enzyme (Re)design: Lessons from Natural Evolution and Computation. Current Opinion in Chemical Biology 2009;13:10–18. 11. Gillam EMJ. Engineering Cytochrome P450 Enzymes. Chemical Research in Toxicology 2008;21:220–231. 12. Girvan HM, Dunford AJ, Neeli R, Ekanem IS, Waltham TN, Joyce MG, Leys D, Curtis RA, Wil¬liams P, Fisher K, Voice MW, Munro AW. Flavocytochrome P450 BM3 mutant W1046A is a NADH-dependent fatty acid hydroxylase: implications for the mechanism of electron transfer in the P450 BM3 dimer. Arch Biochem Biophys 2011;507:75-85. 13. Golynskiy MV and Seelig B. De novo Enzymes: From Computational Design to mRNA Display. Trends in Biotechnology 2010;28:340–345 14. Guengerich, F.P. Cytochrome P450: Structure, Mechanism, and Biochemistry. Plenum Press 1995. 15. Hilvert D, Toscano MD and Woycechowsky KJ. Minimalist Active-Site Redesign: Teach¬ing Old Enzymes New Tricks. Wiley Online Library 2007;46:3212 – 3236. 16. Hrycay EG, Bandiera SM. The Monooxygenase, Peroxidase, and Peroxygenase Properties of Cytochrome P450. Archives of Biochemistry and Biophysics 2012;522:71–89 CHAPTER 14

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17. Julsing MK, Cornelissen S, Buhler B, Schmid A. Heme-Iron Oxygenases: Powerful Industrial Biocatalysts? Current Opinion in Chemistry and Biology 2008;12:177-186. 18. Jung SJ, Lauchli R, Arnold FH. Cytochrome P450: Taming a Wild Type Enzyme. Current Opinion in Biotechnology 2011;22:1–9. 19. Kim DH, Ahn T, Jung HC, et al,. Generation of the Human Metabolite Piceatannol from the Anticancer-Preventive Agent Resveratrol by Bacterial Cytochrome P450 BM3. Drug Metabolism and Disposition 2009;37:932–936.

28. McLaughlin LA, Paine MJ, Kemp CA, Maréchal JD, Flanagan JU, Ward CJ, Sutcliffe MJ, Roberts GC, Wolf CR. Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding. The Journal of Biological Chemistry 2005;280:38617-24. 29. Munro AW, Girvan HM, Mason, AE, et al,. What Makes a P450 Tick? Trends in Biochemical Sciences 2013; 38:140-50. 30. Nair NU, Denard CA, Zhao H. Engineering of Enzymes for Selective Catalysis. Current Organic Chemistry 2010;14:1870-1882.

20. Kim DH, Kim KH, Liu KH, Jung HC, et al,. Generation of Human Metabolites of 7-ethoxycoumarin by Bacterial Cytochrome P450 BM3. Drug Metabolism and Disposition 2008;36: 2166–2170.

31. Nannemann DP, Birmingham WR, Scism RA and Bachmann BO. Assessing Directed Evolution Methods for the Generation of Biosynthetic Enzymes with Potential in Drug Biosynthesis. Future Medicinal Chemistry 2011;3: 809–819.

21. Kim KH, Kang JY, Kim DH, et al,. Generation of Human Chiral Metabolites of Simvastatin and Lovastatin by Bacterial CYP102A1 Mutants. Drug Metabolism and Disposition 2011;39:140–150.

32. Nelson DR. Progress in Tracing the Evolutionary Paths of Cytochrome P450. Biochimica et Biophysica Acta 2011;1814:14-18.

22. Kumar S. Engineering Cytochrome P450 Biocatalysts for Biotechnology, Medicine, and Bioremediation. Expert Opinion on Drug Metabolism and Toxicology 2010;6:115–131. 23. Li DN, Pritchard MP, Hanlon SP, Burchell B, Wolf CR, Friedberg T. Competition between cytochrome P-450 isozymes for NADPH-cytochrome P-450 oxidoreductase affects drug metabolism. J Pharmacol Exp Ther 1999;289(2):661- 7. 24. Li X, Zhang Z, Song J. Computational Enzyme Design Approaches with Significant Biological Outcomes: Progress and Challenges. Computational and Structural Biotechnology Journal 2012;2:1-10. 25. Luo XJ, Yu HL, Xu JH. Genomic Data Mining: An Efficient Way to Find New and Better Enzymes. Enzyme Engineering 2012;1:1-4. 26. Lutz S. Beyond Directed Evolution-Semi- Rational Protein Engineering and Design. Current Opinion in Biotechnology 2010;21:734–743. 27. Mandai T, Fujiwara S and Imaoka S. Construction and Engineering of a Thermostable Self-Sufficient Cytochrome P450. Biochemical and Biophysical Research Communications 2009;384:61–65. 238


