yeast: description and structure

CHAPTER 2

YEAST: DESCRIPTION AND STRUCTURE 1*

Montes de Oca, R., 1Salem, A.Z.M., 2Kholif, A.E., 1Monroy, H., 1Pérez, L.S., 1Zamora, J.L. and 1Gutiérez, A.

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Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma del Estado de México Estado de México, Mexico 2 Dairy Science Department, National Research Centre, 33 Bohouth St. Dokki, Giza, Egypt Abstract Yeasts are unicellular eukaryotic fungi with completely different properties from those of bacteria, which are Prokaryotic microorganisms. Yeast contains almost the same organelles of a mature eukaryotic cell. Nucleus, Golgi apparatus, mitochondria, endoplasmic reticulum, vacuole, and cytoskeleton are the most important one. Yeast cell particle size is typically of 5×10μm. The primary method of reproduction is by budding, and occasionally by fission. Yeast can be identified and characterized based on cell morphology, physiology, immunology, and using molecular biology techniques. The natural habitat of yeast may be soil, water, plants, animals, and insects with special habitat of plant tissues. Many commercial products contain a mixture of varying proportions of live and dead S. cerevisiaecells are available for using as feed additives in animals nutrition. Key words: Structure, Yeast INTRODUCTION Saccharomyces cerevisiae yeast is unicellular fungi that divide asexually by budding or fission and whose individual cell size with a large diameter of 5– 10μm and a small diameter of 1–7μm. The cells of S. cerevisiae are pigmented, where cream color may be visualized in surface-grown colonies (Walker and White, 2011). Yeast cell is completely deferred than bacterial cell in both structure and function. Yeast Saccharomyces S. cerevisiae has an extensive history of uses in the area of food processing. It is commonly known as baker’s yeast or brewer’s yeast. S. cerevisiaehas been used for centuries as leavening for bread and as a fermenter of alcoholic beverages and wine production. Yeast also has a new function as natural feed additives in ruminant and non-ruminant animals for manipulating the gastrointestinal tract and the rumen environment. Description and significance Yeasts are fungi, whose common characteristics are predominant or permanent unicellular state. Yeasts are unicellular eukaryotic fungi with

Yeast: Description And Structure

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completely different properties than those of bacteria, which are Prokaryotes (Fig.1). For example, yeasts have a resistant to antibiotics, sulfamides and other anti-bacterial agents. This resistance is genetically and natural, and not liable to be modified or transmitted to other microbes. Moreover, yeast particle size (5×10μm) is also significantly higher than bacteria size (0.5×5μm). The main method of yeast reproduction is primarily by budding, and occasionally by fission, and these do not form spores in or on a fruiting body. Identification and characterization of yeast species may be according to a number of criteria such as cell morphology (e.g., mode of cell division and spore shape), physiology (e.g., sugar fermentation tests), immunology (e.g., immunofluorescence), and molecular biology (e.g., DNA reassociation, ribosomal DNA phylogeny, karyotyping, random amplified polymorphic DNA (RAPD), DNA base composition and hybridization, and amplified fragment length polymorphism (AFLP) of D1/D2 domain sequences of 26S rDNA). Molecular sequence analyses are being increasingly used by yeast taxonomists to categorize new species (Walker, 2009). Among yeast, S. cerevisiaeis of industrially important due to its ability to convert sugars (i.e., glucose, maltose) into ethanol and carbon dioxide (baking, brewing, distillery, liquid fuel industries). S. cerevisiae breaks down glucose through aerobic respiration in presence of oxygen. If oxygen is absent, the yeast will then go through anaerobic fermentation. The net result of this process is two adenosine triphosphate molecules, in addition to two by products; carbon dioxide and ethanol. Another common use of yeast is in the rising of bread. The carbon dioxide that is produce inside the dough causes it to rise and expand. In the baker’s yeast, these have strains that produce dioxide are more prevalent than ethanol and vice versa for brewing industries. Ecology and natural habitats The distribution of yeasts is not as bacteria in the natural environment, but nevertheless these can be isolated from soil, water, plants, animals, and insects (Table 1).

Fig. 1. Yeast cell (Distillique.co.za. 2015. 'Distillique - Basics of Yeast Nutrients'. (http://distillique.co.za/distilling_shop/blog/96-basics-of-yeast-nutrients.)


