From yeast genetics to biotechnology

Roots of classical yeast genetics go back to the early work of Lindegreen in the 1930s, who studied thallism, sporulation and inheritance of wine yeast strains belonging to S. cerevisiae. Consequent mutation and hybridization of heterothallic S. cerevisae strains resulted in the discovery of life cycle and mating type system, as well as construction of the genetic map. Elaboration of induced mutation and controlled hybridization of yeast strains opened up new possibilities for the genetic analysis of technologically important properties and for the production of improved industrial strains, but a big drawback was the widely different genetic properties of laboratory and industrial yeast strains. Genetic analysis and mapping of industrial strains were generally hindered because of homothallism, poor sporulation and/or low spore viability of brewing and wine yeast strains [1, 2]. In spite of this, there are a few examples of the application of sexual hybridization in the study of genetic control of important technological properties, e.g. sugar utilization, flocculation and flavor production in brewing yeast strains [3] or in the improvement of ethanol producing S. cerevisiae strains [4]. Rare mating and application of karyogamy deficient (kar-) mutants also proved useful in strain improvement [5]. Importance of yeasts in biotechnology is enormous. This includes food and beverage fermentation processes where a wide range of yeast species are playing role, but S. cerevisiae is undoubtedly the most important species among them. New biotechnology is aiming to improve these technologies, but besides this, a completely new area of yeast utilization has been emerged, especially in the pharmaceutical and medical areas. Without decreasing the importance of S. cerevisiae, numerous other yeast species, e.g. Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica have gained increasing potentialities in the modern fermentation biotechnology [6]. Developments in yeast genetics, biochemistry, physiology and process engineering provided bases of rapid development in modern biotechnology, but elaboration of the recombinant DNA technique is far the most important milestone in this field. Other molecular genetic techniques, as molecular genotyping of yeast strains proved also very beneficial in yeast fermentation technologies, because dynamics of both the natural and inoculated yeast biota could be followed by these versatile DNA-based techniques.     

酵母生物多样性开发及工业应用

酵母是一类包括酿酒酵母和非常规酵母在内的多种单细胞真菌的总称,其中酿酒酵母是应用较多的重要工业微生物,广泛应用于生物医药、食品、轻工和生物燃料生产等不同生物制造领域.近年来,研究者从不同生态环境中分离了大量的酵母菌株,鉴定了多个新种,也发现了抗逆性不同以及具有多种活性产物合成能力的菌株,证明天然酵母资源具有丰富的生物多...     

The use of lactic acid-producing,malic acid-producing,or malic acid-degrading yeast strains for acidity adjustment in the wine industry

In an era of economic globalization, the competition among wine businesses is likely to get tougher. Biotechnological innovation permeates the entire world and intensifies the severity of the competition of the wine industry. Moreover, modern consumers preferred individualized, tailored, and healthy and top quality wine products. Consequently, these two facts induce large gaps between wine production and wine consumption. Market-orientated yeast strains are presently being selected or developed for enhancing the core competitiveness of wine enterprises. Reasonable biological acidity is critical to warrant a high-quality wine. Many wild-type acidity adjustment yeast strains have been selected all over the world. Moreover, mutation breeding, metabolic engineering, genetic engineering, and protoplast fusion methods are used to construct new acidity adjustment yeast strains to meet the demands of the market. In this paper, strategies and concepts for strain selection or improvement methods were discussed, and many examples based upon selected studies involving acidity adjustment yeast strains were reviewed. Furthermore, the development of acidity adjustment yeast strains with minimized resource inputs, improved fermentation, and enological capabilities for an environmentally friendly production of healthy, top quality wine is presented.     

Chromosome-scale genetic mapping using a set of 16 conditionally stable Saccharomyces cerevisiae chromosomes

We have created a resource to rapidly map genetic traits to specific chromosomes in yeast. This mapping is done using a set of 16 yeast strains each containing a different chromosome with a conditionally functional centromere. Conditional centromere function is achieved by integration of a GAL1 promoter in cis to centromere sequences. We show that the 16 yeast chromosomes can be individually lost in diploid strains, which become hemizygous for the destabilized chromosome. Interestingly, most 2n - 1 strains endoduplicate and become 2n. We also demonstrate how chromosome loss in this set of strains can be used to map both recessive and dominant markers to specific chromosomes. In addition, we show that this method can be used to rapidly validate gene assignments from screens of strain libraries such as the yeast gene disruption collection.     

