Functional And Comparative Genomics Of Saccharomyces And Non Saccharomyces Yeasts Potential For Industrial And Food Biotechnology

Since 1996, when the first Saccharomyces cerevisiae genome sequence was released, a wealth of genomic data has been made available for numerous S. cerevisiae strains, its close relatives, and non-conventional yeast species isolates of diverse origins. Several annotated genomes of interspecific hybrids, both within the Saccharomyces clade and outside, are now also available. This genomic information, together with functional genomics and genome engineering tools, is providing a holistic assessment of the complex cellular responses to environmental challenges, elucidating the processes underlying evolution, speciation, hybridization, domestication, and uncovering crucial aspects of yeasts´ physiological genomics to guide their biotechnological exploitation. S. cerevisiae has been used for millennia in the production of food and beverages and research over the last century and a half has generated a great deal of knowledge of this species. Despite all this, S. cerevisiae is not the best for all uses and many non-conventional yeast species have highly desirable traits that S. cerevisiae does not have. These include tolerance to different stresses (e.g. acetic acid tolerance in Zygosaccharomyces bailii, osmotolerance in Z. rouxii, and thermotolerance in Kluyveromyces marxianus and Ogataea (Hansenula) polymorpha), the capacity of assimilation of diverse carbon sources (e.g. high native capacity to metabolyze xylose and potential for the valorization of agroforest residues by Scheffersomyces (Pichia) stipites), as well as, high protein secretion, fermentation efficiency and production of desirable flavors, capacity to favor respiration over fermentation, high lipid biosynthesis and accumulation, and efficient production of chemicals other than ethanol amongst many. Several non-Saccharomyces species have already been developed as eukaryotic hosts and cell factories. Others are highly relevant as food spoilers or for desirable flavor producers. Therefore, non-conventional yeasts are now attracting increasing attention with their diversity and complexity being tackled by basic research for biotechnological applications. The interest in the exploitation of non-conventional yeasts is very high and a number of tools, such as cloning vectors, promoters, terminators, and efficient genome editing tools, have been developed to facilitate their genetic engineering. Functional and Comparative Genomics of non-conventional yeasts is elucidating the evolution of genome functions and metabolic and ecological diversity, relating their physiology to genomic features and opening the door to the application of metabolic engineering and synthetic biology to yeasts of biotechnological potential. We are entering the era of the non-conventional yeasts, increasing the exploitation of yeast biodiversity and metabolic capabilities in science and industry. In this collection the industrial properties of S. cerevisiae, in particular uses, are explored along with its closely related species and interspecific hybrids. This is followed by comparisons between S. cerevisiae and non-conventional yeasts in specific applications and then the properties of various non-conventional yeasts and their hybrids.

As a group of microorganisms, yeasts have an enormous impact on food and bev- age production. Scientific and technological understanding of their roles in this p- duction began to emerge in the mid-1800s, starting with the pioneering studies of Pasteur in France and Hansen in Denmark on the microbiology of beer and wine fermentations. Since that time, researchers throughout the world have been engaged in a fascinating journey of discovery and development – learning about the great diversity of food and beverage commodities that are produced or impacted by yeast activity, about the diversity of yeast species associated with these activities, and about the diversity of biochemical, physiological and molecular mechanisms that underpin the many roles of yeasts in food and beverage production. Many excellent books have now been published on yeasts in food and beverage production, and it is reasonable to ask the question – why another book? There are two different approaches to describe and understand the role of yeasts in food and beverage production. One approach is to focus on the commodity and the technology of its processing (e. g. wine fermentation, fermentation of bakery products), and this is the direction that most books on food and beverage yeasts have taken, to date. A second approach is to focus on the yeasts, themselves, and their bi- ogy in the context of food and beverage habitats.

This volume scopes several aspects of non-conventional yeast research prepared by the leading specialists in the field. An introduction on taxonomy and systematics enhances the reader’s knowledge on yeasts beyond established ones such as Saccharomyces cerevisiae. Biotechnological approaches that involve fungal utilization of unusual substrates, production of biofuels and useful chemicals as citric acid, glutathione or erythritol are discussed. Further, strategies for metabolic engineering based on knowledge on regulation of gene expression as well as sensing and signaling pathways are presented. The book targets researchers and advanced students working in Microbiology, Microbial Biotechnology and Biochemistry.