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Table 1. Natural yeast habitats (Walker, 2009) Habitat Animal

Description Several non-pathogenic yeasts are associated with the intestinal tract and skin of warm-blooded animals;yeasts (e.g., Candida albicans) are opportunistic pathogen to humans and animals; yeasts are commensally associated with insects acting as important vectors in the natural distribution Atmosphere A few viable yeast cells may be expected per cubic meter of air. Generally, Cryptococcus, Debaryomyces spp., Rhodotorula, and Sporobolomyces are dispersed by air from layers above soil surfaces Yeasts are fairly ubiquitous in buildings. e.g., Aureobasidium Built Environment pullulans is common on damp household wallpaper and S. cerevisiae is readily isolated from surfaces in wineries Interface between soluble nutrients of plants and the septic Plants world are common niches for yeasts; spread of yeasts on the phyllosphere is aided by insects. The presence of some organic compounds on the surface and decomposing areas creates conditions favorable for growth of yeasts Soil may only be a reservoir for the long-term survival of Soil yeast, rather than a habitat for growth. Yeasts are ubiquitous in cultivated soils (nearly 10 000 cells/g of soil) and are found only in the upper, aerobic soil layers (10–15cm). Lipomyces and Schwanniomyces are isolated exclusively from soil Yeasts predominate in surface layers of fresh and salt waters, Water but are not present in great numbers (nearly 1000 cells/L). Most aquatic yeast isolates are of red pigmented genera (Rhodotorula). The species Debaryomyces hansenii is a halotolerant yeast that can grow in nearly saturated brine solutions Plant tissues (i.e. leaves, flowers, and fruits) are preferred yeast habitats, but a few species are found commensally or in parasitic relationships with animals. Several species of yeast may be isolated from specialized or extreme environments, with high sugar or salt concentrations (i.e., low water potential), with low temperature, and with low oxygen availability. Types of Saccharomyces Saccharomyces is a genus in the kingdom of fungi that includes many species of yeast. The cell of yeast is a saprophytic unicellular fungi cell, where many members of this genus are considered very important in food production specially the brewer's yeast or baker's yeast (Table 2). Taxonomy and characterization S. cerevisiae is yeast that can exist either as a single-celled organism or as pseudo-mycelia(Table 3). The yeast cells reproduce by multilateral budding.


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Each cell produces one to four ellipsoidal, smooth-wall edascospores. Growth characteristics and physiological traits especially the ability to ferment individual sugars, is the main differentiation between S. cerevisiaeand other yeasts. This phenomenon is basis of clinical identification of yeast using commercially available diagnostic kits, which classify the organism through analysis of the ability of the yeast to utilize distinct carbohydrates as sole sources of carbon (Rosini et al., 1982). Table 2. Saccharomyces species (Walker, 2009) Saccharomyces bayanus Saccharomyces boulardii Saccharomyces bulderi Saccharomyces cariocanus Saccharomyces cariocus Saccharomyces cerevisiae Saccharomyces chevalieri Saccharomyces dairenensis Saccharomyces ellipsoideus Saccharomyces eubayanus Saccharomyces exiguus Saccharomyces florentinus

Saccharomyces kluyveri Saccharomyces martiniae Saccharomyces monacensis Saccharomyces norbensis Saccharomyces paradoxus Saccharomyces pastorianus Saccharomyces spencerorum Saccharomyces turicensis Saccharomyces unisporus Saccharomyces uvarum Saccharomyces zonatus

Table 3. Taxonomic hierarch of yeast S. cerevisiae Domain: Kingdom: Division Subdivision Class: Order: Family: Subfamily Genus Species

Eukarya Fungi Ascomycota Saccharomycotina Saccharomycetes Saccharomycetales Saccharomycetaceae Saccharomyetoideae Saccharomyces cerevisiae

The initial classification was based principally on the morphological characteristics with specific physiological and biochemical traits used to differentiate between isolates with similar morphological traits. As a result of the application of newer molecular techniques, the taxonomy of Saccharomyces is subject to greater scrutiny. In addition, the large heterogeneous species, S. cerevisiae, may be divided into four distinct species based on DNA homology. None of the four organisms or other closely related species has been associated with pathogenicity to humans or has been shown to have adverse effects on the environment. The four species are S. cerevisiae, Saccharomyces bayanus (also known as Saccharomyces uvarum), Saccharomyces pasteurianus (also known as Saccharomyces carlsbergensis), and Saccharomyces paradoxus. All these four yeast represent industrially important species.