Cell surface display of highly pathogenic avian influenza virus hemagglutinin on the surface of Pichia pastoris cells using α‐agglutinin for production of oral vaccines

Yeast is an ideal organism to express viral antigens because yeast glycosylate proteins more similarly to mammals than bacteria. Expression of proteins in yeast is relatively fast and inexpensive. In addition to the convenience of production, for purposes of vaccination, yeast has been shown to have natural adjuvant activity making the expressed proteins more immunogenic when administered along with yeast cell wall components. Development of genetic systems to display foreign proteins on the surface of yeast via fusion to glycosylphosphatidylinositol‐anchored (GPI) proteins has further simplified the purification of recombinant proteins by not requiring harsh treatments for cellular lysis or protein purification. We have expressed the hemagglutinin protein from a highly pathogenic avian influenza (HPAI) virus [A/Egret/HK/757.2/02], subtype H5N1, on the surface of the yeast strain Pichia pastoris, as an anchored C‐terminal fusion with the Saccharomyces cerevisiae GPI‐anchored cell wall protein, α‐agglutinin. Surface expression of the hemagglutinin fusion protein was demonstrated by immunofluorescence microscopy. Functionally, the fusion protein retained hemagglutinin agglutinating activity, and oral vaccination with the yeast resulted in production of virus neutralizing antibodies. This study represents the first steps in the generation of a yeast‐based vaccine for protection against highly pathogenic strains of avian influenza. Published 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Apoptosis and lipoapoptosis in the fission yeast Schizosaccharomyces pombe

Yeasts being simple eukaryotes are established genetic systems that are often employed to solve important biological questions. Recently, it has become evident that certain cell death programs exist in these unicellular organisms. For example, it has been shown recently that strains of the fission yeast Schizosaccharomyces pombe deficient in triacylglycerol synthesis undergo cell death with prominent apoptotic markers. This minireview is intended to discuss key developments that have rendered fission yeast useful both as a tool and as a model for apoptosis and lipoapoptosis research. It is attempted to delineate a putative signaling pathway leading to the execution of lipoapoptosis in the fission yeast. Although in its infancy, apoptosis research in the fission yeast promises exciting breakthroughs in the near future.     

A role for the mismatch repair system during incipient speciation in Saccharomyces

The cause of reproductive isolation between biological species is a major issue in the field of biology. Most explanations of hybrid sterility require either genetic incompatibilities between nascent species or gross physical imbalances between their chromosomes, such as rearrangements or ploidy changes. An alternative possibility is that genomes become incompatible at a molecular level, dependent on interactions between primary DNA sequences. The mismatch repair system has previously been shown to contribute to sterility in a hybrid between established yeast species by preventing successful meiotic crossing-over leading to aneuploidy. This system could also promote or reinforce the formation of new species in a similar manner, by making diverging genomes incompatible in meiosis. To test this possibility we crossed yeast strains of the same species but from diverse historical or geographic sources. We show that these crosses are partially sterile and present evidence that the mismatch repair system is largely responsible for this sterility.     

Genetic mapping of quantitative phenotypic traits in Saccharomyces cerevisiae

Saccharomyces cerevisiae has become a favorite production organism in industrial biotechnology presenting new challenges to yeast engineers in terms of introducing advantageous traits such as stress tolerances. Exploring subspecies diversity of S. cerevisiae has identified strains that bear industrially relevant phenotypic traits. Provided that the genetic basis of such phenotypic traits can be identified inverse engineering allows the targeted modification of production strains. Most phenotypic traits of interest in S. cerevisiae strains are quantitative, meaning that they are controlled by multiple genetic loci referred to as quantitative trait loci (QTL). A straightforward approach to identify the genetic basis of quantitative traits is QTL mapping which aims at the allocation of the genetic determinants to regions in the genome. The application of high-density oligonucleotide arrays and whole-genome re-sequencing to detect genetic variations between strains has facilitated the detection of large numbers of molecular markers thus allowing high-resolution QTL mapping over the entire genome. This review focuses on the basic principle and state of the art of QTL mapping in S. cerevisiae. Furthermore we discuss several approaches developed during the last decade that allow down-scaling of the regions identified by QTL mapping to the gene level. We also emphasize the particular challenges of QTL mapping in nonlaboratory strains of S. cerevisiae.     

