THE METABOLIC PATHWAY ENGINEERING ...

21 Escherichia coli as a Well-Developed Host for Metabolic Engineering 21.1 Why Escherichia coli? ��21-1 21.2 Fundamentals of Metabolic Engineering of E. coli....................21-3 Metabolism and Products • Genetic Elements and Tools • Commonly Used Strains and Strain Improvements • Cultivation Technology

Eva Nordberg Karlsson, Louise Johansson, Olle Holst, and Gunnar Lidén Lund University

21.3 M etabolic Engineering of a Specific Pathway— The Shikimate Pathway ��21-12

Increase of the Flux into the Pathway • Supply of Precursors • Minimization of By-Products • Comparison with Metabolic Engineering of an Artificial Pathway—1,3-Propanediol Production

21.4 Concluding Remarks ��21-18 References ��21-18

21.1 Why Escherichia coli? In any encyclopedia concerning industrial applications of microorganisms, Escherichia coli will be included. Although few people would disagree on the importance of the organism today, it is a highly legitimate question to ask why a bacterium originally isolated from human intestines has become an important production host for as diverse products as precursors for plastics and therapeutic proteins? It has been stated [1] that if you “mention E. coli to the man in the street, he’s most likely to make some references to a dodgy burger and the resulting diarrhea.” However, even if there are pathogenic strains, E. coli is actually one of the dominating species in the bowel of healthy individuals. Furthermore, to most applied scientists E. coli is looked upon as a well-known and useful microbial model system. An important reason is that E. coli early became known for its ease of growth on synthetic media, and its fast doubling times [2], making it an attractive system to handle. The development leading to the current importance of E. coli is the result of many factors, some of which are summarized in Figure 21.1. Starting from the beginning, E. coli was first cultured from faeces of healthy individuals in 1885 by the German pediatrician Theodor Escherich. At that time he named the newly isolated organism “Bacterium coli” [3], to reflect the fact that it was a bacterium present in the colon (hence the name “coli”), but it was later renamed Escherichia coli [2,4]. The ease of cultivation was certainly one of the factors that promoted its distribution between scientists, who exchanged available strains for the elucidation of microbial and biochemical phenomena and pathways. In the 1940s and 1950s there was a large development in bacterial genetics, biochemistry, and physiology. During this time-period, Jacques Monod and coworkers at the Pasteur institute performed their classic studies on the lac-operon in E. coli, which resulted in the explanation of “diauxie” (or diauxic 21-1 © 2010 by Taylor & Francis Group, LLC

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Developing Appropriate Hosts for Metabolic Engineering

Discovery, 1855 ”Bacterium coli” (T. Escherich) Indicator of sanitary conditions in food and water (“coliform” test) to alert fecal contamination/ possible pathogen presence, 1892 Renamed Escherichia coli, 1919 ”The phage group” established by Delbrueck and Luria, in early 1940s to work on genes in a less complex system than Drosophila Bacterial conjugation, 1946 (Lederberg) Coordinated gene regulation (Diauxic growth, lac-operon, enzyme induction) by Monod and Jacob (1940-50s) Lederberg suggests the term ”plasmid” for extrachromosomal hereditary elements, 1952 Purification of plasmids. Bacterial antibiotics resistance genes shown to be carried on plasmids. 1968 (Cohen) Discovery of restriction enzymes and ligases, EcoR1 isolated by Boyer, 1970 First recombinant DNA with bacterial genes, 1972 (Berg) First cloning of DNA-fragment in plasmid (pSC101), 1973 (Boyer and Cohen) Humulin, Genentech´s human insulin licensed, 1978 Metabolic engineering for phenylalanine production, 1987 (Miller et al.)

Complete sequence of E. coli K12 genome, 1997 (Blattner et al.) Production of 1,3-propandiol in engineered E. coli, 2003 (Nakamura and Whited)

Figure 21.1 A brief overview of historical developments leading to the position of Escherichia coli as a model organism for many scientists, and a “workhorse” in molecular biology.

growth), i.e., the shift of growth from one substrate (glucose) to another (lactose) upon depletion of the former where growth on the respective substrate was separated by an adaptation or “lag”-phase [5]. Based on the E. coli model system it was also, for the first time proposed that the ability to utilize lactose was coupled to the formation of an enzyme, β-galactosidase (called lactase at that time). Fundamental questions on the relation between gene, enzyme, and inductive substrate were raised, leading to an understanding of © 2010 by Taylor & Francis Group, LLC

