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Dimethyl sulfide (DMS) is a sulfur containing volatile that enhances general fruity aroma and imparts aromatic notes in wine. The most important precursor of DMS is S-methylmethionine (SMM), which is synthesized by grapes and can be metabolized by the yeast S. cerevisiae during wine fermentation. Precursor molecules left after fermentation are chemically converted to DMS during wine maturation, meaning that wine DMS levels are determined by the amount of remaining precursors at bottling. To elucidate SMM metabolism in yeast we performed quantitative trait locus (QTL) mapping using a population of 130 F2-segregants obtained from a cross between two wine yeast strains, and we detected one major QTL explaining almost 30% of trait variation. Within the QTL, gene YLL058W and SMM transporter gene MMP1 were found to influence SMM metabolism, from which MMP1 has the bigger impact. We identified and characterized a variant coding for a truncated transporter with superior SMM preserving attributes. A population analysis with 85 yeast strains from different origins revealed a significant association of the variant to flor strains and minor occurrence in cheese and wine strains. These results will help selecting and improving S. cerevisiae strains for the production of wine and other fermented foods containing DMS such as cheese or beer.

Fungal Oligopeptide Transporters (Fot) Fot1, Fot2 and Fot3 have been found in Saccharomyces cerevisiae wine strains, but not in strains from other environments. In the S. cerevisiae wine strain EC1118, Fot1 and Fot2 are responsible for a broader range of oligopeptide utilization in comparison with strains not containing any Fot. This leads to better fermentation efficiency and an increased production of desirable organoleptic compounds in wine. Despite the benefits associated with Fot activity in S. cerevisiae within the wine environment, little is known about this family of transporters in yeast. The presence of Fot1, Fot2 and Fot3 in S. cerevisiae wine strains is due to horizontal gene transfer from the yeast Torulaspora microellipsoides, which harbors Fot2Tm, FotX and FotY proteins. Sequence analyses revealed that Fot family members have a high sequence identity in these yeast species. In this work, we aimed to further characterize the different Fot family members in terms of subcellular localization, gene expression in enological fermentation and substrate specificity. Using CRISPR/Cas9, we constructed S. cerevisiae wine strains containing each different Fot as the sole oligopeptide transporter to analyze their oligopeptide preferences by phenotype microarrays. The results of oligopeptide consumption show that Fot counterparts have different di-/tripeptide specificities, suggesting that punctual sequence divergence between FOT genes can be crucial for substrate recognition, binding and transport activity. FOT gene expression levels in different S. cerevisiae wine strains during enological fermentation, together with predicted binding motifs for transcriptional regulators in nitrogen metabolism, indicate that these transporters may be under the control of the Nitrogen Catabolite Repression (NCR) system. Finally, we demonstrated that Fot1 is located in the yeast plasma membrane. This work contributes to a better understanding of this family of oligopeptide transporters, which have demonstrated a key role in the utilization of oligopeptides by S. cerevisiae in enological fermentation., This project has received funding from the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie Grant Agreement No. 764927

Hybrids between species or diverged lineages contain fundamentally novel genetic combinations but an impaired meiosis often makes them evolutionary dead ends. Here, we explored to what extent and how an aborted meiosis followed by a return-to-growth (RTG) promotes recombination across a panel of 20 yeast diploid backgrounds with different genomic structures and levels of sterility. Genome analyses of 284 clones revealed that RTG promoted recombination and generated extensive regions of loss-of-heterozygosity in sterile hybrids with either a defective meiosis or a heavily rearranged karyotype, whereas RTG recombination was reduced by high sequence divergence between parental subgenomes. The RTG recombination preferentially occurred in regions with local sequence homology and in meiotic recombination hotspots. The loss-of-heterozygosity had a profound impact on sexual and asexual fitness, and enabled genetic mapping of phenotypic differences in sterile lineages where linkage or association analyses failed. We propose that RTG gives sterile hybrids access to a natural route for genome recombination and adaptation.One sentence summaryAborting meiosis followed by a return to mitotic growth promotes evolution by genome wide-recombination in sterile yeast hybrids.