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Cellular morphology and structure S. cerevisiae are eukaryotic cells that contain all major organelles that are also common to animal cells like nucleus, endoplasmic reticulum, mitochondria, Golgi apparatus, vacuole, cytoskeleton with all three major components, and many others organelles. Although, the complex-I is absent from S. cerevisiae cell, the respiratory process can be continued as a results of a simple NADHdehydrogenase encoded by the gene NDI1. Generally, yeast is unicellular, globose with elongate shape. Multilateral budding is typical and pseudohyphae are rudimentary. True hyphae are absent. Glucan is a major component of cell walls, as well as mannoproteins. Colonies of Saccharomyces grow rapidly and mature nearly in three days. Cells are characterized with flat, smooth, moist, glistening or dull, with cream to tannish cream color. Cell is able to use nitrate and ability to ferment various carbohydrates. When Saccharomyces grow on some media such as V-8 medium, Gorodkowa medium, or acetate ascospor agar, it produces ascospores, which are globose and located in asci that contain 1-4 ascospores. Asci do not rupture at maturity. Most Saccharomyces species are heterothallic, but a few are homothallic. If occurs, vegetative cells act as asci. The result of the sexual reproduction is four ascospores, which formed during meiosis. Once, the ascospores released, these new formed ascospores germinate produce haploid strains. Mating between haploid cells must occur to return to the diploid state. Both of haploid and diploid phases are morphologically similar, but with larger cells for diploid. In the asexual reproduction, bud grows to reach the size of the mother cell while nuclear division occurs. The separation occurs after a nucleus is passed to the daughter cells. Saccharomyces are heterotrophes, obtaining energy from glucose. They utilize both respiratory and fermentative metabolism. Approximately, 98% of glucose is metabolized during fermentation, while 2% of it is made into cell materials. However, the anaerobic metabolism yields more energy, about 10% of the glucose can be converted to cell material. This phenomenon is known as the Pasteur’s effect. Saccharomyces have an active glucose transport system, where glucose metabolization occurred through the glycolytic pathway. The glycolytic pathway is effective, when glucose present in low concentrations and will be repressed, when the concentrations are high. In case of repression, glucose enters the cell via a constitutive facilitated diffusion system. Moreover, high glucose concentrations may also suppress respiration in favor of fermentation, even when oxygen is available. This is known as the Crabtree effect or catabolite repression. Genome structure International Collaboration for the Yeast Genome Sequencing stated that S. cerevisiae was the first eukaryotic genome that was completely sequenced. Chromosomes of Saccharomyces contain a single linear double-stranded DNA with few repeated sequences caused mainly by the encoding of ribosomal RNA. Less than 5% of sequences have introns. In 1996, the Saccharomyces genome sequence was released in the public domain after that, regular updates have been maintained at Saccharomyces Genome Database. Another important S. cerevisiae database is maintained by the Munich Information Center for Protein


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Sequences (MIPS). The genome has about 12,156,677 base pairs with 6,275 genes about 5,800 are believed to be true functional genes. Genes are compactly organized on 16 chromosomes. It is estimated that yeast have at least 31% of its genes homologous with that of humans (Herskowitz, 1988). Yeast genes are classified using gene symbols or systematic names (Fig. 2).

Fig. 2. Yeast chromosome (Millar and Grunstein, 2006)