Isolation by genetic and physiological characteristics of a fuel-ethanol fermentative Saccharomyces cerevisiae strain with potential for genetic manipulation

Fuel ethanol fermentation process is a complex environment with an intensive succession of yeast strains. The population stability depends on the use of a well-adapted strain that can fit to a particular industrial plant. This stability helps to keep high level of ethanol yield and it is absolutely required when intending to use recombinant strains. Yeast strains have been previously isolated from different distilleries in Northeast Brazil and clustered in genetic strains by PCR-fingerprinting. In this report we present the isolation and selection of a novel Saccharomyces cerevisiae strain by its high dominance in the yeast population. The new strain, JP1 strain, presented practically the same fermentative capacity and stress tolerance like the most used commercial strains, with advantages of being highly adapted to different industrial units in Northeast Brazil that used sugar cane juice as substrate. Moreover, it presented higher transformation efficiency that pointed out its potential for genetic manipulations. The importance of this strain selection programme for ethanol production is discussed.     

Advances in quantitative trait analysis in yeast

Understanding the genetic mechanisms underlying complex traits is one of the next frontiers in biology. The budding yeast Saccharomyces cerevisiae has become an important model for elucidating the mechanisms that govern natural genetic and phenotypic variation. This success is partially due to its intrinsic biological features, such as the short sexual generation time, high meiotic recombination rate, and small genome size. Precise reverse genetics technologies allow the high throughput manipulation of genetic information with exquisite precision, offering the unique opportunity to experimentally measure the phenotypic effect of genetic variants. Population genomic and phenomic studies have revealed widespread variation between diverged populations, characteristic of man-made environments, as well as geographic clusters of wild strains along with naturally occurring recombinant strains (mosaics). Here, we review these recent studies and provide a perspective on how these previously unappreciated levels of variation can help to bridge our understanding of the genotype-phenotype gap, keeping budding yeast at the forefront of genetic studies. Not only are quantitative trait loci (QTL) being mapped with high resolution down to the nucleotide, for the first time QTLs of modest effect and complex interactions between these QTLs and between QTLs and the environment are being determined experimentally at unprecedented levels using next generation techniques of deep sequencing selected pools of individuals as well as multi-generational crosses.     

Influence of yeast quality on performance of gnotobiotically grown Artemia

Using axenically grown Artemia, a model system was developed to evaluate the effect of bacteria on the survival and development of this crustacean. Two strains of baker's yeast (Saccharomyces cerevisiae) were used in all experiments as feed for Artemia: a wild-type strain and its mnn9 mutant, defective in the synthesis of mannoproteins in the outer cell wall. The genetic background, yeast growth phase and growth medium appeared to be important parameters determining the quality of yeast cells as feed for Artemia. A strong positive correlation between Artemia performance and the yeast cell wall chitin and glucan content was obtained, while the mannoprotein content was negatively correlated. Mnn9 yeast cells grown till exponential phase in minimal medium proved to be excellent feed for Artemia, yielding an average 95% survival and 4-mm growth after 6 days at 28 °C, which is comparable to the best results obtained with algal feed. The standard growth test yields highly reproducible results and can become an excellent tool to study the mode of action of bacteria. Furthermore, yeast cell viability and the method used to kill/sterilize the cells are important parameters influencing nauplii performance.     

Yeast mutant with efficient secretion identified by a novel secretory reporter, Cluc

Yeast is an important host for the production of pharmaceutical or industrial proteins by virtue of its genetic information and easy handling. A number of heterologous proteins have been produced and purified from yeast cell cultures as secreted forms. Here, we describe a novel screening system of Saccharomyces cerevisiae and its application to improve the secretion efficiency of yeast. In our system, a natural secretory luciferase from Cypridina noctiluca is used as a reporter enzyme. The accumulation of enzymatically active luciferase in culture medium makes it possible to screen many samples simultaneously in a simple and sensitive assay. Using this system, we have discovered that the deletion mutant of MON2, which encoded a scaffold protein for vesicle formation located at the late Golgi, secreted luciferase highly efficiently to the extracellular space. Thus, we conclude that this new reporter assay is useful for the improvement and screening of yeast secretory strains.     