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Escherichia coli as a Well-Developed Host for Metabolic Engineering

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Table 21.1 Pros and Cons of E. coli as a Production Organism Advantages Rapid growth on a multitude of sugars Possible to grow to high cell densitites Well established molecular tools (fully sequenced organism, many plasmids available) No need for complex medium (i.e., vitamin addition) Well developed cultivation strategies Industrial acceptance due to large experience

Disadvantages Overflow metabolism giving acetate production at high glucose fluxes Limited capacity for synthesis of large proteins No protein glycosylation A relatively high maintenance requirement. Due to its facultative metabolism, the product formation may change in large scale operation in zones of oxygen depletion PTS sugar transport system Endotoxin production

the phenomenon of enzyme induction [6]. The parallel discovery of bacterial conjugation in E. coli 1946 by Lederberg [7,8] allowed an early genetic analysis that led to identification of the genes involved in lactose metabolism. By the end of the 1960s the main questions surrounding gene expression and regulation of the genes in the E. coli lac-operon had been solved, and can be said to have laid a foundation for the field of molecular biology [9]. During the years to come the increased understanding of genetic elements and DNA-modifying enzymes, led to development of molecular biology techniques. E. coli was again chosen as the model system, in the pioneering work by Stanley Cohen and Herbert Boyer, who with the help of restriction-endonucleases, and purified plasmids managed to performed the first successful cloning of a recombinant plasmid, which was transformed for expression in an E. coli host strain [10]. The new techniques constituted the basis for a symbiotic relationship between science and technology [11] that quickly resulted in commercial products in the pharmaceutical sector. Not surprisingly, the first products were recombinant proteins. The possibility of producing a target protein by microbial cultivation opened up entirely new possibilities in terms of supplying human proteins/peptides in amounts allowing therapeutic applications. In 1978 the first “molecular biology based” biotechnology company, Genentech, licensed human insulin from a gene cloned in E. coli to the company Eli Lilly, one of the major producers of insulin. Soon to follow (the same year), Genentech got the commission to clone a gene encoding human growth hormone, hGH, Genotropin (originally named Somatonorm), from Kabi-Gen, a Swedish pharmaceutical company (today part of Pfizer). This product finally reached the market in 1985 [12]. The speed of molecular biology development has since then accelerated and recombinant proteins may now be produced in a range of host organisms. E. coli maintains a strong position as a host for protein production, despite limitations in terms of e.g., protein secretion, glycosylation, and folding. The disadvantages are balanced by advantages such as the ease of cultivation, the possibility to achieve very high intracellular titers of recombinant protein (up to 30% of the dry weight) in E. coli and—not least—the substantial amount of genetic tools and practical experience of handling E. coli today accumulated in academia and industry (Table 21.1). The interest in E. coli as a host for metabolite production began with the isolation of high-yielding threonine producing E. coli strains, and the use of E. coli for amino acid production carried into the metabolic engineering era with the development of phenylalanine and tryptophan production [13,14]. One of the most spectacular recent achievement in terms of metabolic engineering of any microorganism is the engineering of E. coli for direct production of 1,3-propanediol from glucose carried out by Genencor in collaboration with Du Pont [15]. The establishment of large-scale commodity chemical production with metabolically engineered E. coli is certainly a milestone in the development of biotechnology.

21.2 Fundamentals of Metabolic Engineering of E. coli 21.2.1 Metabolism and Products To enable targeted metabolic engineering, a detailed knowledge of metabolic pathways and control elements for gene expression is necessary. A factor to keep in mind is that alterations of the metabolic © 2010 by Taylor & Francis Group, LLC

