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Co-culture conditions are beneficial for study due to the advances which arise from symbiotic interactions and which cannot be replicated under pure culture conditions. Here, the focus is on the connection between two fungi – a yeast, Saccharomyces cerevisiae, and a filamentous fungus, Penicillium chrysogenum – in a yeast immobilization system termed’ yeast biocapsules’, where the yeast and filamentous fungus are strongly attached to one another, forming spherical structures. This co-culture condition hinders filamentous fungal biomass growth, while immobilization of yeast cells continues to increase. The effect of the co-culture condition on endometabolites or intracellular metabolites were tracked during the beginning and end of the yeast biocapsule formation period, and metabolites analyzed by Gas Chromatography-Mass Spectrometry Detector (GC-MSD). Distinct metabolite profiles were found between single culture conditions, involving each organism separately, and with the co-culture condition, where there were differences in 54 endometabolites. Specifically, co-culture condition compounds such as fructose, glycolic acid and glyceric acid were present in higher concentrations at the end of biocapsule formation. These results shed light on the mechanisms and biochemical impact of the interaction between the yeast and filamentous fungus and serve as a basis to apply and further develop yeast biocapsules as a new biotechnological tool with benefits for industry.

In this chapter, we characterize the academy as a “complacent system” resistant but not impervious to change, and we discuss how to overcome that resistance in the pursuit of diversity, equity, and inclusion. We outline best practices for disruption drawing upon what we learned during the implementation of the NSF ADVANCE program on our campus. We then offer some thoughts on the steps ahead, including resources for sustainability.

Advice on how to build a more-inclusive academic community is emerging; however, this chapter suggests that such advice warrants “a grain of salt” depending on two circumstances: (1) the organizational culture needing to be “fixed,” and (2) the existence of extra-organizational factors that may shape how transformation can proceed. First, the existing organizational culture affects the processes needed to achieve a more-inclusive community, and defines what “more inclusive” will look like. Programs shown to be effective at one institution might not be effective at another. External factors may also affect local culture. For example, a long-standing ban on affirmative action programs and quota systems at the University of California meant that, even though other institutions found them to be effective, replicating those programs was not an option. The second concern derives from the nature of change needed. Barriers to inclusion are deeply rooted in historical traditions, ideologies, and social practices outside of any single organization, and often these barriers are applied unconsciously. This means genuine cultural transformation will occur only if the organizational community as a whole is committed to that change.

Achieving a diverse and inclusive community requires establishing a culture of genuine equality for all members. Our purpose in writing this book is to share our collective knowledge about how to challenge the forces that enable and sustain discrimination in the workplace as informed by our experience developing and implementing the UC Davis ADVANCE program. The program’s goal is to create an inclusive academic community that reflects and serves the diverse population of California. In this introductory chapter, we emphasize the need to move from affirmative action programs, which have played an important role in fostering diversity, to a focus on institutional transformation, which requires not only changes in policy but also shifts in academic culture. Much of what we cover in this book is broadly applicable beyond academia and will interest those wanting to understand and address challenges to diversity, equity, and inclusion in their own organizations. Since workplaces differ in their goals, priorities, and culture, the book is not a “how to” manual but rather a collective effort to share with readers the approaches we took, the information we gathered, what we observed and experienced, and the lessons we learned along the way.

Addressing barriers to inclusion requires understanding the nature of the problem at the institutional level. Data collection and assessment are both crucial for this aim. In this chapter, we describe two important classes of data: (1) data on diversity that define the potential nature of the issues at stake and the need for change, and (2) data on assessing the usefulness of new programs, processes, and policies in creating a more diverse institution. Both sets of data are important for effective decision-making. At the same time, data analyses can be challenging because issues of equity and inclusion are complex and determining the basis of comparison or the “ideal” diversity target can be difficult. Nevertheless, data gathering and analysis are critical to assess progress and to provide a basis for both accountability and efficacy. Moreover, the ability to document that a problem indeed exists will help justify the need for change and, ideally, spur corrective action.

Ideally, higher education systems are meritocracies in which advancement or promotion is based on demonstrated accomplishment and scholarly impact. “Merit” is believed to be associated with innate intellectual ability, dedication to learning and knowledge generation, mastery of a field of study, and recognition by others of comparable training and academic standing. Evaluations of accomplishment are dutifully (and often wishfully) believed to be wholly objective despite an abundance of evidence to the contrary. Unfortunately, implicit bias and other barriers to inclusion are pervasive within meritocracies. For members of marginalized groups, their social identity may diminish how their accomplishments are perceived and valued; conversely, the accomplishments of those with privileged identities may be over-valued. Moreover, what counts as “valuable” is itself not objective or neutral but rather reflects socially-constructed and culturally-specific priorities. Because academic merit and reward systems, as well as local cultures, can intentionally as well as unintentionally reinforce and hence perpetuate bias and barriers to inclusion, one of our UC Davis ADVANCE initiatives centered on review of all policies and practices affecting faculty advancement. We appraised the potential for bias in hiring, promotion, progression, and retention of faculty. We also evaluated the importance of culture in replicating barriers to inclusion.