A plasmid model to study genetic recombination in yeast

Chimeric plasmids have been constructed containing two heteroallelic mutant copies of the yeast HIS3 gene as an inverted repetition. Intramolecular exchange events between these two allelic mutant copies are capable of generating a wild-type allele. Plasmids containing two mutant heteroalleles have been transformed into appropriate his3^? yeast strains, and the frequency of exchange events generating His⁺ prototrophs has been measured during mitotic division. After 20 generations of growth under nonselective conditions, between 0.1 and 1 % of the transformed yeast cells become His⁺ prototrophs. This percentage decreases at least ten-fold in a strain with a rad52 mutation. Plasmid molecules having undergone exchange events have been isolated from yeast cells and have been examined after transfer to Escherichia coli. Physical examination shows that less than 10 % of the plasmids having undergone genetic exchange have also undergone an internal reciprocal recombination event as evidenced by reorientation of linked restriction sites. The remainder of the plasmids having undergone genetic exchange do not exhibit reciprocal recombination. Characterization of the individual allelic copies within a plasmid having undergone exchange reveals that in 24 of 25 examples only one of the two HIS3 copies has become wild type, and that either copy is equally likely to become wild type. We conclude that the model plasmid we have constructed undergoes intramolecular genetic exchange events and will be useful for studying genetic recombination.     

Transgenic wine yeast technology comes of age: is it time for transgenic wine?

Saccharomyces cerevisiae is the main yeast responsible for alcoholic fermentation of grape juice during wine making. This makes wine strains of this species perfect targets for the improvement of wine technology and quality. Progress in winemaking has been achieved through the use of selected yeast strains, as well as genetic improvement of wine yeast strains through the sexual and pararexual cycles, random mutagenesis and genetic engineering. Development of genetically engineered wine yeasts, their potential application, and factors affecting their commercial viability will be discussed in this review.     

Direct iterative protein profiling (DIPP) - an innovative method for large-scale protein detection applied to budding yeast mitosis

The budding yeast Saccharomyces cerevisiae is a major model organism for important biological processes such as mitotic growth and meiotic development, it can be a human pathogen, and it is widely used in the food-, and biotechnology industries. Consequently, the genomes of numerous strains have been sequenced and a very large amount of RNA profiling data is available. Moreover, it has recently become possible to quantitatively analyze the entire yeast proteome; however, efficient and cost-effective high-throughput protein profiling remains a challenge. We report here a new approach to direct and label-free large-scale yeast protein identification using a tandem buffer system for protein extraction, two-step protein prefractionation and enzymatic digestion, and detection of peptides by iterative mass spectrometry. Our profiling study of diploid cells undergoing rapid mitotic growth identified 86% of the known proteins and its output was found to be widely concordant with genome-wide mRNA concentrations and DNA variations between yeast strains. This paves the way for comprehensive and straightforward yeast proteome profiling across a wide variety of experimental conditions.     

Engineering of protein secretion in yeast: strategies and impact on protein production

Yeasts combine the ease of genetic manipulation and fermentation of a microorganism with the capability to secrete and modify foreign proteins according to a general eukaryotic scheme. Their rapid growth, microbiological safety, and high-density fermentation in simplified medium have a high impact particularly in the large-scale industrial production of foreign proteins, where secretory expression is important for simplifying the downstream protein purification process. However, secretory expression of heterologous proteins in yeast is often subject to several bottlenecks that limit yield. Thus, many studies on yeast secretion systems have focused on the engineering of the fermentation process, vector systems, and host strains. Recently, strain engineering by genetic modification has been the most useful and effective method for overcoming the drawbacks in yeast secretion pathways. Such an approach is now being promoted strongly by current post-genomic technology and system biology tools. However, engineering of the yeast secretion system is complicated by the involvement of many cross-reacting factors. Tight interdependence of each of these factors makes genetic modification difficult. This indicates the necessity of developing a novel systematic modification strategy for genetic engineering of the yeast secretion system. This mini-review focuses on recent strategies and their advantages for systematic engineering of yeast strains for effective protein secretion.     