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pathways to improve cell properties and survival, is a natural adaptation process in bacteria, allowing the possibility to colonize different natural habitats. This flexibility is an asset also in metabolic engineering work. The principal advantage of E. coli as a host organism in terms of its metabolism is its ability of rapid growth on many different sugars with the addition of only mineral salts. The fact that only mineral salts are needed as nutrients considerably increases the range of commercially interesting end products. The metabolic drawbacks include the overflow metabolism—i.e., the formation of acetate also aerobically at high glycolytic fluxes [16], and a rather substantial maintenance energy requirement— giving a nongrowth associated consumption of the carbon source. The last fact requires careful considerations in the process design. E. coli is a facultative aerobe, i.e., it is capable of forming fermentative end products and to generate ATP also anaerobically. The anaerobic metabolism provides pathway flexibility, but may also complicate process scale-up in aerobic processes due to oxygen concentration gradients in large scale reactors [17]. A fundamental question to be asked is—what kind of products are likely to be produced efficiently in this host organism? A summary of already established commercial products as well as currently investigated new potential products from metabolically engineered E. coli is given in Table 21.2. From a market perspective the products range from therapeutic compounds—in the very high price end—down to commodity chemicals such as 1,3-propanediol. Also from a metabolic engineering perspective, the products span a wide range, with some products being achieved with only modest changes in the normal metabolism, whereas others require the insertion of entire pathways. The metabolism of E. coli can be broken down into principal blocks as shown in Figure 21.2. In terms of smaller metabolites, products to be formed are likely to be derived from the 12 central precursor metabolites formed in the catabolism. These can either be converted into a normal metabolite in E. coli, or a metabolite which is only formed in E. coli after introduction of an artificial pathway. Table 21.2 Examples of Products Obtained in Metabolically Engineered E. coli and the Principal Modifications Made Product Class Primary metabolites

Amino acids

Compound

Use

Ethanol

Fuel

Lactic acid

Materials

Succinic acid

Food, Materials

Tryptophan

Pharma

Principal Metabolic Modifications Required Overexpression of PDC (encoding pyruvate decarboxylase) and adhB (encoding alcohol dehydrogenase) from Zymomonas mobilis Deletion of pathways giving anaerobic by-products, i.e., deletion of fumarate reductase (deletion of frdABCD), alcohol dehydrogenase (deletion of adhE), and pyruvate formate lyase (deletion of pflB). Interesting difference in strain background, where an optically purer grade of D-lactate for polylactate production was obtained in strain B (KO11) Minimize drainage of NADH in undesired reductions, such as formation of lactate (deletion of ldhA) and ethanol (deletion of adhE). Increase carbon dioxide binding reactions, e.g., carboxylation of pyruvate (heterologous expression of pyc, Lactococcus lactis) or carboxylation of PEP (heterologous expression of PEPC, e.g., from Actinobacillus succinogenes). Provide an alternative nonreductive pathway for succinate production via activation of the glyoxylate pathway. This can be achieved by inactivation of the repressor encoded by iclR. Minimize acetate formation by deletion of ackA and pta. Modification of feed-back resistance in the first step in the aromatic pathway (mutation of e.g., aroH, aroG, and aroF), modifications in the PTS sugar uptake system, overexpression of the Trp operon (trpEDCBA) (catalyzing the reaction steps downstream of chorismate), deletion of tnaA encoding tryptophanase to minize the breakdown of tryptophan.

Reference 18

19,20

21,22

23,24

(Continued)

© 2010 by Taylor & Francis Group, LLC

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Table 21.2 (Continued) Product Class

Compound

Use

Phenylalanine

Food

Intermediates in pathways

Shikimic acid

Pharma

Secondary metabolites

Amorpha-4,11diene (an isoprenoid)

Pharma

Monomers

1,3-propanediol Materials

Polymers

Polyhydroxyalkanoates

Materials

Recombinant proteins

Insulin hGH

Pharma

DNA

plasmid DNA

Pharma (gene therapy)

Principal Metabolic Modifications Required Similar modifications as for overproduction of tryptophan with respect to sugar transport and feed-back inhibition in the upper part of the aromatic pathway. Modification of feed-back resistance in pheA encoding prephenate dehydratase, i.e., the dedicated step into L-phenylalanine formation from chorismate. Similar modifications as for overproduction of aromatic amino acids with respect to sugar transport and feed-back inhibition in the upper part of the aromatic pathway. Deletion of shikimate kinase (aroK, aroL). Insertion of the entire mevalonate-dependent isoprenoid pathway (8 genes), mainly from S. cerevisiae (atoB acetoacetyl-CoA-thiolase (E.coli), HMGS, HMGR (truncated), ERG12, ERG8, MVD1 (S. cer), idi - IPP isomerase (E. coli), ispA - FPP synthase (E. coli). Insertion of a codon-optimized variant of ADS (amorphadiene synthase). Insertion of heterogeneous genes encoding glycerol-3phosphate dehydrogenase (GPD1, S. cerevisiae) glycerol-3phosphate phosphatase (GPP2, S. cerevisiae), glycerol dehydratase (dhaB1-B3, Klebsiella pneumoniae), and 1,3 propanediol oxidoreductase*. Deletion of genes encoding glycerol kinase (glpK) and glycerol dehydrogenase (gldA). Change of uptake system, i.e., deletion of the PTS system and creation of an ATP-coupled uptake via overexpression of galactose permease (galP) and glucokinase (glk). Furthermore, insertion of reactivation factors for the glycerol dehydratase (dhaBX, orfX, and Klebsiella pneumoniae). Expression of PHA synthase phaC from Aeromonas acromogenes, phaJ from A. hydrophila (encoding enoyl-CoA hydratase) and phbB from Ralstonia eutropha. The first successful recombinant insulin production method was based on a two-chain method. A-and B-chains were produced separately as fusion-proteins, and were extracted from inclusion bodies. For hGH, a first production combined an E. coli expressed peptide with a chemically synthesized. Later hGH was correctly processed in E. coli after periplasmic production. Supported replication of ColE1 plasmids. Decreasing RNA degradation.