Because of its unique genetic structure, S. cerevisiae is a useful tool in research field. At Woolford Laboratory at Carnegie Mellon University, scientists have used it to study pathways of ribosome assembly with better understanding of the genetic structure not just of S. cerevisiae, but to certain general genetic processes. Like other eukaryotes, the 40S ribonucleoprotein contains one 18S rRNA and 32 ribosomal proteins come from a single 35S transcript synthesized by polymerase I, on the contrary, pre-RNA is transcribed by polymerase III. After transcribtion, the pre-RNA is packaged in a 90S RNP. All of these processes are mediated by enzymes of endoribionucleases and exoribionucleases. Following these steps, 66S particles are released into the nucleoplasm, mature, and then are exported to the cytoplasm of the cell. They have also discovered that there are still other steps before ribosomal subunits are able to facilitate protein synthesis. Moreover, they have noted non-ribosomal molecules, which are necessary for some processes such as rearrangement of rRNA structure as well as RNA cleavage and processing. Moreover, Alices-Villanueva (1997) studied the TRP1 RI circle plasmid of chromosome IV of S. cerevisiae species. The TRP1 gene in plasmid (contains Autonomously Replicating Sequence; ARS) codes for an enzyme required in the synthesis of tryptophan. The ARS allows the plasmid to replicate independently of chromosomal DNA. Alices-Villanueva created two versions of this gene, where one of them with only a strong promoter, while the


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other with both a strong and a weak promoter (Alices-Villanueva, 1997). Rates of expression are higher for the gene with both promoters, which gives evidence to the proposed hypothesis. S. cerevisiae contains an acidic cytoplasmic protein named Gir2. This protein lacks extensive secondary structure (Alves and Castilho, 2005) with sensitivity to proteolysis. Kelberg (2005) discovered another new gene in S. cerevisiae called HIM1 on the right arm of chromosome IV. They stated that when mutations occur in HIM1, there was an increase both in spontaneous mutation rate and in overall frequencies of mutations. Saccharomyces cerevisiae life cycle Growth in yeast cells is synchronized with the growth of buds.The buds reach the size of the mature cells by the time it separates from the parent cell. In case of rapidly growing yeast cultures, yeast cells can be seen to have buds, where bud formation occupies the whole yeast cell cycle. Both of mother and daughter cells can start budding before the cell separation occurs (Fig. 3). S. cerevisiae can reproduce as sexually or asexually. It can indefinitely reproduce both as diploids (2n) and as haploids (1n) in which new daughter cells arise mitotically as buds that grow in size and eventually split from the mother cell. This fact greatly facilitates genetic analysis. The transition between haploid and diploid phases of the life cycle is accomplished by mating of two haploids to form a diploid zygote and by meiosis, where one diploid cell undergoes premeiotic S-phase and two meiotic divisions resulting in four haploid cells, which are enclosed in ascospore walls. Haploid cells occur in two mating types, a and ά (Fig. 4). Both of them can reproduce mitotically as stable haploid cells. Or they can engage in sexual reproduction, in which cells of opposite mating types communicate with each another by proteins known as pheromones. Both mating and meiosis are controlled genetically by the mating type locus of which two alleles exist, corresponding to the two sexes. Tetrad analysis of the meiotic products, which is impossible to perform in higher organisms, is one of the most convenient ways of genetic analysis. The cell life cycle in yeast normally consists of the following stages – G1, S, G2, and M – which are the normal stages. In the G1 phase of the cell cycle, S. cerevisiae cells have the options for cell differentiation, where haploid cells can mate with partner cells of the opposite sex or form stationary (G0) cells, and these have the ability to age. Diploid cells can undergo meiosis or form stationary cells. Furthermore, these can be transformed into pseudohyphae and those can age.


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Fig. 3. Saccharomyces cerevisiae mitotic cell cycle (Cosma, 2004)

Fig. 4. Haploid yeast cells be ‘a’ or ‘ά’ mating type (Lodish et al., 2000)

The pheromones induce dissimilar cells to undergo cell fusion followed by nuclear fusion. The new formed zygote has a single diploid nucleus and buds to produce diploid progeny. Diploid yeast cells also propagate as stable diploid cells by mitotic division. Starvation, however, induces those to undergo meiosis and sporulation, which allows the yeast cells to ‘reshuffle’ their genes, when