33. Parikh A, Guengerich FP. Expres¬sion, purification, and characterization of a cata¬lytically active human cytochrome P450 1A2:rat NADPH-cytochrome P450 reductase fusion protein. Elsevier 1997;9:346-54. 34. Otyepkaa M, Berkaa K, Anzenbacher P. Is There a Relationship Between the Substrate Preferences and Structural Flexibility of Cytochromes P450. Current Drug Metabolism 2012;13:130-142. 35. Omura T. Structural diversity of cytochrome P450 enzyme system. J Biochem 2010;147:297-306. 36. Pantazes RJ, Grisewood MJ, Maranas CD. Recent Advances in Computational Protein Design. Current Opinion in Structural Biology 2011;21:447– 572. 37. Park S, Stowell XF, Wang W, et al,. Computational Protein Design and Discovery. Annual Reports on the Progress of Chemistry Section C 2004;100:195–236. 38. Pleiss J. Protein Design in Metabolic Engineering and Synthetic Biology. Current Opinion in Biotechnology 2011;22:611–617. 39. Rabe KS, Gandubert VJ, Spengler M, et al,. Engineering and Assaying of Cytochrome P450 Bio-


catalysts. Analytical and Bioanalytical Chemistry 2008;392:1059–1073.

putational and Structural Biotechnology Journal 2012;2:3.

40. Richter F, Leaver-Fay A and Khare SD, et al,. De Novo Enzyme Design Using Rosetta3. PLoS One 2011;6: e19230.

45. Tiwari M, Singh R, Kim I, Lee J. Computational Approaches for Rational Design of Proteins with Novel Functionalities. Computational and Structural Biotechnology Journal 2012;2.

41. Schuler MA. P450s in Plant Insect Interactions. Biochimica et Biophysica Acta 2011;1814: 36-45. 42. Schumachera SD, Jose J. Expression of active human P450 3A4 on the cell surface of Escherichia coli by Autodisplay. Journal of Biotechnology 2012;161:113– 120. 43. Singh RK, Tiwari MK, Singh R, Lee JK. From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes. International Journal of Molecular Sciences 2013;14:1232-1277.

46. Traylor MJ, Chai J, Clark DS. Si¬multaneous Measurement of CYP1A2 Activity, Regioselectivity, and Coupling: Implications for Environmental Sensitivity of Enzyme Substrate Binding. Archives of Biochemistry and Biophysics 2011;505:186– 193. 47. Urlacher VB, Girhard M. Cytochrome P450 Monooxygenases: An Update on Perspectives for Synthetic Application. Trends in Biotechnology 2012;30: 26-36.

44. Steiner S and Schwab H. Recent Advances in Rational Approaches for Enzyme Engineering. Com-


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CHAPTER 15 DNA SEQUENCING TECHNOLOGY: THE CLINICAL POTENTIAL Tommaso Beccari, Maria Rachele Ceccarini, Michela Codini, Mariapia Viola Magni CONTENTS Summary ............................................................................................................................... 243

1. History ......................................................................................................................... 243

2. First Generation DNA Sequencing ............................................................................... 244

3. Second Generation Sequencing .................................................................................... 244

4. Sequencing By Synthesis ............................................................................................... 246

4.1 Roche GS-FLX 454 .................................................................................................... 246

4.2 Illumina MiSeq ........................................................................................................... 247

4.3 Ion Torrent ................................................................................................................ 248

5. Sequencing By Ligation ................................................................................................ 249

5.1 Life Technologies’ SOLiD ........................................................................................... 249

6. Third Generation Sequencing Technologies .................................................................. 250

7. Single-Molecule Sequencing ......................................................................................... 250