Whole-genome comparison reveals novel genetic elements that characterize the genome of industrial strains of Saccharomyces cerevisiae

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species.     

The winemaker's bug: From ancient wisdom to opening new vistas with frontier yeast science

The past three decades have seen a global wine glut. So far, well-intended but wasteful and expensive market-intervention has failed to drag the wine industry out of a chronic annual oversupply of roughly 15%. Can yeast research succeed where these approaches have failed by providing a means of improving wine quality, thereby making wine more appealing to consumers? To molecular biologists Saccharomyces cerevisiae is as intriguing as it is tractable. A simple unicellular eukaryote, it is an ideal model organism, enabling scientists to shed new light on some of the biggest scientific challenges such as the biology of cancer and aging. It is amenable to almost any modification that modern biology can throw at a cell, making it an ideal host for genetic manipulation, whether by the application of traditional or modern genetic techniques. To the winemaker, this yeast is integral to crafting wonderful, complex wines from simple, sugar-rich grape juice. Thus any improvements that we can make to wine, yeast fermentation performance or the sensory properties it imparts to wine will benefit winemakers and consumers. With this in mind, the application of frontier technologies, particularly the burgeoning fields of systems and synthetic biology, have much to offer in their pursuit of "novel" yeast strains to produce high quality wine. This paper discusses the nexus between yeast research and winemaking. It also addresses how winemakers and scientists face up to the challenges of consumer perceptions and opinions regarding the intervention of science and technology; the greater this intervention, the stronger the criticism that wine is no longer "natural." How can wine researchers respond to the growing number of wine commentators and consumers who feel that scientific endeavors favor wine quantity over quality and "technical sophistication, fermentation reliability and product consistency" over "artisanal variation"? This paper seeks to present yeast research in a new light and a new context, and it raises important questions about the direction of yeast research, its contribution to science and the future of winemaking.     

Truth in wine yeast

Evolutionary history and early association with anthropogenic environments have made Saccharomyces cerevisiae the quintessential wine yeast. This species typically dominates any spontaneous wine fermentation and, until recently, virtually all commercially available wine starters belonged to this species. The Crabtree effect, and the ability to grow under fully anaerobic conditions, contribute decisively to their dominance in this environment. But not all strains of Saccharomyces cerevisiae are equally suitable as starter cultures. In this article, we review the physiological and genetic characteristics of S. cerevisiae wine strains, as well as the biotic and abiotic factors that have shaped them through evolution. Limited genetic diversity of this group of yeasts could be a constraint to solving the new challenges of oenology. However, research in this field has for many years been providing tools to increase this diversity, from genetic engineering and classical genetic tools to the inclusion of other yeast species in the catalogues of wine yeasts. On occasion, these less conventional species may contribute to the generation of interspecific hybrids with S. cerevisiae. Thus, our knowledge about wine strains of S. cerevisiae and other wine yeasts is constantly expanding. Over the last decades, wine yeast research has been a pillar for the modernisation of oenology, and we can be confident that yeast biotechnology will keep contributing to solving any challenges, such as climate change, that we may face in the future.     

Identification of mutations in the asporogenic yeast Candida tropicalis using intrageneric fusion of protoplasts

The possibility of genetic identification of mutations in asporogenic yeast by the technique of intrageneric fusion of yeast protoplasts of Candida tropicals and Saccharomyces cerevisiae has been demonstrated for Candida tropicals strains G5-9 (Ade- Leu-) and G32-4 (Leu-). The mutations to auxotrophy ade- in the strain G5-9 and leu- in G32-4 of Candida tropicals are allelic to ade2 and leu1 mutations in the genes of Saccharomyces cerevisiae yeast. The allelic character of adenine auxotrophy mutation in Candida tropicals and ade2 mutation in Saccharomyces cerevisiae is confirmed by the absence of AIR-carboxylase activity in cellular extract from the strain G5-9.