Fungi possess extraordinary strength in attachment to biotic and abiotic surfaces. This review focuses on adhesion mechanisms of yeast and filamentous fungi and the proposed combination of the adhesive forces of both organisms in an immobilization system called yeast biocapsules, whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum. The natural adherent properties of each organism, one multicellular and another unicellular, allow yeast to be fixated securely on the filamentous fungi and complete alcoholic fermentation. Following alcoholic fermentation, the hyphae become an inert support for yeast cells while maintaining shape and integrity. Biocapsules have been used successfully in both wine and bioethanol production. Investigation of the potential genes involved in fungal-yeast fusion suggests that natural hydrophobic interactions of both organisms play a major role. Analysis of the possible mechanisms involved in fungus and yeast adhesion, future perspectives on improving yeast immobilization, and proposed applications of the biocapsules are explored.

Schizosaccharomyces japonicus UCD2489 was evaluated for potential use as a starter culture in wine-making. Laboratory-scale fermentations of Trebbiano grape juice were set up to compare fermentation kinetics of pure cultures of Sch. japonicus, immobilized Sch. japonicus cells, a commercial strain of Saccharomyces cerevisiae (EC1118), and mixed cultures of both species. The fermentation kinetics of the sequential and coinoculated fermentations were largely driven by the presence of S. cerevisiae. UCD2489 cell immobilization resulted in a significant reduction in ethanol levels in mixed fermentations compared with EC1118. Acetic acid levels similar to those of EC1118 in pure culture were produced when fermentations were coinoculated. The ability of UCD2489 to consume malic acid was adversely affected by EC1118, particularly in the coinoculated fermentation, suggesting that acid levels could be manipulated by adjusting the relative ratios of the two yeasts and the timing of inoculation with S. cerevisiae. Depending upon the inoculation conditions used, Sch. japonicus produced a quantity of glycerol ~2-fold higher than those released by S. cerevisiae. The analyses of volatile compounds showed increases in aroma-impacting compounds such as ethyl acetate in all Sch. japonicus wines, and acetaldehyde in the free-cell coinoculated fermentation that exceed reported sensory thresholds for these compounds, and for other important aroma compounds such as isoamyl acetate, hexyl acetate, phenyl ethyl acetate, ethyl isobutyrate, and ethyl butyrate. Polysaccharide release by UCD2489 was ~4.7-fold greater than that of S. cerevisiae alone. Reduction of induced wine protein haze was correlated with the concentration of polysaccharides. Our findings suggest that Sch. japonicus could be useful in wine production to reduce acidity and final ethanol levels and to increase glycerol, volatile compounds, and active polysaccharides with potential beneficial enhancement of protein stability.

A novel method of yeast immobilization, called “biocapsules”, has been developed in which cells of the yeast Saccharomyces cerevisiae become attached to the hyphae of the fungus Penicillium chrysogenum, remaining adhered following loss of viability of this fungus. Yeast immobilization facilitates higher cell densities than traditional fermentation methods, improves yield, and allows the reuse of the biocatalyst. Yeast cells may adhere to each other via specific cell-surface molecular interactions (flocculation) or may attach to surfaces (biofilm formation), and the roles of these two distinct attachment mechanisms in biocapsule formation is unknown. To elucidate the influence of biofilm formation versus flocculation on the yeast-fungus coimmobilization, a screening of selected strains from the Viticulture and Enology Department collection at the University of California, Davis, was carried out, and their ability to flocculate and form biofilm was quantified. Eighteen yeast strains capable of flocculation and biofilm formation were identified in this screening. Strains displaying differential capabilities in flocculation or biofilm formation and two control strains were further evaluated for their ability to specifically immobilize with P. chrysogenum. Seven strains exhibiting different patterns of flocculation and biofilm formation were identified. Biofilm-forming yeast strains displayed higher rates of immobilization with P. chrysogenum and formed more consistent biocapsules. In contrast, strains able to flocculate developed smaller, inconsistent biocapsules. Although the size and number of biocapsules formed varied by yeast strain, the total mass of biocapsules generated was similar for all strains. These results shed light on parameters that influence yeast-fungus coimmobilization, which may lead to an improvement of biocapsule consistency and advance the field of application for this new immobilization system.