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conditions are poor, perhaps enabling those to find a combination more suitable for survival in the environment. Generally, meiosis reduces the diploid nucleus to four haploid nuclei, which become encapsulated in four haploid spores. The nutrient depletion induces meiosis and sporulation, while the subsequent availability of nutrients promotes spore germination and gamete production. Sex in yeast is determined by the mating type locus (designated as MAT) on chromosome III. There are two mating types: ‘a’ and Mating ability segregates 2a: 2α in tetrads derived from MATa/MATα heterozygous diploids, indicating that the ‘a’and mating types are specified by alleles of a single locus (MAT). MATa or MATα cells mate efficiently with cells of the opposite sex. Heterozygous MATa/MATa diploids are sterile, but it is possible to derive MATa/MATa or MAT α/MATα diploid cells. These diploid cells will mate with other cells of the opposite mating type, either haploids or diploids. The ability to mate is thus determined by the genetic configuration at the MAT locus and as such is not related to ploidy. Saccharomyces cerevisiae commercial applications Most commercial products contain a mixture of varying proportions of live and dead S. cerevisiae cells. Those with a predominance of live cells are sold as live yeasts, while others containing more dead cells and the growth medium are sold as yeast cultures (Newbold and Rode, 2006). Examples include Yea-sacc (Alltech Inc.), Diamond V Yeast culture (Diamond V, Mills Inc.), and Levucell SC-20 (Lallemand Animal Nutrition). In addition to its use in food processing, S. cerevisiae is widely used for the production of macromolecular cellular components such as lipids, proteins, enzymes, and vitamins (Bigelis, 1985; Stewart and Russell, 1985). S. cerevisiae has been regarded having GRAS status by FDA. Furthermore, the National Institutes of Health in its Guidelines for Research Involving Recombinant DNA Molecules considers S. cerevisiae a safe organism. The abundance of information on S. cerevisiae, derived from its role in industrial applications, has positioned S. cerevisiae as a primary model for the genetic manipulation. Conclusions Yeast, as a eukaryotic cell can be used in many different applications rather than the use in bread backing and wine industries. Utilization of yeast as feed additives in animal nutrition as safe and natural feed additives is an area of research interest, where, it can be proved to be efficient in improving animal performance. REFERENCES Alices-Villanueva, H. 1997. Investigation of The Influence of Selected Gene Promoter Strength on Yeast Acentric Ring Plasmid Copy Number (Doctoral dissertation).University of Hawaii. Alves, V. S. and Castilho, B. A. 2005. Gir2 is an intrinsically unstructured protein that is present in Saccharomyces cerevisiae as a group of heterogeneously electrophoretic migrating forms. Biochemical and Biophysical Research Communications, 332(2), 450-5.


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Bigelis, R. 1985. Primary metabolism and industrial fermentations. In: J. W. Bennet, L. L. Lasure (Eds.),Gene Manipulations in Fungi. (pp. 357), New York, NY: Academic Press. Cosma, M. P. 2004. Daughter‐specific repression of Saccharomyces cerevisiae HO: Ash1 is the commander. EMBO reports, 5, 921 – 1013. Herskowitz, I. 1988. Life cycle of the budding yeast Saccharomyces cerevisiae. Microbiolgy Reviews, 52: 536–53. Kelberg, E. P. 2005. HIM1, a new yeast Saccharomyces cerevisiae gene playing a role in control of spontaneous and induced mutagenesis.Mutat Research, 578, 64-78. Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., and Darnell, J. 2000.Mutations: Types and Causes. In: W. H. Freeman, Molecular Cell Biology. New York: Available from: http://www.ncbi.nlm.nih.gov/ books/NBK21578/. Millar, C. B. and Grunstein, M. 2006. Genome-wide patterns of histone modifications in yeast. Nature Reviews Molecular Cell Biology, 7, 657-666. Newbold, C. J., and Rode, L. M. 2006.Dietary additives to control methanogenesis in the rumen. International Congress Series, 1293, 138–147. Rosini, G., Federici, F., Vaughn, A. E., and Martini, A. 1982. Systematics of the species of the yeast genus Saccharomyces associated with the fermentation industry. European Journal of Applied Microbiology and Biotechnology, 15, 188-193. Stewart, G. C., and Russell, I. 1985. The biology of Saccharomyces. In: A. L. Demain, N. A. Solomon, (Eds.), Biology of industrial organisms (pp. 511536). Menlo Park, California: Benjamin Cummins Publishers. Walker, G. M. 2009. Yeasts. In: M. Schaechter (Ed.) Desk Encyclopedia of Microbiology. (pp. 1174-1187) 2nd ed. London: Elsevier/Academic Press. Walker, G. M., and White, N. A. 2011.Introduction to Fungal Physiology. In: Kavanagh, K. (ed), Fungi: Biology and Applications (pp. 1-36). West Sussex, UK: John Wiley and Sons Ltd.