The mating-type switching endonuclease HO plays a central role in the natural life cycle of Saccharomyces cerevisiae, but its evolutionary origin is unknown. HO is a recent addition to yeast genomes, present in only a few genera close to Saccharomyces. Here we show that HO is structurally and phylogenetically related to a family of unorthodox homing genetic elements found in Torulaspora and Lachancea yeasts. These WHO elements home into the aldolase gene FBA1, replacing its 3' end each time they integrate. They resemble inteins but they operate by a different mechanism that does not require protein splicing. We show that a WHO protein cleaves Torulaspora delbrueckii FBA1 efficiently and in an allele-specific manner, leading to DNA repair by gene conversion or NHEJ. The DNA rearrangement steps during WHO element homing are very similar to those during mating-type switching, and indicate that HO is a domesticated WHO-like element., eLife digest In the same way as a sperm from a male and an egg from a female join together to form an embryo in most animals, yeast cells have two sexes that coordinate how they reproduce. These are called “mating types” and, rather than male or female, an individual yeast cell can either be mating type “a” or “alpha”. Every yeast cell contains the genes for both mating types, and each cell’s mating type is determined by which of those genes it has active. Only one mating type gene can be ‘on’ at a time, but some yeast species can swap mating type on demand by switching the corresponding genes ‘on’ or ‘off’. This switch is unusual. Rather than simply activate one of the genes it already has, the yeast cell keeps an inactive version of each mating type gene tucked away, makes a copy of the gene it wants to be active and pastes that copy into a different location in its genome. To do all of this yeast need another gene called HO. This gene codes for an enzyme that cuts the DNA at the location of the active mating type gene. This makes an opening that allows the cell to replace the ‘a’ gene with the ‘alpha’ gene, or vice versa. This system allows yeast cells to continue mating even if all the cells in a colony start off as the same mating type. But, cutting into the DNA is risky, and can damage the health of the cell. So, why did yeast cells evolve a system that could cause them harm? To find out where the HO gene came from, Coughlan et al. searched through all the available genomes from yeast species for other genes with similar sequences and identified a cluster which they nicknamed “weird HO” genes, or WHO genes for short. Testing these genes revealed that they also code for enzymes that make cuts in the yeast genome, but the way the cell repairs the cuts is different. The WHO genes are jumping genes. When the enzyme encoded by a WHO gene makes a cut in the genome, the yeast cell copies the gene into the gap, allowing the gene to ‘jump’ from one part of the genome to another. It is possible that this was the starting point for the evolution of the HO gene. Changes to a WHO gene could have allowed it to cut into the mating type region of the yeast genome, giving the yeast an opportunity to ‘domesticate’ it. Over time, the yeast cell stopped the WHO gene from jumping into the gap and started using the cut to change its mating type. Understanding how cells adapt genes for different purposes is a key question in evolutionary biology. There are many other examples of domesticated jumping genes in other organisms, including in the human immune system. Understanding the evolution of HO not only sheds light on how yeast mating type switching evolved, but on how other species might harness and adapt their genes.

Horizontal gene transfer: one of the key mechanisms involved into the adaptation of yeast to their environment. ESF-EMBO Symposium, Comparative genomics of eukaryotic microbes: Genomes in flux, and flux between genomes

Horizontal gene transfer: one of the key mechanisms involved into the adaptation of yeast to their environment. ESF-EMBO Symposium, Comparative genomics of eukaryotic microbes: Genomes in flux, and flux between genomes

Exploring [i]S. cerevisiae[/i] domestication from the analysis of their genome and from an experimental evolution approach in the grape must.. 29. International Conference on Yeast Genetics and Molecular Biology-ICYGMB18

Diversity of oligopeptide transporters in industrial yeasts. 7. Conference on Physiology of Yeast and Filamentous Fungi

The Darwinian fitness of wine strains of [i]Saccharomyces cerevisiae[/i] adapted toin the grape must environment. ISSY 34

Horizontal gene transfer (HGT) and introgression are increasingly recognized as a prominent source of adaptation in eukaryotes. Over the past decade, several cases of HGT from prokaryotes to the yeast Saccharomyces strains have been demonstrated. In addition, we have recently identified eukaryote-to-eukaryote gene transfers and some of them have been shown to be involved in the adaptation of the yeast S. cerevisiae to environmental conditions (1;2;3). Three large genomic regions, (A, B and C), were acquired by wine yeasts strains from more distant yeast species (1;3). The yeasts Zygosaccharomyces bailii and Torulaspora microellipsoides, species found in wine fermentations, were identified as the donors of regions B and C, respectively (1;3). We obtained evidence for the amplification of the region B (17 kb) in the genome of S. cerevisiae wine strains (4). The organization of this region differ considerably between strains and the identification of an autonomously replicating sequence functional in S. cerevisiae strongly suggest an expansion mechanism in yeast genomes involving an extrachromosomal circular DNA molecule. We also showed that region C have undergone several rearrangements and carries genes playing a key role in the adaptation of wine yeasts to the nitrogen-limited wine fermentation environment (3). The analysis of 82 S. cerevisiae strains from various ecological origins allowed identification of 33 HGT or introgression for which we have proposed potential donors using phylogenetic topology test. Among these events, we observed, the replacement of a GAL cluster in cheese strains which enables a faster switch from glucose to galactose and improves growth speed in a media containing a mix of the two hexoses such as fermented milk products. These data support the vision of customized yeast genomes associated to specific ecological niches and highlight the key role of HGT in the adaptation of fungal species to their environments.1. Novo et al. 2009, PNAS, 106: 16333-16338.2. Damon et al 2011, ISME Journal 9: 67.3. Marsit et al 2015, Mol. Biol. Evol., 32:1695–1707 4. Galeote et al 2011, PLoS One 6: e17872.

Saccharomyces cerevisiae strains are genetically diverse, largely as a result of human efforts to develop strains specifically adapted to various fermentation processes. These adaptive pressures from various ecological niches have generated behavioral differences among these strains, particularly in terms of their nitrogen consumption capacities. In this work, we characterize this phenotype by the specific quantity of nitrogen consumed under oenological fermentation conditions using a new approach. Indeed, unlike previous studies, our experiments were conducted in an environment containing excess nitrogen, eliminating the nitrogen limitation/starvation factor that is generally observed in fermentation processes. Using these conditions, we evaluated differences in the nitrogen consumption capacities for a set of five strains from diverse origins. The strains presented extremely different phenotypes and variations in their capacities to take up nitrogen from a wine fermentation environment. These variations reflect the differences in the nitrogen uptake capacities between wine and non-wine strains. Finally, the strains differed in their ability to adapt to the nitrogen composition of the environment, leading to variations in the cellular stress states, fermentation performances and the activity of the nitrogen sensing signaling pathway.

Mixed fermentations using a controlled inoculation of Starmerella bacillaris and Saccharomyces cerevisiae starter cultures represent a feasible way to modulate wine composition that takes advantage of both the phenotypic specificities of the non-Saccharomyces strain and the ability of S. cerevisiae to complete wine fermentation. However, according to the composition of grape juices, the consumption by Starm. bacillaris of nutrients, in particular of nitrogen sources, during the first stages of the process may result in depletions that further limit the growth of S. cerevisiae and lead to stuck or sluggish fermentations. Consequently, understanding the preferences of non-Saccharomyces yeasts for the nitrogen sources available in grape must together with their phenotypic specificities is essential for an efficient implementation of sequential wine fermentations with Starm. bacillaris and S. cerevisiae species. The results of our study demonstrate a clear preference for ammonium compared to amino acids for the non-Saccharomyces species. This finding underlines the importance of nitrogen sources, which modulate the functional characteristics of inoculated yeast strains to better control the fermentation process and product quality., Over the last few years, the potential of non-Saccharomyces yeasts to improve the sensory quality of wine has been well recognized. In particular, the use of Starmerella bacillaris in mixed fermentations with Saccharomyces cerevisiae was reported as an appropriate way to enhance glycerol formation and reduce ethanol production. However, during sequential fermentation, many factors, such as the inoculation timing, strain combination, and physical and biochemical interactions, can affect yeast growth, the fermentation process, and/or metabolite synthesis. Among them, the availability of yeast-assimilable nitrogen (YAN), due to its role in the control of growth and fermentation, has been identified as a key parameter. Consequently, a comprehensive understanding of the metabolic specificities and the nitrogen requirements would be valuable to better exploit the potential of Starm. bacillaris during wine fermentation. In this study, marked differences in the consumption of the total and individual nitrogen sources were registered between the two species, while the two Starm. bacillaris strains generally behaved uniformly. Starm. bacillaris strains are differentiated by their preferential uptake of ammonium compared with amino acids that are poorly assimilated or even produced (alanine). Otherwise, the non-Saccharomyces yeast exhibits low activity through the acetaldehyde pathway, which triggers an important redistribution of fluxes through the central carbon metabolic network. In particular, the formation of metabolites deriving from the two glycolytic intermediates glyceraldehyde-3-phosphate and pyruvate is substantially increased during fermentations by Starm. bacillaris. This knowledge will be useful to better control the fermentation process in mixed fermentation with Starm. bacillaris and S. cerevisiae. IMPORTANCE Mixed fermentations using a controlled inoculation of Starmerella bacillaris and Saccharomyces cerevisiae starter cultures represent a feasible way to modulate wine composition that takes advantage of both the phenotypic specificities of the non-Saccharomyces strain and the ability of S. cerevisiae to complete wine fermentation. However, according to the composition of grape juices, the consumption by Starm. bacillaris of nutrients, in particular of nitrogen sources, during the first stages of the process may result in depletions that further limit the growth of S. cerevisiae and lead to stuck or sluggish fermentations. Consequently, understanding the preferences of non-Saccharomyces yeasts for the nitrogen sources available in grape must together with their phenotypic specificities is essential for an efficient implementation of sequential wine fermentations with Starm. bacillaris and S. cerevisiae species. The results of our study demonstrate a clear preference for ammonium compared to amino acids for the non-Saccharomyces species. This finding underlines the importance of nitrogen sources, which modulate the functional characteristics of inoculated yeast strains to better control the fermentation process and product quality.

We report here the genome sequence of the ascomycetous yeast Torulaspora microellipsoides CLIB 830T. A reference genome for this species, which has been found as a donor of genetic material in wine strains of Saccharomyces cerevisiae, will undoubtedly give clues to our understanding of horizontal transfer mechanisms between species in the wine environment.

Adaptation of Yeast to Anthropogenic Environments Using Comparative Genomics. 27. International Conference on Yeast Genetics and Molecular Biology (ICYGMB)

In this article, we review some of the best-studied fungi used as food sources, in particular, the cheese fungi, the truffles, and the fungi used for drink fermentation such as beer, wine, and sake. We discuss their history of consumption by humans and the genomic mechanisms of adaptation during artificial selection.

Saccharomyces cerevisiae has been used to perform wine fermentation for several millennia due to its endurance and unmatched qualities. Nevertheless, at the moment of inoculation, wine yeasts must cope with specific stress factors that still challenge wine makers by slowing down or compromising the fermentation process. To better assess the role of genetic and environmental factors that govern multistress resistance during the wine fermentation lag phase, we used a factorial plan to characterise the individual and combined impact of relevant stress factors on eight S. cerevisiae and two non-S. cerevisiae wine yeast strains that are currently commercialised. The S. cerevisiae strains are very genetically diverse, belonging to the wine and flor groups, and frequently contain a previously described XVIVIII translocation that confers increased resistance to sulphites. We found that low temperature and osmotic stress substantially affected all strains, promoting considerably extended lag phases. SO2 addition had a partially temperature-dependent effect, whereas low phytosterol and thiamine concentrations impacted the lag phase in a strain-dependent manner. No major synergic effects of multistress were detected. The diversity of lag-phase durations and stress responses observed among wine strains offer new insights to better control this critical step of fermentation.

Summary Nitrogen and lipids are key nutrients of grape must that influence the production of fermentative aromas by wine yeast, and we have previously shown that a strong interaction exists between these two nutrients. However, more than 90% of the acids and higher alcohols (and their acetate ester derivatives) were derived from intermediates produced by the carbon central metabolism (CCM). The objective of this study was to determine how variations in nitrogen and lipid resources can modulate the contribution of nitrogen and carbon metabolisms for the production of fermentative aromas. A quantitative analysis of metabolism using 13C‐labelled leucine and valine showed that nitrogen availability affected the part of the catabolism of N‐containing compounds, the formation of α‐ketoacids from CCM and the redistribution of fluxes around these precursors, explaining the optimum production of higher alcohols occurring at an intermediate nitrogen content. Moreover, nitrogen content modulated the total production of acids and higher alcohols differently, through variations in the redox state of cells. We also demonstrated that the phytosterol content, modifying the intracellular availability of acetyl‐CoA, can influence the flux distribution, especially the formation of higher alcohols and the conversion of α‐ketoisovalerate to α‐ketoisocaproate.

During fermentative growth in natural and industrial environments, Saccharomyces cerevisiae must redistribute the available nitrogen from multiple exogenous sources to amino acids in order to suitably fulfill anabolic requirements. To exhaustively explore the management of this complex resource, we developed an advanced strategy based on the reconciliation of data from a set of stable isotope tracer experiments with labeled nitrogen sources. Thus, quantifying the partitioning of the N compounds through the metabolism network during fermentation, we demonstrated that, contrary to the generally accepted view, only a limited fraction of most of the consumed amino acids is directly incorporated into proteins. Moreover, substantial catabolism of these molecules allows for efficient redistribution of nitrogen, supporting the operative de novo synthesis of proteinogenic amino acids. In contrast, catabolism of consumed amino acids plays a minor role in the formation of volatile compounds. Another important feature is that the α-keto acid precursors required for the de novo syntheses originate mainly from the catabolism of sugars, with a limited contribution from the anabolism of consumed amino acids. This work provides a comprehensive view of the intracellular fate of consumed nitrogen sources and the metabolic origin of proteinogenic amino acids, highlighting a strategy of distribution of metabolic fluxes implemented by yeast as a means of adapting to environments with changing and scarce nitrogen resources. IMPORTANCE A current challenge for the wine industry, in view of the extensive competition in the worldwide market, is to meet consumer expectations regarding the sensory profile of the product while ensuring an efficient fermentation process. Understanding the intracellular fate of the nitrogen sources available in grape juice is essential to the achievement of these objectives, since nitrogen utilization affects both the fermentative activity of yeasts and the formation of flavor compounds. However, little is known about how the metabolism operates when nitrogen is provided as a composite mixture, as in grape must. Here we quantitatively describe the distribution through the yeast metabolic network of the N moieties and C backbones of these nitrogen sources. Knowledge about the management of a complex resource, which is devoted to improvement of the use of the scarce N nutrient for growth, will be useful for better control of the fermentation process and the sensory quality of wines.

Prix de la meilleure conférence; Stress resistance during wine fermentation and development of optimised wine yeasts. 33. International Specialised Symposium on Yeasts Exploring and Engineering Yeasts for Industrial Application

Horizontal Gene Transfer is a key mechanism permitting adaptation of [i]S. cerevisiae[/i][i][/i] to its ecological niches. Reunión anual conjunta genética y evolución

BGPI : équipe 5; The molecular and evolutionary processes underlying fungal domestication remain largely unknown despite the importance of fungi to bioindustry and for comparative adaptation genomics in eukaryotes. Wine fermentation and biological aging are performed by strains of S. cerevisiae with, respectively, pelagic fermentative growth on glucose, and biofilm aerobic growth utilizing ethanol. Here, we use environmental samples of wine and flor yeasts to investigate the genomic basis of yeast adaptation to contrasted anthropogenic environments. Phylogenetic inference and population structure analysis based on single nucleotide polymorphisms (SNPs) revealed a group of flor yeasts separated from wine yeasts. A combination of methods revealed several highly differentiated regions between wine and flor yeasts, and analyses using codon-substitution models for detecting molecular adaptation identified sites under positive selection in the high affinity transporter gene ZRT1. The Cross Population Composite Likelihood Ratio (XP-CLR) revealed selective sweeps at three regions, including in the hexose transporter gene HXT7, the yapsin gene YPS6 and the membrane protein coding gene MTS27. Our analyses also revealed that the biological aging environment has led to the accumulation of numerous mutations in proteins from several networks, including Flo11 regulation and divalent metal transport. Together, our findings suggest that the tuning of FLO11 expression and zinc transport networks are a distinctive feature of the genetic changes underlying the domestication of flor yeasts. Our study highlights the multiplicity of genomic changes underlying yeast adaptation to man-made habitats, and reveals that flor/wine yeast lineage can serve as a useful model for studying the genomics of adaptive divergence.

Background: During must fermentation thousands of volatile aroma compounds are formed, with higher alcohols, acetate esters and ethyl esters being the main aromatic compounds contributing to floral and fruity aromas. The action of yeast, in particular Saccharomyces cerevisiae, on the must components will build the architecture of the wine flavour and its fermentation bouquet. The objective of the present work was to better understand the molecular and metabolic bases of aroma production during a fermentation process. For such, comparative transcriptomic and metabolic analysis was performed at two time points (5 and 50 g/L of CO2 released) in fermentations conducted by four yeast strains from different origins and/or technological applications (cacha double dagger a, sake, wine, and laboratory), and multivariate factorial analyses were used to rationally identify new targets for improving aroma production.Results: Results showed that strains from cacha double dagger a, sake and wine produced higher amounts of acetate esters, ethyl esters, acids and higher alcohols, in comparison with the laboratory strain. At fermentation time T1 (5 g/L CO2 released), comparative transcriptomics of the three S. cerevisiae strains from different fermentative environments in comparison with the laboratory yeast S288c, showed an increased expression of genes related with tetracyclic and pentacyclic triterpenes metabolism, involved in sterol synthesis. Sake strain also showed upregulation of genes ADH7 and AAD6, involved in the formation of higher alcohols in the Ehrlich pathway. For fermentation time point T2 (50 g/L CO2 released), again sake strain, but also VL1 strain, showed an increased expression of genes involved in formation of higher alcohols in the Ehrlich pathway, namely ADH7, ADH6 and AAD6, which is in accordance with the higher levels of methionol, isobutanol, isoamyl alcohol and phenylethanol observed.Conclusions: Our approach revealed successful to integrate data from several technologies (HPLC, GC-MS, microarrays) an, Ines Mendes is recipient of a fellowship from the Portuguese Science Foundation, FCT (SFRH/BD/74798/2010). This work was supported by FCT through grant (PTDC/AGR-ALI/121062/2010) and by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI)., info:eu-repo/semantics/publishedVersion

Yeast cryotolerance brings some advantages for wine fermentations, including the improved aromatic complexity of white wines. Naturally cold tolerant strains are generally less adept at wine fermentation but fermentative fitness can potentially be improved through hybridization. Here we studied the potential of using hybrids involving Saccharomyces eubayanus and a S. cerevisiae wine strain for low-temperature winemaking. Through screening the performance in response to variable concentrations of sugar, nitrogen and temperature we isolated one hybrid strain that exhibited the superior performance. This hybrid strain was propagated and dried in pilot scale and tested for the fermentation of Macabeu and Sauvignon blanc grape musts. We obtained highly viable active dry yeast, which was able to efficiently ferment the grape musts with superior production of aroma active volatiles, in particular, 2-phenylethanol. The genome sequences of the hybrid strains revealed variable chromosome inheritance among hybrids, particularly within the S. cerevisiae sub-genome. With the present paper, we expand the knowledge on the potentialities of using S. eubayanus hybrids in industrial fermentation at beverages other than lager beer.

Aim: In the context of climate change, adaptation of enological practices and implementation of novel techniques are major challenges for winemakers. The potential interventions are linked in particular with the alcohol content and the global acidity of wine. Here, we review current microbiological and technological strategies to overcome such issues.Methods and results: Reducing ethanol concentration poses a number of technical and scientific challenges, in particular looking for specific yeast strains with lower alcohol yield. Several non-genetically modified organism (GMO) strains – S. cerevisiae or interspecific hybrids of the Saccharomyces genus – have yet been developed using different strategies, and some of them allow decreasing the final ethanol concentration by up to 1%. Several membrane-based technologies have also been developed not only to reduce the ethanol content of wines but also to increase the acidity and more generally to control the wine pH. New strategies are also proposed to improve the control of winemaking, especially the management of alcoholic fermentation of sugar-rich musts and the control of oxidation during the process.Conclusion: Reducing ethanol of wines and increasing their acidity are good examples of novel techniques of interest in the context of climate change. Other strategies are still under study to adapt winemaking practices to changes in grape composition.Significance and impact of the study: [Membrane-based technologies can be used to reduce the ethanol content of wines or to increase the acidity. Microbiological strategies will also be soon available for winemakers.

Yeasts from the Saccharomyces complex have been used for millennia for the production of fermented food and alcoholic beverages. The availability of large genomic datasets during the past decade has provided new insights into the genetic and phenotypic diversity, population structure and evolutionary history of these yeasts. Studies of these datasets have shown that man-made environments have led to several distinct domesticated variants. Comparative genomics approaches have revealed domestication fingerprints and indicated divergent regions that may explain the adaptation of strains to different ecological niches. In addition, the genetic basis of several technological traits of S. cerevisiae has been elucidated through QTL mapping, and strains improved for various industrial traits have been developed through hybridization or evolutionary engineering. The expansion of large-scale genomic and high-throughput phenotypic data on these strains will provide a unique resource for understanding their adaptation to their ecological niches and for elucidating the missing links between genotype and phenotype, paving the way for strain improvement.

Transport of glucose and fructose in the yeast Saccharomyces cerevisiae plays a crucial role in controlling the rate of wine fermentation. In S. cerevisiae, hexoses are transported by facilitated diffusion via hexose carriers (Hxt), which prefer glucose to fructose. However, utilization of fructose by wine yeast is critically important at the end of fermentation. Here, we report the characterization of a fructose transporter recently identified by sequencing the genome of the commercial wine yeast strain EC1118 and found in many other wine yeasts. This transporter is designated Fsy1p because of its homology with the Saccharomyces pastorianus fructose/H+ symporter Fsy1p. A strain obtained by transformation of the V5 hxt1-7Δ mutant with FSY1 grew well on fructose, but to a much lesser extent on glucose as the sole carbon source. Sugar uptake and symport experiments showed that FSY1 encodes a proton-coupled symporter with high affinity for fructose (K m 0.24±0.04 mM). Using real-time RT-PCR, we also investigated the expression pattern of FSY1 in EC1118 growing on various carbon sources. FSY1 was repressed by high concentrations of glucose or fructose and was highly expressed on ethanol as the sole carbon source. The characteristics of this transporter indicate that its acquisition could confer a significant advantage to S. cerevisiae during the wine fermentation process. This transporter is a good example of acquisition of a new function in yeast by horizontal gene transfer.

In Saccharomyces cerevisiae, the oxidative stress-activated zinc cluster protein Stb5p activates genes involved in NADPH production and most genes of the pentose phosphate (PP) pathway. To gain insight into the role of Stb5p, we studied the behaviour of stb5 deletion mutants during aerobic and anaerobic growth on glucose. stb5 mutants were auxotrophic for methionine and pyrimidine nucleotides. The methionine auxotrophy phenotype was air dependent, suggesting an impaired aerobic NADPH status. Consistent with this, the acetate level was reduced and the α-ketoglutarate level was increased in the stb5 mutant. stb5 cells also required pyrimidine nucleotides for aerobic and anaerobic growth, consistent with a reduction in 5-phosphoribosyl-1-pyrophosphate production caused by a reduced flux through the PP pathway. Strains overexpressing STB5 could not grow on glucose. This growth defect was restored by overproduction of an NADPH-butanediol dehydrogenase, which reoxidizes the excess NADPH in the oxidative PP pathway. These findings suggest a major role for the transcription factor Stb5p in maintaining a basal flux through the PP pathway to meet the NADPH requirements for aerobic growth, and to provide the nucleotide precursors. Our data also demonstrate the potential use of a system based on overproduction of this transcription factor to increase flux through the PP pathway.

Appl. Environ. Microbiol. ISI Document Delivery No.: 443KL Times Cited: 14 Cited Reference Count: 45 Ehsani, Maryam Fernandez, Maria R. Biosca, Josep A. Julien, Anne Dequin, Sylvie Ministry of Education and Science [BMC-2003-09606] This work was supported in part by a grant from the Ministry of Education and Science (BMC-2003-09606), Spain. Amer soc microbiology Washington; International audience; Engineered Saccharomyces cerevisiae strains overexpressing GPD1, which codes for glycerol-3-phosphate dehydrogenase, and lacking the acetaldehyde dehydrogenase Ald6 display large-scale diversion of the carbon flux from ethanol toward glycerol without accumulating acetate. Although GPD1 ald6 strains have great potential for reducing the ethanol contents in wines, one major side effect is the accumulation of acetoin, having a negative sensory impact on wine. Acetoin is reduced to 2,3-butanediol by the NADH-dependent 2,3-butanediol dehydrogenase Bdh1. In order to investigate the influence of potential factors limiting this reaction, we overexpressed BDH1, coding for native NADH-dependent Bdh1, and the engineered gene BDH1 221,222,223, coding for an NADPH-dependent Bdh1 enzyme with the amino acid changes 221 EIA 223 to 221 SRS 223, in a glycerol-overproducing wine yeast. We have shown that both the amount of Bdh1 and the NADH availability limit the 2,3-butanediol dehydrogenase reaction. During wine fermentation, however, the major limiting factor was the level of synthesis of Bdh1. Consistent with this finding, the overproduction of native or engineered Bdh1 made it possible to redirect 85 to 90% of the accumulated acetoin into 2,3-butanediol, a compound with neutral sensory characteristics. In addition, the production of diacetyl, a compound causing off-flavor in alcoholic beverages, whose production is increased in glycerol-overproducing yeast cells, was decreased by half. The production of higher alcohols and esters, which was slightly decreased or unchanged in GPD1 ald6 cells compared to that in the control cells, was not further modified in BDH1 cells. Overall, rerouting carbons toward glycerol and 2,3-butanediol represents a new milestone in the engineering of a low-alcohol yeast with desirable organoleptic features, permitting the decrease of the ethanol contents in wines by up to 3 degrees.

The most important dogma in white-wine production is the preservation of the wine aroma and the limitation of the oxidative action of oxygen. In contrast, the aging of Sherry and Sherry-like wines is an aerobic process that depends on the oxidative activity of flor strains of Saccharomyces cerevisiae. Under depletion of nitrogen and fermentable carbon sources, these yeast produce aggregates of floating cells and form an air–liquid biofilm on the wine surface, which is also known as velum or flor. This behavior is due to genetic and metabolic peculiarities that differentiate flor yeast from other wine yeast. This review will focus first on the most updated data obtained through the analysis of flor yeast with -omic tools. Comparative genomics, proteomics, and metabolomics of flor and wine yeast strains are shedding new light on several features of these special yeast, and in particular, they have revealed the extent of proteome remodeling imposed by the biofilm life-style. Finally, new insights in terms of promotion and inhibition of biofilm formation through small molecules, amino acids, and di/tripeptides, and novel possibilities for the exploitation of biofilm immobilization within a fungal hyphae framework, will be discussed.

Redox homeostasis is a fundamental requirement for the maintenance of metabolism, energy generation, and growth in Saccharomyces cerevisiae . The redox cofactors NADH and NADPH are among the most highly connected metabolites in metabolic networks. Changes in their concentrations may induce widespread changes in metabolism. Redox imbalances were achieved with a dedicated biological tool overexpressing native NADH-dependent or engineered NADPH-dependent 2,3-butanediol dehydrogenase, in the presence of acetoin. We report that targeted perturbation of the balance of cofactors (NAD + /NADH or, to a lesser extent, NADP + /NADPH) significantly affected the production of volatile compounds. In most cases, variations in the redox state of yeasts modified the formation of all compounds from the same biochemical pathway (isobutanol, isoamyl alcohol, and their derivatives) or chemical class (ethyl esters), irrespective of the cofactors. These coordinated responses were found to be closely linked to the impact of redox status on the availability of intermediates of central carbon metabolism. This was the case for α-keto acids and acetyl coenzyme A (acetyl-CoA), which are precursors for the synthesis of many volatile compounds. We also demonstrated that changes in the availability of NADH selectively affected the synthesis of some volatile molecules (e.g., methionol, phenylethanol, and propanoic acid), reflecting the specific cofactor requirements of the dehydrogenases involved in their formation. Our findings indicate that both the availability of precursors from central carbon metabolism and the accessibility of reduced cofactors contribute to cell redox status modulation of volatile compound formation.

Background Fermentative aromas play a key role in the organoleptic profile of young wines. Their production depends both on yeast strain and fermentation conditions. A present-day trend in the wine industry consists in developing new strains with aromatic properties using adaptive evolution approaches. An evolved strain, Affinity™ ECA5, overproducing esters, was recently obtained. In this study, dynamics of nitrogen consumption and of the fermentative aroma synthesis of the evolved and its ancestral strains were compared and coupled with a transcriptomic analysis approach to better understand the metabolic reshaping of Affinity™ ECA5. Results Nitrogen assimilation was different between the two strains, particularly amino acids transported by carriers regulated by nitrogen catabolite repression. We also observed differences in the kinetics of fermentative aroma production, especially in the bioconversion of higher alcohols into acetate esters. Finally, transcriptomic data showed that the enhanced bioconversion into acetate esters by the evolved strain was associated with the repression of genes involved in sterol biosynthesis rather than an enhanced expression of ATF1 and ATF2 (genes coding for the enzymes responsible for the synthesis of acetate esters from higher alcohols). Conclusions An integrated approach to yeast metabolism—combining transcriptomic analyses and online monitoring data—showed differences between the two strains at different levels. Differences in nitrogen source consumption were observed suggesting modifications of NCR in the evolved strain. Moreover, the evolved strain showed a different way of managing the lipid source, which notably affected the production of acetate esters, likely because of a greater availability of acetyl-CoA for the evolved strain. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0434-6) contains supplementary material, which is available to authorized users.

Butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae belongs to the superfamily of the medium-chain dehydrogenases and reductases and converts reversibly R -acetoin and S -acetoin to (2 R ,3 R )-2,3-butanediol and meso -2,3-butanediol, respectively. It is specific for NAD(H) as a coenzyme, and it is the main enzyme involved in the last metabolic step leading to (2 R ,3 R )-2,3-butanediol in yeast. In this study, we have used the activity of Bdh1p in different forms—purified enzyme, yeast extracts, permeabilized yeast cells, and as a fusion protein (with yeast formate dehydrogenase, Fdh1p)—to transform several vicinal diketones to the corresponding diols. We have also developed a new variant of the delitto perfetto methodology to place BDH1 under the control of the GAL1 promoter, resulting in a yeast strain that overexpresses butanediol dehydrogenase and formate dehydrogenase activities in the presence of galactose and regenerates NADH in the presence of formate. While the use of purified Bdh1p allows the synthesis of enantiopure (2 R ,3 R )-2,3-butanediol, (2 R ,3 R )-2,3-pentanediol, (2 R ,3 R )-2,3-hexanediol, and (3 R ,4 R )-3,4-hexanediol, the use of the engineered strain (as an extract or as permeabilized cells) yields mixtures of the diols. The production of pure diol stereoisomers has also been achieved by means of a chimeric fusion protein combining Fdh1p and Bdh1p. Finally, we have determined the selectivity of Bdh1p toward the oxidation/reduction of the hydroxyl/ketone groups from (2 R ,3 R )-2,3-pentanediol/2,3-pentanedione and (2 R ,3 R )-2,3-hexanediol/2,3-hexanedione. In conclusion, Bdh1p is an enzyme with biotechnological interest that can be used to synthesize chiral building blocks. A scheme of the favored pathway with the corresponding intermediates is proposed for the Bdh1p reaction.

Flor yeasts of Saccharomyces cerevisiae have been extensively studied for biofilm formation, however the lack of specific haploid model strains has limited the application of genetic approaches such as gene knockout, allelic replacement and Quantitative Trait Locus mapping for the deciphering of the molecular basis of velum formation under biological ageing. The aim of this work was to construct a set of flor isogenic haploid strains easy to manipulate genetically. The analysis of the allelic variations at 12 minisatellite loci of 174 Saccharomyces cerevisiae strains allowed identifying three flor parental strains with different phylogenic positions. These strains were characterized for sporulation efficiency, growth on galactose, adherence to polystyrene, agar invasion, growth on wine and ability to develop a biofilm. Interestingly, the inability to grow on galactose was found associated with a frameshift in GAL4 gene that seems peculiar of flor strains. From these wild flor strains, isogenic haploid strains were constructed by deleting HO gene with a loxP-KanMX-loxP cassette followed by the removal of the kanamycin cassette. Haploid strains obtained were characterized for their phenotypic and genetic properties and compared with the parental strains. Preliminary results showed that the haploid strains represent new tools for genetic studies and breeding programs on biofilm formation.The inability to metabolize galactose was found to be a useful selectable marker in flor yeast strain development programs.The inability to metabolize galactose was found to be a useful selectable marker in flor yeast strain development programs.

The yeast Saccharomyces cerevisiae is one of the most important microorganisms for food and drink production and it is as well a model for biology. Surprisingly, the bases of yeast population structure have been unraveled only recently [1, 2] and the first genomic population approaches have failed to point adaptation to ecological niches. However, as many groups present highly contrasted lifestyle (e.g. anaerobic growth on grape must for wine yeast, aerobic growth as a biofilm on ethanol and glycerol for flor strains, growth on low amount of sugars for oak strains) different adaptations are expected. In this project we have obtained high quality genome sequences of 82 yeast strains: 35 from wine environment (27 wine strains, 8 flor strains) as well as 47 other strains from rum, fermented dairy product, bakery including 8 from oaks trees. Our genomic data enable us to delineate specific genetic groups corresponding to the different ecological niches, and indicate different life cycles. We first detected the amplification of several genes with critical function in their environment (e.g. CUP1 for wine yeast, IMA2 and SUC2 for bakery yeast…). We then established a catalog of genes potentially impacted per population. Several tests revealed a non-neutral evolution at several loci, and especially the presence of selective sweep for wine yeast, suggesting positive selection. The comparison of flor and wine yeast revealed allelic variation associated to growth under velum, as well as the fructophily of flor strains, or a specific metal homeostasis. Last among the three large chromosomal regions originated from horizontal gene transfer (HGT) from distant yeasts first discovered in EC1118 [3] were encountered mainly in wine or flor yeasts but the complete region C was only found among flor strains. These genomic data represent a unique resource for understanding the adaptation of yeast to wine making niches and thus for elucidating the bases of technological properties.