The ability of Lachancea thermotolerans strains to ferment brewer's wort has been investigated. Initial fermentations with three L. thermotolerans strains compared the use of maltose and maltotriose, as well as production of glycerol and lactic acid and pH evolution over the course of the fermentation. The most promising strain was subsequently tested for additional traits important for beer production, including pitching rate, generational capacity, foam stability, hop tolerance, vicinal diketone production, oxygen requirement and flocculation. These tests suggest that L. thermotolerans may be a good choice for producing sour beers in a single fermentation step without the use of lactic acid bacteria. Copyright © 2016 The Institute of Brewing & Distilling

Interdelta sequence typing was used to investigate the genetic diversity of 54 Chinese indigenous wine strains of Saccharomyces cerevisiae selected on the basis of preliminary sequence analysis from 349 strains that were isolated previously from 15 spontaneous fermentations in Shanshan, Xinjiang, and Qing Tongxia, Ningxia, China. Of the 54 strains tested, 78% (42/54) were confirmed as genetically distinct. Dendrograms based on strain similarity revealed differences in the genetic relationships of Xinjiang yeast populations between table and wine grape varieties, in addition to differences between red and white grape varieties in Ningxia (Dice coefficients of 0.448 and 0.674, respectively). When data from Saccharomyces strains collected from California, France, Italy, northern Europe, and Spain were included in the analysis, the dendrogram revealed five groups containing 51, 4, 48, 3, and 1 strain, respectively. Ningxia and Xinjiang provinces displayed local specific S. cerevisiae biotas that show a clear separation from other strains. Cluster XJ19 isolated from Xinjiang displayed a high level of similarity with UCD587, UCD2515, and UCD2516 from California. Clusters XJ2, XJ7, XJ20, and XJ3, also isolated from Xinjiang, had a lower degree of similarity with other Chinese indigenous genotypes and strains from other regions. This study compares, for the first time, the genetic diversity and relationships between indigenous S. cerevisiae wine strains collected from Xinjiang and Ningxia provinces in China with wine strains from different geographic regions.

Summary Goals: The ability of the yeast Brettanomyces to produce negative aroma attributes from grape phenolic precursors is well known. However, this yeast synthesizes a broad array of aroma-active compounds in wines, some of which are valued or deemed positive in certain wine styles or matrices. To better understand the spectrum of positive and negative aroma traits associated with the presence of Brettanomyces in wine, we created an aroma wheel to categorize and describe the variety of aroma impacts of this yeast on the basis of analyses of aroma-active compound production from spiked precursor compounds in a model controlled environment. Key Findings: The Brettanomyces aroma wheel categorizes many of the aroma descriptors associated with wine spoilage by Brettanomyces . Terms on the wheel may categorize aroma-active compounds generally associated with microbial infection of both Brettanomyces and lactic acid bacteria (LAB). There is no evidence for a Brettanomyces strain that produces only positive characters in wine. Impact and Significance: A comprehensive Brettanomyces aroma wheel was generated with different strains under controlled product–precursor conditions and tested for broader utility in commercial winemaking. Thirty commercial wines described by wine critics using two or more of the identical or similar terms to those found on the Brettanomyces aroma wheel were obtained from a retail outlet. All wines purchased had evidence of presence of Brettanomyces , LAB, or both. The inner circle, or general categories of the wheel, were used more commonly by critics in describing wines and the specific terms (outer wheel) less generally used as commercial wine descriptors. The terms presented on the aroma wheel can therefore be used to determine if a wine is likely to have sensory characteristics contributed by Brettanomyces or LAB. This appeared to be particularly true for the general categories “savory,” “woody,” “spicy,” “floral,” “earthy,” “chemical,” and “animal.” Thus, the wheel can be used to identify wines with a strong, but not necessarily negative, microbial signature.

Summary Aim: There is significant variety in the choice of commercial yeast strains available for wine production. Yeast hybrids represent one class of commercial yeasts. The purpose of this review is to define what yeast hybrids are, to describe how they arise in nature and in the laboratory, and to discuss the features of these yeasts that differentiate them from non-hybrid strains. Key Themes: How does mating occur normally in yeast? What is a hybrid yeast strain? How do hybrid strains form in nature? How are hybrid wine strains constructed in the laboratory? Are natural hybrids of wine yeast found in nature? What are the features of laboratory-generated wine yeast hybrids? What are the impacts of hybrid strains on wine composition? Impact and Significance: Yeast influence wine aroma and flavor profiles in multiple ways1. In addition to the formation of ethanol, yeast can directly produce both positive and negative aroma impact compounds, such as esters, higher alcohols, higher aldehydes, and sulfur-volatile compounds. Some yeast strains can produce factors that enhance mouthfeel, and many can modify varietal aroma through enzymatic and chemical mechanisms. Yeast mannoproteins and polysaccharides may also affect wine stability. Last, yeast end products can alter the wine matrix and therefore the sensory expression of wine characters. Yeast hybrids combine traits of Saccharomyces cerevisiae and other species in this genus, and thereby expand the metabolic repertoire of the strain. These yeasts can create novel characters in wine as well as conduct fermentation under nonstandard conditions.