7.1 Heliscope™ Single Molecule Sequencer ....................................................................... 250

7.2 Pacific Bioscience ........................................................................................................ 250

7.3 Nanopore DNA Sequencer ......................................................................................... 251

8. Complete Genomics ..................................................................................................... 251

9. Single-Cell Sequencing ................................................................................................. 252

10. Clinical Application of HT-NGS ................................................................................ 253

10.1 Genetic mutations in Mendelian Disorders ............................................................... 253

10.2 Epigenetics ................................................................................................................ 253

10.3 Genetics in Common Diseases .................................................................................. 254

10.4 Cancer Research and Biomarkers .............................................................................. 254

10.5 Discovering Noncoding RNAs .................................................................................. 255

10.6 Pathology-Important Cells ........................................................................................ 255

11. Conclusions ................................................................................................................ 255

Review Questions and Answers ............................................................................................. 256 Further Readings ................................................................................................................... 257 DNA SEQUENCING TECHNOLOGY: THE CLINICAL POTENTIAL

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Summary

T

he ability to sequence complete genomes has changed the nature of biological and biomedical research. Sequences and other genomic data have the potential to lead to remarkable improvements in many facets of human life and society, including the understanding, diagnosis, treatment and prevention of disease; advantages in agriculture, environmental science and remediation; the understanding of evolution of the ecology system and moreover the evolutionary history of the human species. The rapid speed of sequencing, attained with modern DNA sequencing technology, has provided collections of information to use in different fields. With its unprecedented throughput, scalability, and speed, next-generation sequencing (NGS) enables researchers to study biological systems at a level never before possible. In 2007, a single sequencing run could produce a maximum of around one gigabase (Gb) of data. By 2011, that rate has nearly reached a terabase (Tb) of data in a single sequencing run (nearly a 1000x increase in four years). With the ability to rapidly generate large volumes of sequencing data, NGS enables researchers to move quickly from an idea to full data sets in a matter of hours or days. Researchers can now sequence more than five human genomes in a single run, producing data in roughly one week, for a reagent cost of less than $5,000 per genome (up-to-date in 2014). By comparison, the first human genome required approximately ten years to sequence using Sanger technology and an additional three years to finish the analysis.

1. History Since the time DNA was discovered as the code to all biological life on Earth, man has sought to unravel its mysteries. If the genetic code could be sequenced or “read”, the origins of life itself may be revealed. Although this thought might not be entirely true, the efforts to date have certainly revolutionized the biological field. DNA SEQUENCING TECHNOLOGY: THE CLINICAL POTENTIAL

The human genome project (HGP), declared completed in April 2003, but a working draft also available and published in February 2001, was undertaken with the aim of sequencing the 3 billion nucleotides of human DNA and identifying all the genes; the functional elements of DNA. Moreover, it was expected that the knowledge gleaned from the genome would result in the development of novel diagnostic assays, targeted therapies and improved ability to predict the onset, severity as well as progression of diseases and thus will have a major impact on medical practice. Currently, genetic information is being used to identify mutations in rare as well as undiagnosed genetic disorders and select the therapy best suited for a genotype. This has been made possible by post HGP development of many parallelized high throughput technologies such as microarrays and High-Throughput Next Second Generation Sequencing (HT-NGS). HT-NGS is one of the great challenges of today’s genomic research. For future applications, indepth genome sequence information and analysis for most of the mammals, including humans, is necessary to fully understand genome variation of economic traits, genetic susceptibility to diseases, and pharmagenomics of drug responses. The past decade has witnessed devolution in the field of human genomic research. Today, a more global approach is being embraced which has not only given rise to the field of systems biology, but has also touched all areas of biological and medical research, as well as bringing them closer together and blurring the lines that previously defined them as individual disciplines of research. Horizons and expectations have broadened due to the technological advances in the field of genomics, especially HTNGS and its wide range of applications such as: chromatin immunoprecipitation coupled to DNA microarray (ChIP-chip) or sequencing (ChIP-seq), RNA sequencing (RNA-seq), whole genome genotyping, de novo assembling and re-assembling of genome, genome wide structural variation, mutation detection and carrier screening, detection of inherited disorders and complex human diseases, DNA library preparation, paired ends and genomic CHAPTER 15

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captures, sequencing of mitochondrial genome and personal genomics.