The Saccharomyces cerevisiae deletion collection has revolutionized yeast genetics by allowing genome-wide deletion screens to be performed in pooled cultures. The inclusion of unique, 20-bp DNA sequences known as "tags" or "barcodes" in each strain allows all ~6000 yeast deletion mutants to be analyzed in a single culture by using a microarray to detect changes in tag abundance. Here we discuss improvements and extensions made to the pooled-yeast-deletion assay. This includes a redesign of the barcode array that is used for detecting the tags included in the deletion collection. Our analysis of this barcode array examines whether it is better to use available microarray surface area on a single feature for each probe, or to instead include multiple smaller features. It also covers aspects of data analysis such as the correction of hybridization defects and array saturation, tag-sequence repairs, and estimation of background hybridization. In addition to these microarray changes, we also examine the impact of experimental design on the quality of pooled growth data. Each step in both the experimental protocol and the data-analysis pipeline is examined for possible improvement. Finally, we discuss preliminary experiments testing the application of pooled fitness profiling to the study of epistasis. Data are shown that demonstrate the feasibility of a pooled-double-mutant fitness assay that is compatible with existing barcode array technology. This assay has the potential to become a valuable functional-genomics tool that may provide an improved understanding of pathway relationships in yeast.

Enzymes in Food Biotechnology: Production, Applications, and Future Prospects presents a comprehensive review of enzyme research and the potential impact of enzymes on the food sector. This valuable reference brings together novel sources and technologies regarding enzymes in food production, food processing, food preservation, food engineering and food biotechnology that are useful for researchers, professionals and students. Discussions include the process of immobilization, thermal and operational stability, increased product specificity and specific activity, enzyme engineering, implementation of high-throughput techniques, screening to relatively unexplored environments, and the development of more efficient enzymes. Explores recent scientific research to innovate novel, global ideas for new foods and enzyme engineering Provides fundamental and advanced information on enzyme research for use in food biotechnology, including microbial, plant and animal enzymes Includes recent cutting-edge research on the pharmaceutical uses of enzymes in the food industry

White Wine Technology addresses the challenges surrounding white wine production. The book explores emerging trends in modern enology, including molecular tools for wine quality and analysis of modern approaches to maceration extraction, alternative microorganisms for alcoholic fermentation, and malolactic fermentation. The book focuses on the technology and biotechnology of white wines, providing a quick reference of novel ways to increase and improve overall wine production and innovation. Its reviews of recent studies and technological advancements to improve grape maturity and production and ways to control PH level make this book essential to wine producers, researchers, practitioners, technologists and students. Covers trends in in both traditional and modern enology technologies, including extraction, processing, stabilization and ageing technologies Examines the potential impacts of climate change on wine quality Provides an overview of biotechnologies to improve wine freshness in warm areas and to manage maturity in cold climates Includes detailed information on hot topics such as the use of GMOs in wine production, spoilage bacteria, the management of oxidation, and the production of dealcoholized wines

New and Future Developments in Microbial Biotechnology and Bioengineering: Microbes in Soil, Crop and Environmental Sustainability reviews the exploitation of microbial biodiversity in soil with respect to nutrient-use efficiency, also discussing the improvement and maintenance of certain physical and chemical conditions in soil that can provide economic and environmental benefits toward agricultural sustainability. The utilization of microbes ranges from applications in biotechnology, marginal land restoration, the formulation of microbial inoculants, the enhancement of crop productivity, and the mitigation of global warming gases. Finally, various uses for microbial resources in crop disease management, bioenergy production, and income based on microbial cultivation are explored. Highlights the developments and achievements of microbial resources and their role in the sustainable management of soil fertility and agriculture productivity Outlines the role of microbial resource and biotechnology in sustainability to industry, agriculture, forest and management of environment Provides up-to-date information on the application of microbial resources and the role of biotechnology to meet the ever increasing demand of food, soil and plant productivity management Outlines enhancement in productivity through interventions of microbial bio-agents and eco-friendly technology

At a fundamental research level, the yeasts offer valuable opportunities for modelling regulatory and metabolic processes in multicellular eukaryotic organisms: this volume deals with the multifunctional chromosome regulatory proteins, topoisomerase and nuclear transport. A combination of biochemical and genetic approaches applied to the yeast translation system is also currently yielding a wealth of data, while the mating pheromone signal transduction pathway in yeasts provides a valuable analogue of the signal transduction components used by multicellular organisms, including receptors, G proteins, protein kinases and transcription factors. With a well-established history of fermantation studies, yeasts remain the first-choice vehicle for production of heterologous eukaryotic proteins. Interest is diversifying, as an increasing number of non-Saccharomyces species are now being utilised for the production of specific heterologous proteins. Molecular biologists, microbiologists and biochemical geneticists will find this volume an authoritative and valuable update on a vibrant area of research.