The efficiency and efficacy of alcoholic fermentation by yeast is crucial for the winemaking process. Sluggish or arrested fermentations can negatively affect winery operations and wine quality. Here, we present a novel mechanism by which problem fermentations can arise. Yeast can induce a prion known as [GAR+] that allows the cell to circumvent glucose repression of alternative carbon substrates. We have confirmed that Saccharomyces cerevisiae strain UCD932 can spontaneously generate the [GAR+] phenotype and that this phenotype exhibits the genetic traits of a prion. Differences were observed in the fermentative behavior of UCD932 wild-type [gar−] versus [GAR+] yeasts in laboratory-scale model juice fermentations. To further understand these differences, fermentations were performed in Chardonnay juice to study the interaction of the [GAR+] prion and presence of sulfur dioxide (SO2) on fermentation kinetics, bacterial community composition, and volatile compound production. Cells harboring the [GAR+] prion displayed reduced fermentation capacity, which was especially evident in the absence of SO2. Presence of SO2 and fermentation time had the most significant effects on the types of bacteria present in the fermentation. However, [GAR+] yeasts without added SO2 were especially sensitive to bacterial competition. This difference was also reflected in the bacterial and volatile profiles of the finished wine. We hypothesize that the bacterial induction of the [GAR+] prion by yeast during fermentation is another possible mechanism by which stuck or sluggish fermentations may become established.

The analysis of gene regulation and changes in transcript profiles for important model organisms in their natural environments provides critical insights into their biology and genome evolution. Such research allows the processes of adaptation to stressors present in changing environments to be delineated and the physiology of various non-proliferative states to be defined. In the context of the wine yeast, Saccharomyces cerevisiae, where there is extensive biodiversity among wild strains, changes in gene expression can be correlated with phenotypes that are important to survival and dominance in natural environments. Research in this area is leading to a comprehensive understanding of the changing physiology of yeast during the conversion of grape juice to wine. Transcript analysis is also being used to define the factors underlying complex phenotypes in wine yeast. These phenotypes are under the control of multiple independently segregating genes. Such analyses have underscored the central role that alterations in gene expression and regulation play in phenotypic expression and the development of novel phenotypes. The degree of plasticity or conservation of the regulation of metabolic pathways and cellular processes provides a basis for the elucidation of strain evolution and environmental selection.

A survey of 95 Brettanomyces strains was undertaken to identify strains that consistently give positive aroma characteristics (e.g., spicy, fruity, and floral). These strains were grown in a defined medium, and five human evaluators rated each strain according to aroma quality, and their ratings were coupled with a solid phase micro-extraction with gas chromatography (SPME GC-MS) analysis. None of the strains yielded universally positive aromas for the evaluators. A further characterization of nine of these strains grown with both aromatic amino acids (phenylalanine, tryptophan, and tyrosine) and hydroxycinnamic acids (caffeic, p-coumaric, and ferulic acids) indicated that low levels of compounds that were most important in differentiating the strains may contribute to a positive sensorial perception of Brettanomyces strain aromas under these conditions. To define the components associated with positive Brettanomyces character, the volatile aroma compounds produced by five Brettanomyces bruxellensis strains were analyzed. Compound detecting in SPME GC-MS was coupled with olfactory analysis (SPME GC-MS-O), using nine individual evaluators to identify compounds most associated with Brettanomyces character and to assess the breadth of descriptive terms used by different individuals for the same compound. Twenty-two compounds were identified as having an impact on aroma, including the well-known ethylphenols and vinylphenols, as well as several fatty acids, alcohols, esters, terpenes, and an aldehyde.