2. First Generation DNA Sequencing First generation sequencing was originally developed by Sanger in 1975, who introduced the concept of DNA sequencing in his pioneering Croonian lecture. Sanger’s method, based on the chainterminating dideoxynucleotide analogues was developed in parallel with the Maxam and Gilbert’s chemical degradation DNA sequencing technique in which terminally labelled DNA fragments were chemically cleaved at specific bases and separated by gel electrophoresis. From these first-generation methods, Sanger sequencing ultimately prevailed given that it was technically less complex and more amenable to being scaled up. For the Sanger sequencing practiced today, during sample preparation, different-size fragments of DNA are generated each starting from the same location (Figure 1).

mixtures are loaded into separate lanes of a gel and electrophoresis is used to separate the DNA fragments (Molecular Biology of the Cell, Fifth Edition).

Each fragment ends with a particular base that is labelled with one of four fluorescent dyes corresponding to that particular base. Then all the fragments are distributed in the order of their length via capillary electrophoresis (Figure 2). This method results in a read length that is less than 800 bases on average, but may be extended to over 1000 bases. While fully automated implementations of this approach were the mainstay from the original sequencing of the human genome, their chief limitation was the small amount of DNA that could be processed per unit time, referred to as throughput, as well as high cost, resulting in it taking roughly 10 years and three billion dollars to sequence the first human genome.

Figure 2. Sanger sequencing read display: Chromatograms after capillary electrophoresis. Comparison of two high quality mitochondrial DNA sequences showing sharp and well spaced peaks.

3. Second Generation Sequencing

Figure 1. The Sanger method: Single-stranded DNA is mixed with a primer and split into four aliquots, each containing DNA polymerase, four deoxyribonucleotide triphosphates and a replication terminator. Each reaction proceeds until a replication-terminating nucleotide is added. The

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Commercial Second-generation sequence (SGS) emerged in 2005 in response to the low throughput and high cost of first-generation methods. To address this problem, SGS tools achieve much higher throughout by sequencing a large number of DNA molecules in parallel. Several reviews of what was first called “next-generation” or, more precisely, SGS technologies have appeared and this technique was classified as a combination of a synchronized



reagent wash of nucleoside triphosphate (NTPs) with a synchronized optical detection method. The SGS platforms can generate about five hundred million bases of raw sequence (Roche’s 454) to billions of bases in a single run (Illumina, SOLiD). These novel methods rely on parallel, cyclic interrogation of sequences from spatially separated clonal amplicons: 26 μm oil-aqueous emulsion bead (Roche: pyrosequencing chemistry), 1 μm clonal bead (SOLiD: sequencing by sequential ligation of oligonucleotide probes), clonal bridge (Illumina: sequencing by reversible dye terminators). The dramatic drop in cost seen in 2008 is the result of transitioning from First-generation Sanger sequencing to Second-generation platforms installed in sequencing centres (i.e. Roche’s 454, Illumina, and SOLiD). With most SGS technologies, tens of thousands of identical strands of DNA molecules are anchored to a given location to be read in a process consisting of successive washing and scanning operations. The ‘wash-and-scan’ sequencing process involves sequentially flooding with reagents, such as labelled nucleotides, incorporating nucleotides into the DNA strands, stopping the incorporation reaction, washing out the excess reagent, scanning to identify the incorporated bases and finally treating the newly incorporated bases to prepare the DNA templates for the next ‘wash-and-scan’ cycle. This cycle is repeated until the reaction is no longer viable. The array of DNA anchor locations can have a very high density of DNA fragments, leading to extremely high overall throughput and a resultant low cost per identified base when such instruments are run at high capacity. For example, Illumina’s HiSeq 2000 instrument can generate upwards of 300 or more gigabases (GB) of sequence data in a single run. The time-to-result for these SGS methods is generally long (typically taking multiple days), due to the large number of scanning and washing cycles required. Furthermore, because step yields for the addition of each base are less than 100%, a population of molecules becomes more asynchronous as each base is added. This loss of synchronicity (called dephasing) causes an increase in noise and sequencing errors as the read extends, effectively limiting the read length produced by the DNA SEQUENCING TECHNOLOGY: THE CLINICAL POTENTIAL