Reference 25

26,27

28

15

29,30

31–33

34

* The source for the oxidoreductase used in the final construct was in fact the E. coli gene yqhD.

The changes made to obtain the products shown in Table 21.2 can be said to principally fall into the following categories:

1. Modification of substrate uptake 2. Increase of the yield of the essential precursor molecule(s) from the substrate 3. Insertion of genes enabling the synthesis of the desired product molecule from precursor molecule(s) 4. Modification(s)—typically gene deletions—aiming at decreasing the formation of by-products

The initial modification made is very often of the third category, i.e., primarily one makes sure that the product of interest is indeed synthesized from a suitable precursor. Ethanol production from pyruvate can for example be obtained in E. coli by providing a heterologous pyruvate decarboxylase and an


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G6P R5P F6P E4P G3P 3-PG PEP Pyruvate AcCoA OA SuccCoA α-KG

Glucose Lactose Sucrose Xylose Galactose

Substrates Transport

Substrates Fuelling reactions

Precursor metabolites

Secretion

Proteins

Ethanol Lactic acid Succinic acid 1,3 propanediol

Metabolites Transport

Inherent or Engineered pathways

Building blocks Biosynthetic reactions Polymerization

Macromolecules Assemblance

Amino acids RNA-nucleotides DNA-nucleotides Fatty acids UDP-glucose UDP-N-acetylglucoseamine

Proteins RNA DNA Lipids LPS Peptidoglycan

Biomass (more cells)

Figure 21.2 A schematic breakdown of metabolism, showing principal precursors and potential product formation.

alcohol dehydrogenase [18]. The introduction of genes encoding the necessary enzymes for converting the precursor to product may be sufficient in some cases, but normally also other modifications are necessary. With respect to sugar uptake, many sugars—including glucose, mannose, and fructose—are taken up via the phosphoenol pyruvate: sugar phosphotransfer system (PTS). This results—as a net effect—in an intracellular phosporylated sugar molecule produced at the expense of the conversion of one molecule of phosphoenol pyruvate (PEP) to pyruvate [35]. Since the uptake is coupled to phosphorylation, the consumption of one PEP is not necessarily disadvantageous from an energetic viewpoint [36]. However, inactivation of PTS components has been shown efficient to avoid catabolite repression, and thereby allow uptake of several sugars simultaneously [37]. Furthermore, use of the PTS system decreases the maximum yield of PEP from glucose from two to one. This is a drawback for products derived from PEP, such as aromatic amino acids as will be discussed later. An alternative in such cases is to introduce a facilitated glucose transporter, by, e.g., expression of the glf gene encoding a glucose facilitator from the bacterium Zymomonas mobilis, and a glucose kinase [38]. To decrease the by-product formation is in some cases a question of fine-tuning, but may in other cases in fact be the main—or only—modification made. As an example it can be mentioned that production of lactate in E. coli is obtained by deleting the competing pathways in the fermentative metabolism, i.e., deletion of the enzymes catalyzing formation of ethanol, acetate, formate, and succinate (cf. Table 21.2).

21.2.2 Genetic Elements and Tools To implement the gene insertions or deletions in the E. coli metabolic pathways, tools for cloning, transformation, and control of gene expression are needed. As indicated in Table 21.3, a wealth of alternatives is available. A difference in metabolic engineering, compared to genetic engineering for recombinant protein production, is the interest in both inserting and deleting genes as well as both up- and down


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Table 21.3 Selected Promoter Elements Used for Gene Expression in E. coli Promoter

Source

lac (lacUV5, lac(TS)) araBAD

E. coli E. coli

trp

E. coli

phoA

E. coli

recA cspA

E. coli E. coli

cadA

E. coli

Nalidixic acid Low temperature shift, 