The use of sulfur dioxide (SO2) as an antimicrobial in winemaking is a well-established, common practice. Although much is known about the antimicrobial effects of SO2 at single concentrations, little is known about its effects on microbial growth dynamics across a range of concentrations or when used in conjunction with yeast inoculation. Using high-throughput marker-gene sequencing, we investigated the cumulative impacts of yeast inoculation and SO2 treatments across a broad concentration range (0 to 150 mg/L SO2) on the bacterial and fungal communities in wine fermentations. Our results indicated a dose-dependent effect of SO2, with lactic acid bacteria and Gluconobacter proliferating in fermentations with

Eight non-Saccharomyces wine strains, previously selected for their ability to modulate the final concentrations of various volatile compounds and to persist with Saccharomyces cerevisiae in mixed inocula fermentations of grape juice, have been analyzed in the present work to test their ability to release mannoproteins. The eight strains were members of different genera originally isolated from grape: Hansensiaspora osmophila, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia fermentans, Saccharomycodes ludwigii, Starmerella bacillaris, Torulaspora delbrueckii and Zygosaccharomyces florentinus. A synthetic polysaccharide-free grape juice, was used to characterize the mannoproteins released during the alcoholic fermentation. Mannoproteins profiles were characterized by gel electrophoresis and carbohydrate composition was analyzed both by HPLC and by mass spectrometry. The eight non-Saccharomyces yeasts demonstrated a higher capacity to release polysaccharides compared to S. cerevisiae. The proteins released by the eight yeast strains showed a wide variety of protein sizes, ranging from 25 kDa to greater than 250 kDa. The mass spectrometric profile of the N-glycans ranged from 1600 to 4000 Da and was characteristic for each strain. Detailed investigation of the degree of polymerization of released N-glycans revealed variable composition from 8 to 15 units of monosaccharides.

Two molecular methods, interdelta polymorphism fingerprinting of genomic DNA and COX1 intron polymorphism fingerprinting of mitochondrial DNA, were used to characterize 52 strains of Saccharomyces identified to species by 26S rDNA sequencing only in order to assess the potential of these techniques for strain typing and genetic analysis individually and in combination. Two laboratory isolates derived from the same parentage served as a control. Forty-seven S. cerevisiae strains representing a mix of commercial strains and vineyard or winery isolates were used. Five of these strains have been given the commercial designation S. cerevisiae race bayanus. Three strains were identified as S. bayanus, S. kudriavzevii, and S. servazzii from their 26S rDNA sequence and thus may be hybrids. Forty-four genetic patterns were found among the strains by interdelta polymorphism fingerprinting and 47 genetic patterns were found by COX1 intron polymorphism fingerprinting. Each method allowed differentiation of the majority of strains but grouped strains into different clusters. Strains were clustered into eight groups using both techniques in combination. The two laboratory strains clustered together but were in a larger grouping of wine strains. Saccharomyces bayanus, S. kudriavzevii, and S. servazzii were separated from each other but each clustered with S. cerevisiae strains and could not be differentiated from S. cerevisiae by either method. Three of the S. cerevisiae race bayanus strains clustered together but the other two were in different groupings. Of the 10 commercial strains, 904 (French Red) was grouped alone, while 522 (Montrachet) and 905 (Premier Cuvee) were grouped with wild isolates from different areas. Thus both methods reveal patterns of strain similarity but neither method differentiated strains by species or by origin.

The production of volatile metabolic products was determined for five Brettanomyces bruxellensis isolates from wine when grown in a defined medium with and without the hydroxycinnamic acids caffeic, coumaric, and ferulic or the aromatic amino acids phenylalanine, tryptophan, and tyrosine. The aim of the study was to determine the relationship between substrates and end products and to define strain differences in the production of volatile compounds. In the presence of coumaric and ferulic acids, all strains produced very similar metabolic products, primarily 4-ethylphenol (4EP) and 4-ethylguaiacol (4EG), respectively. There was a more pronounced effect of strain with the other substrates, and a variety of compounds with the potential to impact wine aroma were detected. Growth of Brettanomyces in the defined medium, with and without coumaric acid, under varying oxygen concentrations was also studied to determine the effect of this compound on growth parameters. The highest concentrations of 4EP were found under anaerobic conditions. Coumaric acid also had a significant positive affect on growth of Brettanomyces at 25% air saturation levels. At full aeration, coumaric acid addition showed little to no impact on growth or 4EP formation. Significantly higher concentrations of acetic acid were formed in the presence of coumaric acid, suggesting that 4EP formation may aid in the recycling of oxidized cofactor NAD+.