most widely used SGS systems to significantly less than the average read length achieved by Sanger sequencing. Furthermore, in order to generate this large number of DNA molecules, PCR amplification is required. The amplification process can introduce errors in the template sequence as well as amplification bias. The effects of these amplification cycles are that neither the sequences nor the frequencies in which they appear are always faithfully preserved making it unreliable. In addition, the process of amplification increases the complexity and time associated with the sample preparation. Finally, the massive high throughput rates per run achieved by SGS technologies generate mountains of highly informative data that challenge data storage and informatics operations, especially in light of the shorter reads (compared with Sanger sequencing) that make alignment and assembly processes challenging. SGS technologies have led the way in revolutionizing the field of genomics and beyond, motivating an astonishing number of scientific advances. In particular these technologies have provided a large impetus for de novo sequencing, resequencing, exome sequencing, transcriptome profiling, methylation profiling, and metagenomics studies. Additionally, in a few instances, SGS systems have been used in clinical settings for diagnostic purposes. Using the whole-genome sequencing approach, Welch et al. showed that in a patient suffering from a rare form of acute promyelocytic-leukaemia (APL), a large chunk of Chr. 15 (chromosome 15) was inserted into the second intron of the retinoic acid receptor alpha (RARA) gene on Chr. 17 resulting in a fusion oncogene. This knowledge was further used to decide on an appropriate medical management strategy for the patient [alltrans retinoic acid (ATRA) consolidation instead of stem cell transplant]. Another example is whole-genome sequencing of skin (normal) and bone marrow (leukaemia) DNA of a patient with early-onset breast and ovarian cancer. It was found that the patient had a novel deletion mutation (3-kb heterozygous deletion encompassing exons 7–9) in the TP53 gene of the skin cells and a homozygous deletion of the same region in leukaemia cells which may have conferred high cancer susceptibility. Recently, in another study CHAPTER 15

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Mellmann et al. used the Ion Torrent Personal Genome Machine (PGM) for rapid characterization of the highly virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 during an outbreak in Germany. These examples clearly highlight the immense value of SGS technology in understanding genetic variations underlying pathological conditions, provided its current limitations such as high cost of sequencing, complexity of data management and analysis are addressed appropriately. Another important concern for clinical application of SGS is the requirement for a low error rate (less than 1%, which is close to the approximately 1% error rate typically reported for Sanger sequencing) in data generated. Nevertheless there are sequencing applications and aspects of genome biology that are at present beyond the reach of current sequencing technologies, leaving fertile ground for additional innovation in this area.

4. Sequencing By Synthesis The idea of sequencing by synthesis has been around for some time and is the basis for several second-generation sequencing technologies including Roche’s 454 sequencing platform and Illumina’s line of sequencing system. 454’s pyrosequencing method uses an enzyme cascade to produce light from a pyrophosphate released during nucleotide incorporation. Illumina’s fluorescently labelled sequencing by synthesis techniques employs fluorescently labelled nucleotides with reversible termination chemistry and modified polymerases for improved incorporation of nucleotide analogues. These sequencing by synthesis methods increased throughput compared to first-generation sequencing methods; however, optical imaging is needed to detect each sequencing step. Since an intricate optics system can increase the overall cost of a sequencing system, the next logical advancement in the sequencing field has been to abandon the use of optics for a less expensive approach to detection. Based on these observations new methods of detection have been proposed to measure the temperature or pH change in microstructures. Since both changes are by-products of nucleotide incorporation in a DNA polymerization reaction, the need 246

for optical detection of light produced by the luciferase enzyme was eliminated. Like pyrosequencing, the thermo-sequencing method requires sequential cycles in which one of the four nucleotides is introduced to the system, followed by measurement of nucleotide incorporation by heat detection. Between each cycle, the system is regenerated by thorough washing of reaction wells to minimize residual NTPs and, therefore, reduce error accumulation.

4.1 Roche GS-FLX 454 The Roche GS-FLX 454 Genome Sequencer was the first commercial platform introduced in 2004 as the 454 Sequencer. The second complete genome of an individual (James D. Watson) was sequenced with this platform. The 454 Genome Sequencer uses sequencing-by-synthesis technology known as pyrosequencing. The key procedure in this approach is emulsion PCR in which singlestranded DNA binding beads are encapsulated by vigorous vortexing into aqueous micelles containing PCR reactants surrounded by oil for emulsion PCR amplification. During the pyrosequencing process, light emitted from phosphate molecules during nucleotide incorporation is recorded as the polymerase synthesizes the DNA strand.

Figure 3. Roche 454: In emulsion PCR a reaction mixture consisting of an oil–aqueous emulsion is created to encapsulate bead–DNA complexes into single aqueous droplets. PCR amplification is performed within these droplets to create beads containing several thousand copies of the same template sequence (From Transcriptomics & Functional Genomics News Letter No 11, Ken Laing, St George’s University of London, with permission).



The beads carrying amplified fragments are deposited in picotiter wells such that a single bead with a unique fragment is present in each well. Additional beads with enzymes (polymerase, sufurylase and luciferase) and other reagents are added to the wells. The picotiter plate is then flooded with unlabeled nucleotides in a predetermined order. A nucleotide complementary to the nucleotide in the fragment to be sequenced gets incorporated in the strand being synthesized and a pyrophosphate is released (Figure 3). The pyrophosphate and activated sulphate are converted to ATP in the presence of sulfurylase, which generates light in a reaction catalyzed by luciferase, that is captured by a charge coupled device (CCD) camera. The intensity of generated light is proportional to the number of nucleotides incorporated (Figure 4).

Figure 4. Pyrosequencing technology: a system that allows real time detection of sequencing events using reliable chemistry and robust detection mechanisms. It uses an enzymecascade system, consisting of four enzymes and specific substrates to produce light upon nucleotide incorporation during DNA synthesis. The amount of light produced is proportional to the number of incorporated nucleotides (adapted from Metzker ML, Nature Reviews Genetics. 2010; 11, 31-46).

Initially, the 454 Sequencer had a read length of 100 bp but now can produce an average read length of 400 bp. The maximum ~600 bp capacity of 454 systems approaches half the current Sanger sequencing capacities (~1200 bp). At 600 bp, the 454 Sequencer has the longest short reads among all the NGS platforms; and generates ~400–600 Mb of sequence reads per run; critical for some applications such DNA SEQUENCING TECHNOLOGY: THE CLINICAL POTENTIAL

as RNA isoform identification in RNA-seq and de novo assembly of microbes in metagenomics. Raw base accuracy reported by Roche is very good (over 99%); however, the reported relatively error-prone raw data sequence, especially associated with insertion-deletions, is a major concern. Low yields of sequence reads could translate into a much higher cost if additional coverage is needed to define a genetic mutation (the relevant details are in Table 1).

4.2 Illumina MiSeq The basic goal of Illumina is: to generate large numbers of unique polonies (polymerase generated colonies) that can be simultaneously sequenced. These parallel reactions occur on the surface of a “flow cell” (basically a water-tight microscope slide) which provides a large surface area for many thousands of parallel chemical reactions. The DNA sample of interest is sheared to appropriate size using a compressed air device known as a nebulizer (average ~800 bps) or by an enzymatic process (average ~200-300bps). End-polished DNA fragments can be ligated to two unique adapters. Ligated fragments of the size range of 150-200bp are isolated via gel extraction and amplified using limited cycles of PCR. The single-stranded DNA fragments bind to the adapters that are ligated on the surface of the flow cell (as shown in Table 1). In contrast to the Roche 454 and ABI methods that use a bead-based emulsion PCR to generate polonies, Illumina utilizes a unique “bridged” amplification reaction that occurs on the surface of the flow cell. The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapterligated fragments are bound to the surface of the flow cell exposed to reagents for polymerase-based extension. Priming occurs as the free/distal end of a ligated fragment “bridges” to a complementary oligonucleotide on the surface. Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface (Figure 5a). This process occurs in what is referred to as Illumina’s CHAPTER 15

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“cluster station”, an automated flow cell processor. A flow cell containing millions of unique clusters is now loaded into the sequencer for automated cycles of extension and imaging (Figure 5b).

the fluorescent emission identifies which of the four bases was incorporated at that position. This cycle is repeated, one base at a time, generating a series of images each representing a single base extension of a specific cluster. Base calls are derived with an algorithm that identifies the emission colour over time. At this time reports of useful Illumina reads range from 26 to 50 bases.

