Beyond S. cerevisiae for winemaking: Fermentation-related trait diversity in the genus Saccharomyces.

Saccharomyces cerevisiae is the yeast of choice for most inoculated wine fermentations worldwide. However, many other yeast species and genera display phenotypes of interest that may help address the environmental and commercial challenges the wine industry has been facing in recent years. This work aimed to provide, for the first time, a systematic phenotyping of all Saccharomyces species under winemaking conditions. For this purpose, we characterized the fermentative and metabolic properties of 92 Saccharomyces strains in synthetic grape must at two different temperatures. The fermentative potential of alternative yeasts was higher than expected, as nearly all strains were able to complete fermentation, in some cases more efficiently than commercial S. cerevisiae strains. Various species showed interesting metabolic traits, such as high glycerol, succinate and odour-active compound production, or low acetic acid production, compared to S. cerevisiae. Altogether, these results reveal that non-cerevisiae Saccharomyces yeasts are especially interesting for wine fermentation, as they may offer advantages over both S. cerevisiae and non-Saccharomyces strains. This study highlights the potential of alternative Saccharomyces species for winemaking, paving the way for further research and, potentially, for their industrial exploitation.

Genetic bases for the metabolism of the DMS precursor S-methylmethionine by Saccharomyces cerevisiae.

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.

Adaptation of S. cerevisiae to Fermented Food Environments Reveals Remarkable Genome Plasticity and the Footprints of Domestication.

The budding yeast Saccharomyces cerevisiae can be found in the wild and is also frequently associated with human activities. Despite recent insights into the phylogeny of this species, much is still unknown about how evolutionary processes related to anthropogenic niches have shaped the genomes and phenotypes of S. cerevisiae. To address this question, we performed population-level sequencing of 82 S. cerevisiae strains from wine, flor, rum, dairy products, bakeries, and the natural environment (oak trees). These genomic data enabled us to delineate specific genetic groups corresponding to the different ecological niches and revealed high genome content variation across the groups. Most of these strains, compared with the reference genome, possessed additional genetic elements acquired by introgression or horizontal transfer, several of which were population-specific. In addition, several genomic regions in each population showed evidence of nonneutral evolution, as shown by high differentiation, or of selective sweeps including genes with key functions in these environments (e.g., amino acid transport for wine yeast). Linking genetics to lifestyle differences and metabolite traits has enabled us to elucidate the genetic basis of several niche-specific population traits, such as growth on galactose for cheese strains. These data indicate that yeast has been subjected to various divergent selective pressures depending on its niche, requiring the development of customized genomes for better survival in these environments. These striking genome dynamics associated with local adaptation and domestication reveal the remarkable plasticity of the S. cerevisiae genome, revealing this species to be an amazing complex of specialized populations.

QTL mapping of volatile compound production in Saccharomyces cerevisiae during alcoholic fermentation.

BACKGROUND: The volatile metabolites produced by Saccharomyces cerevisiae during alcoholic fermentation, which are mainly esters, higher alcohols and organic acids, play a vital role in the quality and perception of fermented beverages, such as wine. Although the metabolic pathways and genes behind yeast fermentative aroma formation are well described, little is known about the genetic mechanisms underlying variations between strains in the production of these aroma compounds. To increase our knowledge about the links between genetic variation and volatile production, we performed quantitative trait locus (QTL) mapping using 130 F2-meiotic segregants from two S. cerevisiae wine strains. The segregants were individually genotyped by next-generation sequencing and separately phenotyped during wine fermentation. RESULTS: Using different QTL mapping strategies, we were able to identify 65 QTLs in the genome, including 55 that influence the formation of 30 volatile secondary metabolites, 14 with an effect on sugar consumption and central carbon metabolite production, and 7 influencing fermentation parameters. For ethyl lactate, ethyl octanoate and propanol formation, we discovered 2 interacting QTLs each. Within 9 of the detected regions, we validated the contribution of 13 genes in the observed phenotypic variation by reciprocal hemizygosity analysis. These genes are involved in nitrogen uptake and metabolism (AGP1, ALP1, ILV6, LEU9), central carbon metabolism (HXT3, MAE1), fatty acid synthesis (FAS1) and regulation (AGP2, IXR1, NRG1, RGS2, RGT1, SIR2) and explain variations in the production of characteristic sensorial esters (e.g., 2-phenylethyl acetate, 2-metyhlpropyl acetate and ethyl hexanoate), higher alcohols and fatty acids. CONCLUSIONS: The detection of QTLs and their interactions emphasizes the complexity of yeast fermentative aroma formation. The validation of underlying allelic variants increases knowledge about genetic variation impacting metabolic pathways that lead to the synthesis of sensorial important compounds. As a result, this work lays the foundation for tailoring S. cerevisiae strains with optimized volatile metabolite production for fermented beverages and other biotechnological applications.