The yeast Saccharomyces cerevisiae has evolved to dominate grape juice fermentation. A suite of cellular properties, rapid nutrient depletion, production of inhibitory compounds and the metabolic narrowing of the niche, all enable a minor resident of the initial population to dramatically increase its relative biomass in the ecosystem. This dominance of the grape juice environment is fueled by a rapid launch of glycolysis and energy generation mediated by transport of hexoses and an efficient coupling of transport and catabolism. Fermentation occurs in the presence of molecular oxygen as the choice between respiratory or fermentative growth is regulated by the availability of sugar a phenomenon known as glucose or catabolite repression. Induction of the [GAR + ] prion alters the expression of the major hexose transporter active under these conditions, Hxt3, reducing glycolytic capacity. Bacteria present in the grape juice ecosystem were able to induce the [GAR + ] prion in wine strains of S. cerevisiae. This induction reduced fermentation capacity but did not block it entirely. However, dominance factors such as the rapid depletion of amino acids and other nitrogen sources from the environment were impeded enabling greater access to these substrates for the bacteria. Bacteria associated with arrested commercial wine fermentations were able to induce the prion state, and yeast cells isolated from arrested commercial fermentations were found to be [GAR + ] thus confirming the ecological relevance of prion induction. Subsequent analyses demonstrated that the presence of environmental acetic acid could lead to [GAR + ] induction in yeast strains under certain conditions. The induction of the prion enabled yeast growth on non-preferred substrates, oxidation and reduction products of glucose and fructose, present as a consequence of bacterial energy production. In native ecosystems prion induction never exceeded roughly 50-60% of the population of yeast cells suggesting that the population retains the capacity for maximal fermentation. Thus, the bacterial induction of the [GAR + ] prion represents a novel environmental response: the query of the environment for the presence of competing organisms and the biological decision to temper glucose repression and dominance and enter a metabolic state enabling coexistence.

© 2016 Elsevier Ltd The present work demonstrates that yeasts belonging to the Schizosaccharomyces genus release a high quantity of polysaccharides of cell wall origin starting from the onset of the alcoholic fermentation. By the end of the alcoholic fermentation, all of the Schizosaccharomyces yeast strains released a quantity of polysaccharides approximately 3–7 times higher than that released by a commercial Saccharomyces cerevisiae yeast strain under the same fermentative conditions of synthetic juice. A higher content of polysaccharide was found in media fermented by Schizosaccharomyces japonicus with respect to that of Schizosaccharomyces pombe. Some of the strains evaluated were also able to produce high levels of pyruvic acid, which has been shown to be an important compound for color stability of wine. The presence of strains with different malic acid consumption patterns along with high polysaccharide release would enable production of naturally modified wines with enhanced mouth feel and reduced acidity. The chemical analysis of the released polysaccharides demonstrated divergence between the two yeast species S. pombe and S. japonicus. A different mannose/galactose ratio and a different percentage of proteins was observed on the polysaccharides released by S. pombe as compared to S. japonicus. Analysis of the proteins released in the media revealed the presence of a glycoprotein with a molecular size around 32–33 kDa only for the species S. japonicus. Mass spectrometry analysis of carbohydrate moieties showed similar proportions among the N-glycan chains released in the media by both yeast species but differences between the two species were also observed. These observations suggest a possible role of rapid MALDI-TOF screening of N-glycans compositional fingerprint as a taxonomic tool for this genus. Polysaccharides release in the media, in particular galactomannoproteins in significant amounts, could make these yeasts particularly interesting also for the industrial production of exogenous polysaccharide preparations.

In the YPH500 laboratory strain background of Saccharomyces cerevisiae , disruption of the SNF3 open reading frame results in the inability to grow at low glucose concentrations. Conversely, in haploid derivatives of wine strains and several other wild laboratory strains, deletion of SNF3 does not lead to a discernable phenotype. Complementation and subsequent tetrad analysis demonstrated that the snf3 suppressor found in wine strains is a dominant gene encoded by a single locus, not allelic to the known dominant suppressor, RGT2-1. HXT7 was identified independently from two low-copy number genomic libraries of strains carrying the novel dominant suppressor as capable of suppressing the snf3Δ phenotype in YPH500. Cloned HXT7 plus 1 kb of promoter was sufficient for suppression. Sequence comparison of HXT7 from the YPH500 background to that of the archetype lab strain, S288C, demonstrated that YPH500-derived strains contain a chimera of the HXT6 promoter and the HXT7 gene ( HXT6 P /7 ). Deletion of HXT7 in S288C results in expression of the snf3 phenotype in this genetic background, while deletion of HXT6 only slightly diminishes growth in the snf3 HXT7 strain. In contrast to cloned HXT7 , expression of HXT6 P /7 cloned in a centromere (CEN)-based plasmid is not sufficient for snf3 suppression. A construct placing HXT6 under the control of the HXT7 promoter also suppresses the snf3 growth defect. Suppression of snf3 in the wine and wild strains of Saccharomyces evaluated is therefore due to the pattern of expression of the Hxt7 protein rather than to differences in the HXT7 and HXT6 coding sequences.