4.3 Ion Torrent

Figure 5a. Illumina clonal expansion by bridge amplification. Solid-phase amplification is composed of two basic steps: initial priming and extending of the single-stranded, single-molecule template, and bridge amplification of the immobilized template with immediately adjacent primers to form clusters (From Transcriptomics & Functional Genomics News Letter No 11, Ken Laing, St George’s University of London, with permission).

Figure 5b. The basis of Illumina sequencing in a flow cell: opening of the bridge allows the release of one end in preparation for sequencing (From Transcriptomics & Functional Genomics News Letter No 11, Ken Laing, St George’s University of London, with permission).

The first cycle of sequencing consists first of a single fluorescent nucleotide incorporation, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base. Any signal above background identifies the physical location of a cluster (or polony), and 248

Ion Torrent’s Personal Genome Machine (PGM) (Ion Torrent, Guilford, CT) is very similar to Roche 454 in terms of sequencing principle, but instead of using imaging to capture the nucleotide incorporation event, it detects the change in pH resulting from H+ ion release upon nucleotide incorporation. It leverages the advances made in the semiconductor industry to enable fast and simple massively parallel sequencing. PGM uses a highdensity array of microwells to perform sequencing and beneath the wells is an ion sensitive layer followed by a proprietary ion sensor which detects changes in pH resulting from release of a hydrogen ion following nucleotide incorporation. Fragments to be sequenced are captured on beads and amplified by emulsion PCR. The beads are then deposited in the microwell such that each well has only one bead carrying a unique amplified fragment. Nucleotides are then added in a predetermined sequence to the wells. A nucleotide complementary to the nucleotide in the fragment being sequenced gets incorporated in the strand being synthesized and a hydrogen ion is released. Upon release of the hydrogen ion, the voltage of the solution changes in that well and is detected by the ion sensor. If two nucleotides are incorporated in a cycle, then the voltage is doubled and the sensor records two nucleotides added. If a nucleotide is not added during a cycle then no voltage change is recorded. Thus, in PGM, fluorescently labeled nucleotides or light are not used for detection. Instead the hydrogen ion released following nucleotide incorporation is used for detecting the incorporation event in a well.



Figure 6. Outline of the sequencing work flow for the SOLiD (From Transcriptomics & Functional Genomics News DNA SEQUENCING TECHNOLOGY: THE CLINICAL POTENTIAL

Life technologies’ SOLiD

Ion Torrent

Disadvantages

Advantages

Throughput

Read Length (bp)

Life Technologies’ SOLiD system (Life Technologies, Carlsbad, CA) uses a unique color space mechanism for sequencing. The fragments to be sequenced are first immobilized on beads and then amplified using emulsion PCR. The beads carrying amplified fragments are arrayed on glass slide to perform the sequencing reaction. The underlying principle of the sequencing reaction is sequencing by synthesis, but instead of using DNA polymerase, SOLiD uses DNA ligase to ligate fluorescently labeled octamers to the fragment being read (Figure 6). The first two nucleotides of the octamer are interrogation bases, the next three nucleotides are degenerate nucleotides and the last three nucleotides are universal nucleotides such as inosine. The octamers carry four different fluorescent labels at the 5’ end, each corresponding to four possible dinucleotide combinations. After ligation, the images are acquired and the fluorescent label removed by cleaving the octamer between the fifth and the sixth nucleotide.

Name

5.1 Life Technologies’ SOLiD

Multiple ligation cycles are conducted to generate the sequence (read length 35–75 bp). A number of aspects are unique to the SOLiD system; DNA ligase is used instead of DNA polymerase to perform the sequencing reaction, the sequence is generated in color space and not base space and each nucleotide is read twice due to dinucleotide encoding in the octamers thereby reducing the error in sequencing. The SOLiD system can generate up to 15 GB data in anywhere between 2 and 7 days (depending on the sequencing application). The error rate in sequence data generated from the SOLiD systems is

current applications of biotechnology

current applications of biotechnology

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