Regional promoter-independent gene silencing is critical in the establishment of cellular identity in Saccharomyces. Domains of transcriptionally silent regions in the genome are associated with certain heritable modifications made to chromatin, such as histone hypoacetylation and methylation. In Saccharomyces cerevisiae, this type of gene repression occurs through the activity of the four Silent Information Regulator, or SIR genes (SIR1-4). From an evolutionary perspective, the SIR genes are unique: except for SIR2, all are specific to budding yeasts. Many other organisms, from Schizosaccharomyces pombe to human, utilize the RNA interference (RNAi) pathway, whereas most budding yeasts lack this pathway entirely. Interestingly, SIR1, SIR3, and SIR4 are also rapidly evolving among Saccharomyces yeasts, providing a model by which to examine the essential principles governing successful silencing across various species and the relationship between rapid sequence evolution and evolution of function. To examine the relationship between gene duplication, extreme sequence divergence, and functional evolution, I studied the SIR1 gene in S. cerevisiae and its most ancestral paralog, KOS3, in the pre-whole-genome-duplication budding yeast, Torulaspora delbrueckii. T. delbrueckii also possesses genes for RNAi, AGO1 and DCR1, allowing us the possibility of exploring how the evolutionary divergence of RNAi and SIR silencing occurred. In the process, I developed genetic tools for T. delbrueckii. To fully characterize SIR1 function in S. cerevisiae and SIR gene function in T. delbrueckii, I utilized chromatin immunoprecipitation followed by deep-sequencing (ChIP-Seq) of tagged Sir proteins in both species. This strategy allowed for the discovery of potential novel functions, as well, revealing functions that may have been gained or lost throughout SIR1's evolution. To identify loci that were directly repressed by Sir proteins, I also generated whole-transcriptome data by performing mRNA-Seq on wild-type and sir mutants in both species. Collectively, these data revealed that though SIR1 in both species is still involved in silencing, its role in that process has dramatically shifted. Previous data suggested that SIR1 is primarily associated with the establishment or nucleation phase of silencing and not involved in telomeric silencing. The Sir1 ChIP data in S. cerevisiae corroborated this assessment. In T. delbrueckii, however, KOS3 was essential for silencing, and was also found at telomeres. Thus, Sir1 in its early evolution had a more essential role in silencing; this role may have changed due to the duplication and diversification of the other Sir complex members. This diversification may be contributing to the continual change in interactions between Sir1 and other Sir complex members across budding yeasts, leading to different mutant phenotypes in each species. Assays of silencer function in T. delbrueckii answered critical questions about when in the phylogeny important shifts in transcription factor binding sites took place. My work showed that the arrival of the Rap1, ORC, and Abf1 binding sites in the silencers of budding yeasts took place prior to the whole-genome duplication event. Analysis of silencer structure also revealed the diversity of chromatin architecture in budding yeasts: S. cerevisiae silent mating type loci have two silencers on either side of each locus, whereas in T. delbrueckii, there appears to be a single silencer on one side of each mating type locus. Transcriptome analysis of RNAi mutants revealed that this pathway in T. delbrueckii does not function in heterochromatic gene silencing, suggesting that this pathway has already been repurposed for some other biological process. The examination of whole-transcriptome data in S. cerevisiae in conjunction with the enrichment patterns of the Sir proteins at telomeres allowed us to evaluate widely accepted models regarding the molecular architecture of heterochromatin and expression at S. cerevisiae telomeres. I established that repression of gene expression at native telomeres is not as widespread as previously thought, and that many genes in proximity to regions of Sir protein enrichment were, in fact, expressed just as equally in wild type as they were in sir mutant genetic backgrounds. However, twenty-one genes were convincingly repressed by Sir proteins, highlighting the complex and individual nature of native telomeres and subtelomeric genes. The sensitivity of RNA-Seq also uncovered a previously under-appreciated class of haploid-regulated genes: genes that were not fully repressed or de-repressed in the diploid a/[alpha]-cell type, but rather weakly repressed or de-repressed. Thus, my work has expanded the set of known a/[alpha]-regulated genes in S. cerevisiae. In conclusion, this dissertation has broadened our understanding of the functional constraints dictating silencing gene evolution across species that diverged prior to and after the whole-genome-duplication event. My data speaks to the actual chromatin architecture and expression state of native S. cerevisiae telomeres, leading to the refinement of existing models and an appreciation for how heterogeneous these regions of the genome can be.