The yeast Dekkera/Brettanomyces bruxellensis can cause enormous economic losses in wine industry due to production of phenolic off-flavor compounds. D. bruxellensis is a distant relative of baker's yeast Saccharomyces cerevisiae. Nevertheless, these two yeasts are often found in the same habitats and share several food-related traits, such as production of high ethanol levels and ability to grow without oxygen. In some food products, like lambic beer, D. bruxellensis can importantly contribute to flavor development. We determined the 13.4 Mb genome sequence of the D. bruxellensis strain Y879 (CBS2499) and deduced the genetic background of several "food-relevant" properties and evolutionary history of this yeast. Surprisingly, we find that this yeast is phylogenetically distant to other food-related yeasts and most related to Pichia (Komagataella) pastoris, which is an aerobic poor ethanol producer. We further show that the D. bruxellensis genome does not contain an excess of lineage specific duplicated genes nor a horizontally transferred URA1 gene, two crucial events that promoted the evolution of the food relevant traits in the S. cerevisiae lineage. However, D. bruxellensis has several independently duplicated ADH and ADH-like genes, which are likely responsible for metabolism of alcohols, including ethanol, and also a range of aromatic compounds.

The availability of the sequence of the Saccharomyces genome in combination with the development of chemical analytical technologies with dynamic ranges sensitive enough to detect volatile aromatic compounds has generated a renewed interest in defining the role of yeast in the generation of wine aroma and flavor. Genetic differences among wine strains are well documented and aroma profiles also appear to vary, implying that specific allelic alterations may exist and impact the production of compounds associated with flavor. Partial or complete sequencing data on several wine strains are available and reveal underlying genetic differences across strains in key genes implicated in flavor formation. This review discusses the current understanding of the roles of Saccharomyces in wine flavor with an emphasis on positive contributions to flavor and highlights the discoveries of the underlying enzymatic and metabolic mechanisms responsible for the yeast contribution to wine quality.

A screen of theSaccharomyces cerevisiaedeletion strain set was performed to identify genes affecting hydrogen sulfide (H2S) production. Mutants were screened using two assays: colony color on BiGGY agar, which detects the basal level of sulfite reductase activity, and production of H2S in a synthetic juice medium using lead acetate detection of free sulfide in the headspace. A total of 88 mutants produced darker colony colors than the parental strain, and 4 produced colonies significantly lighter in color. There was no correlation between the appearance of a dark colony color on BiGGY agar and H2S production in synthetic juice media. Sixteen null mutations were identified as leading to the production of increased levels of H2S in synthetic juice using the headspace analysis assay. All 16 mutants also produced H2S in actual juices. Five of these genes encode proteins involved in sulfur containing amino acid or precursor biosynthesis and are directly associated with the sulfate assimilation pathway. The remaining genes encode proteins involved in a variety of cellular activities, including cell membrane integrity, cell energy regulation and balance, or other metabolic functions. The levels of hydrogen sulfide production of each of the 16 strains varied in response to nutritional conditions. In most cases, creation of multiple deletions of the 16 mutations in the same strain did not lead to a further increase in H2S production, instead often resulting in decreased levels.

In Saccharomyces cerevisiae the process of transport of sugar substrates into the cell comprises a complex network of transporters and interacting regulatory mechanisms. Members of the large family of hexose (HXT) transporters display uptake efficiencies consistent with their environmental expression and play physiological roles in addition to feeding the glycolytic pathway. Multiple glucose-inducing and glucose-independent mechanisms serve to regulate expression of the sugar transporters in yeast assuring that expression levels and transporter activity are coordinated with cellular metabolism and energy needs. The expression of sugar transport activity is modulated by other nutritional and environmental factors that may override glucose-generated signals. Transporter expression and activity is regulated transcriptionally, post-transcriptionally and post-translationally. Recent studies have expanded upon this suite of regulatory mechanisms to include transcriptional expression fine tuning mediated by antisense RNA and prion-based regulation of transcription. Much remains to be learned about cell biology from the continued analysis of this dynamic process of substrate acquisition.

In Saccharomyces cerevisiae the process of transport of sugar substrates into the cell comprises a complex network of transporters and interacting regulatory mechanisms. Members of the large family of hexose (HXT) transporters display uptake efficiencies consistent with their environmental expression and play physiological roles in addition to feeding the glycolytic pathway. Multiple glucose-inducing and glucose-independent mechanisms serve to regulate expression of the sugar transporters in yeast assuring that expression levels and transporter activity are coordinated with cellular metabolism and energy needs. The expression of sugar transport activity is modulated by other nutritional and environmental factors that may override glucose-generated signals. Transporter expression and activity is regulated transcriptionally, post-transcriptionally and post-translationally. Recent studies have expanded upon this suite of regulatory mechanisms to include transcriptional expression fine tuning mediated by antisense RNA and prion-based regulation of transcription. Much remains to be learned about cell biology from the continued analysis of this dynamic process of substrate acquisition.