Specific Phenotypic Traits of Starmerella bacillaris Related to Nitrogen Source Consumption and Central Carbon Metabolite Production during Wine Fermentation.

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. cerevisiaeIMPORTANCE 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.

Integrating transcriptomics and metabolomics for the analysis of the aroma profiles of Saccharomyces cerevisiae strains from diverse origins.

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 (cachaça, 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 cachaça, 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) and using different data analysis methods (PCA, MFA). The results obtained increased our knowledge on the production of wine aroma and flavour, identifying new gene in association to the formation of flavour active compounds, mainly in the production of fatty acids, and ethyl and acetate esters.

Management of Multiple Nitrogen Sources during Wine Fermentation by Saccharomyces cerevisiae.

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.

Exploring the potential of Saccharomyces eubayanus as a parent for new interspecies hybrid strains in winemaking.

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 subgenome. 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.

Yeast multistress resistance and lag-phase characterisation during wine fermentation.

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.

Evolutionary Advantage Conferred by an Eukaryote-to-Eukaryote Gene Transfer Event in Wine Yeasts.

Although an increasing number of horizontal gene transfers have been reported in eukaryotes, experimental evidence for their adaptive value is lacking. Here, we report the recent transfer of a 158-kb genomic region between Torulaspora microellipsoides and Saccharomyces cerevisiae wine yeasts or closely related strains. This genomic region has undergone several rearrangements in S. cerevisiae strains, including gene loss and gene conversion between two tandemly duplicated FOT genes encoding oligopeptide transporters. We show that FOT genes confer a strong competitive advantage during grape must fermentation by increasing the number and diversity of oligopeptides that yeast can utilize as a source of nitrogen, thereby improving biomass formation, fermentation efficiency, and cell viability. Thus, the acquisition of FOT genes has favored yeast adaptation to the nitrogen-limited wine fermentation environment. This finding indicates that anthropic environments offer substantial ecological opportunity for evolutionary diversification through gene exchange between distant yeast species.

Horizontally acquired oligopeptide transporters favour adaptation of Saccharomyces cerevisiae wine yeast to oenological environment.

In the past decade, horizontal gene transfer (HGT) has emerged as a major evolutionary process that has shaped the genome of Saccharomyces cerevisiae wine yeasts. We recently showed that a large Torulaspora microellipsoides genomic island carrying two oligopeptide transporters encoded by FOT genes increases the fitness of wine yeast during fermentation of grape must. However, the impact of these genes on the metabolic network of S. cerevisiae remained uncharacterized. Here we show that Fot-mediated peptide uptake substantially affects the glutamate node and the NADPH/NADP(+) balance, resulting in the delayed uptake of free amino acids and altered profiles of metabolites and volatile compounds. Transcriptome analysis revealed that cells using a higher amount of oligopeptides from grape must are less stressed and display substantial variation in the expression of genes in the central pathways of carbon and nitrogen metabolism, amino acid and protein biosynthesis, and the oxidative stress response. These regulations shed light on the molecular and metabolic mechanisms involved in the higher performance and fitness conferred by the HGT-acquired FOT genes, pinpointing metabolic effects that can positively affect the organoleptic balance of wines.

Metabolic Impact of Redox Cofactor Perturbations on the Formation of Aroma Compounds in Saccharomyces cerevisiae.

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.

Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase.

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 (2R,3R)-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 (2R,3R)-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 (2R,3R)-2,3-butanediol, (2R,3R)-2,3-pentanediol, (2R,3R)-2,3-hexanediol, and (3R,4R)-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 (2R,3R)-2,3-pentanediol/2,3-pentanedione and (2R,3R)-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.

A set of haploid strains available for genetic studies of Saccharomyces cerevisiae flor yeasts.

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.

Diversity of flux distribution in central carbon metabolism of S. cerevisiae strains from diverse environments.

BACKGROUND: S. cerevisiae has attracted considerable interest in recent years as a model for ecology and evolutionary biology, revealing a substantial genetic and phenotypic diversity. However, there is a lack of knowledge on the diversity of metabolic networks within this species. RESULTS: To identify the metabolic and evolutionary constraints that shape metabolic fluxes in S. cerevisiae, we used a dedicated constraint-based model to predict the central carbon metabolism flux distribution of 43 strains from different ecological origins, grown in wine fermentation conditions. In analyzing these distributions, we observed a highly contrasted situation in flux variability, with quasi-constancy of the glycolysis and ethanol synthesis yield yet high flexibility of other fluxes, such as the pentose phosphate pathway and acetaldehyde production. Furthermore, these fluxes with large variability showed multimodal distributions that could be linked to strain origin, indicating a convergence between genetic origin and flux phenotype. CONCLUSIONS: Flux variability is pathway-dependent and, for some flux, a strain origin effect can be found. These data highlight the constraints shaping the yeast operative central carbon network and provide clues for the design of strategies for strain improvement.

Key role of lipid management in nitrogen and aroma metabolism in an evolved wine yeast strain.

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.

A population genomics insight into the Mediterranean origins of wine yeast domestication.

The domestication of the wine yeast Saccharomyces cerevisiae is thought to be contemporary with the development and expansion of viticulture along the Mediterranean basin. Until now, the unavailability of wild lineages prevented the identification of the closest wild relatives of wine yeasts. Here, we enlarge the collection of natural lineages and employ whole-genome data of oak-associated wild isolates to study a balanced number of anthropic and natural S. cerevisiae strains. We identified industrial variants and new geographically delimited populations, including a novel Mediterranean oak population. This population is the closest relative of the wine lineage as shown by a weak population structure and further supported by genomewide population analyses. A coalescent model considering partial isolation with asymmetrical migration, mostly from the wild group into the Wine group, and population growth, was found to be best supported by the data. Importantly, divergence time estimates between the two populations agree with historical evidence for winemaking. We show that three horizontally transmitted regions, previously described to contain genes relevant to wine fermentation, are present in the Wine group but not in the Mediterranean oak group. This represents a major discontinuity between the two populations and is likely to denote a domestication fingerprint in wine yeasts. Taken together, these results indicate that Mediterranean oaks harbour the wild genetic stock of domesticated wine yeasts.

Diversity and adaptive evolution of Saccharomyces wine yeast: a review.

Saccharomyces cerevisiae and related species, the main workhorses of wine fermentation, have been exposed to stressful conditions for millennia, potentially resulting in adaptive differentiation. As a result, wine yeasts have recently attracted considerable interest for studying the evolutionary effects of domestication. The widespread use of whole-genome sequencing during the last decade has provided new insights into the biodiversity, population structure, phylogeography and evolutionary history of wine yeasts. Comparisons between S. cerevisiae isolates from various origins have indicated that a variety of mechanisms, including heterozygosity, nucleotide and structural variations, introgressions, horizontal gene transfer and hybridization, contribute to the genetic and phenotypic diversity of S. cerevisiae. This review will summarize the current knowledge on the diversity and evolutionary history of wine yeasts, focusing on the domestication fingerprints identified in these strains.

Intrastrain genomic and phenotypic variability of the commercial Saccharomyces cerevisiae strain Zymaflore VL1 reveals microevolutionary adaptation to vineyard environments.

The maintenance of microbial species in different environmental conditions is associated with adaptive microevolutionary changes that are shown here to occur within the descendants of the same strain in comparison with the commercial reference strain. However, scarce information is available regarding changes that occur among strain descendants during their persistence in nature. Herein we evaluate genome variations among four isolates of the commercial winemaking strain Saccharomyces cerevisiae Zymaflore VL1 that were re-isolated from vineyards surrounding wineries where this strain was applied during several years, in comparison with the commercial reference strain. Comparative genome hybridization showed amplification of 14 genes among the recovered isolates being related with mitosis, meiosis, lysine biosynthesis, galactose and asparagine catabolism, besides 9 Ty elements. The occurrence of microevolutionary changes was supported by DNA sequencing that revealed 339-427 SNPs and 12-62 indels. Phenotypic screening and metabolic profiles also distinguished the recovered isolates from the reference strain. We herein show that the transition from nutrient-rich musts to nutritionally scarce natural environments induces adaptive responses and microevolutionary changes promoted by Ty elements and by nucleotide polymorphisms that were not detected in the reference strain.

Combined effects of nutrients and temperature on the production of fermentative aromas by Saccharomyces cerevisiae during wine fermentation.

Volatile compounds produced by yeast during fermentation greatly influence the organoleptic qualities of wine. We developed a model to predict the combined effects of initial nitrogen and phytosterol content and fermentation temperature on the production of volatile compounds. We used a Box-Behnken design and response surface modeling to study the response of Lalvin EC1118® to these environmental conditions. Initial nitrogen content had the greatest influence on most compounds; however, there were differences in the value of fermentation parameters required for the maximal production of the various compounds. Fermentation parameters affected differently the production of isobutanol and isoamyl alcohol, although their synthesis involve the same enzymes and intermediate. We found differences in regulation of the synthesis of acetates of higher alcohols and ethyl esters, suggesting that fatty acid availability is the main factor influencing the synthesis of ethyl esters whereas the production of acetates depends on the activity of alcohol acetyltransferases. We also evaluated the effect of temperature on the total production of three esters by determining gas-liquid balances. Evaporation largely accounted for the effect of temperature on the accumulation of esters in liquid. Nonetheless, the metabolism of isoamyl acetate and ethyl octanoate was significantly affected by this parameter. We extended this study to other strains. Environmental parameters had a similar effect on aroma production in most strains. Nevertheless, the regulation of the synthesis of fermentative aromas was atypical in two strains: Lalvin K1M® and Affinity™ ECA5, which produces a high amount of aromatic compounds and was obtained by experimental evolution.