SNF3 encodes a low-glucose sensor in Saccharomyces cerevisiae that regulates the expression of a subset of hexose transporter genes. Deletion of SNF3 prevents rapid adaptation to low glucose concentration. Novel spontaneous suppressor mutants of the snf3Delta phenotype were isolated. The mutations isolated fell into one of two groups: those that increase the expression of transporters regulated by Snf3p, and those that show no detectable effect on the regulation of these genes. The physiologic role of one mutation, rgg2 (restoration of growth on glucose), that did not affect HXT gene expression was assessed by transcriptome analysis. Genes involved in glycogen metabolism and cAMP pathways were affected by the rgg2 mutation, suggesting a cellular role as a regulatory protein. Attempts to clone the wild-type RGG2 allele were unsuccessful. The glycogen phenotype and genetic crossing allowed rgg2 to be identified as an allele of the IRA2 gene. Suppression of the snf3 mutant phenotype by deletion of IRA2 was confirmed. A possible mechanism of the suppression of the snf3 growth defect by mutation of ira2 is discussed.

The production of wine from grapes is one of the world's oldest biotechnological processes. The alcoholic fermentation, the conversion of grape sugars to ethanol, is conducted by yeast of the genus Saccharomyces. In addition to wide-ranging roles in beverage and food processing, Saccharomyces is also a premier research model system because of its genetic tractability. Its genome has been sequenced, and an extensive array of molecular technologies has been developed for the genetic manipulation of this organism. In spite of the availability of these molecular tools, genetically engineered yeast strains are not yet in use in the commercial wine industry. Anxiety over consumer acceptance is one reason genetically modified strains are not employed, but there are other concerns as well. Wine production is unique in many respects. The wine production process is not conducted under sterile conditions, and any modified organism has the potential to become an enduring resident of the winery flora. Further...

The hexokinase PII isozyme has been implicated as an essential component of multiple glucose sensing pathways in the yeast Saccharomyces cerevisiae. Several lines of evidence suggest that the flux through this enzymatic step, but not the levels of substrates, cofactors or products, is the critical process detected by downstream sensing machinery. In spite of intensive research efforts, how the activity of this enzyme is translated into a quantitative signal remains an unresolved question.

Zero trans-influx assays of glucose and xylose were performed using Saccharomyces cerevisiae to investigate transport characteristics under high and low glucose conditions. Under high glucose conditions, most glucose was transported by the low-affinity transporter. The high-affinity transporter was expressed under low glucose conditions, transporting over 50% glucose. Inhibition kinetics revealed that xylose was transported by both high- and low-affinity glucose transporters. Affinities of both glucose transporters for xylose were very low under high glucose condition but increased to a similar level to glucose under low glucose condition. The maximum rate of xylose transport increased by 85%, while an overall maximum glucose transport rate decreased by 42% under low glucose condition, indicating the presence of other transport system for sugars except for glucose. It was suggested that expression of the high-affinity transporter and increased affinity of glucose transporters for xylose under low glucose condition would provide a fermentation strategy for enhancing the productivity of xylitol by recombinant S. cerevisiae harboring the xylose reductase gene.

In experimental science, organisms are usually studied in isolation, but in the wild they compete and cooperate in complex communities. We report a system for cross-kingdom communication by which bacteria heritably transform yeast metabolism. An ancient biological circuit blocks yeast from using other carbon sources in the presence of glucose. [GAR[superscript +]], a protein-based epigenetic element, allows yeast to circumvent this glucose repression and use multiple carbon sources in the presence of glucose. Some bacteria secrete a chemical factor that induces [GAR[superscript +]]. [GAR[superscript +]] is advantageous to bacteria because yeast cells make less ethanol, and is advantageous to yeast because their growth and long-term viability is improved in complex carbon sources. This crosskingdom communication is broadly conserved, providing a compelling argument for its adaptive value. By heritably transforming growth and survival strategies in response to the selective pressures of life in a biological community, [GAR[superscript +]] presents a unique example of Lamarckian inheritance., G. Harold and Leila Y. Mathers Foundation, Howard Hughes Medical Institute

While the heat/chill stability of white wines cannot be predicted from the total protein concentration, this information is useful in assessing the effectiveness of fining treatments. We have adapted the Amido Black assay for use in grape juice and wine. The response of bovine serum albumin (BSA) was similar in water, model wine and model juice. The linear range of the assay (

We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml(-1). PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine.