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The processes of yeast selection for using as wine fermentation starters have revealed a great phenotypic diversity both at interspecific and intraspecific level, which is explained by a corresponding genetic variation among different yeast isolates. Thus, the mechanisms involved in promoting these genetic changes are the main engine generating yeast biodiversity. Currently, an important task to understand biodiversity, population structure and evolutionary history of wine yeasts is the study of the molecular mechanisms involved in yeast adaptation to wine fermentation, and on remodeling the genomic features of wine yeast, unconsciously selected since the advent of winemaking. Moreover, the availability of rapid and simple molecular techniques that show genetic polymorphisms at species and strain levels have enabled the study of yeast diversity during wine fermentation. This review will summarize the mechanisms involved in generating genetic polymorphisms in yeasts, the molecular methods used to unveil genetic variation, and the utility of these polymorphisms to differentiate strains, populations, and species in order to infer the evolutionary history and the adaptive evolution of wine yeasts, and to identify their influence on their biotechnological and sensorial properties. ; This work has been financially supported from the Spanish Government through MINECO and FEDER funds (AGL2013-47300-C3-3-R, AGL2015-67504-C3-3-R, and PCIN-2015-143grants) and from Generalitat Valenciana through PROMETEOII/2014/042 grant. We acknowledge support of the publication fee by the CSIC Open Access Support Initiative through its Unit of Information Resources for Research (URICI) ; Peer reviewed ; Peer Reviewed

Wine fermentation has not significantly changed since ancient times and the most traditional aspects are seen by the market as elements that uplift wine nuances and quality. In recent years, new trends have emerged from the sector in line with consumer preferences, and due to the effects of global climate change on grape ripening. In the first cases, the consumers are looking for wines with less ethanol and fruitier aromas and in the second cases the wineries want to reduce the wine alcohol levels and/or astringency. New yeast starters of alternative Saccharomyces species and their hybrids can help to solve some problems that wineries face. In this article we review several physiological and genetic aspects of S. uvarum and S. kudriavzevii and the hybrids, which are especially relevant during the winemaking process, such as their good fermentative capabilities at low temperatures, resulting in wines with lower alcohol and higher glycerol amounts. ; This work has supported by grants AGL2015-67504-C3-1-R from the Spanish Government and FEDER and Generalitat Valenciana PROMETEO (PROMETEOII/2014/042) to AQ. ; Peer reviewed

7 pages, 2 figures, 3 tables.-- Printed version published May 2009. ; In this study, we performed an analysis of the ability of four Saccharomyces cerevisiae and one S. bayanus var. uvarum strains, isolated from different industrial processes, to ferment increasing amounts of fructose (from 0 to 70%, w/v). Overall yeast growth was estimated by integration of the area under optical density vs. time curves. Subsequently, this parameter was modeled by means of a substrate inhibition model. All strains showed a similar behavior against fructose concentration in spite of their different origins, but with slight differences. The optimum fructose concentrations to stimulate yeast growth were obtained between 4.33 and 6.05%, while the maximum concentrations above which growth was completely inhibited were attained between 59.56 and 63.85%. Statistically, model parameters calculated for wine yeast strains were significantly different than those obtained for yeasts from Agave and table olive fermentations, except for the maximum inhibitory concentration. The methodology used in this work could be useful for the industry in general as a first procedure to select yeast strains with higher fructose preferences or tolerances, and especially for winemakers, where the risk of spoilage increases by the presence of a marked residual fructose concentration in the finished wine. ; This work was supported by Generalitat Valenciana (project GV2008-037) and the Spanish Government (projects AGL2006-12703-CO2-01 and 02/ALI). ; Peer reviewed

Yeasts belonging to the Saccharomyces genus play an important role in human-driven fermentations. The species S. cerevisiae has been widely studied because it is the dominant yeast in most fermentations and it has been widely used as a model eukaryotic organism. Recently, other species of the Saccharomyces genus are gaining interest to solve the new challenges that the fermentation industry are facing. One of these species is S. kudriavzevii, which exhibits interesting physiological properties compared to S. cerevisiae, such as a better adaptation to grow at low temperatures, a higher glycerol synthesis and lower ethanol production. The aim of this study is to understand the molecular basis behind these phenotypic differences of biotechnological interest by using a species-based comparative genomics approach. In this work, we sequenced, assembled and annotated two new genomes of S. kudriavzevii. We used a combination of different statistical methods to identify functional divergence, signatures of positive selection and acceleration of substitution rates at specific amino acid sites of proteins in S. kudriavzevii when compared to S. cerevisiae, and vice versa. We provide a list of candidate genes in which positive selection could be acting during the evolution of both S. cerevisiae and S. kudriavzevii clades. Some of them could be related to certain important differences in metabolism previously reported by other authors such us DAL3 and ARO4, involved in nitrogen assimilation and amino acid biosynthesis. In addition, three of those genes (FBA1, ZIP1, and RQC2) showed accelerated evolutionary rates in Sk branch. Finally, genes of the riboflavin biosynthesis were also among those genes with a significant higher rate of nucleotide substitution and those proteins have amino acid positions contributing to functional divergence. ; This work was funded by grant AGL2015-67504-C3-3-R from the Spanish Government and European Union ERDF-FEDER to EB and by ERA CoBioTech MeMBrane Project PCI2018-093190. LM was supported by the aforementioned grant associated to EB. MM was supported by a Ph.D. student contract ACIF/2015/194 from the Regional Government of Valencia. CT acknowledged a "Juan de la Cierva" postdoctoral contract JCI-2012-14056 from the Spanish Government. ; Peer reviewed

© 2015 Sociedad Española de Microbiologia. All rights reserved. We evaluated the genetic stabilization of artificial intra- (Saccharomyces cerevisiae) and interspecific (S. cerevisiae × S. kudriavzevii) hybrids under wine fermentative conditions. Large-scale transitions in genome size and genome reorganizations were observed during this process. Interspecific hybrids seem to need fewer generations to reach genetic stability than intraspecific hybrids. The largest number of molecular patterns recovered among the derived clones was observed for intraspecific hybrids, particularly for those obtained by rare-mating. Molecular marker analyses revealed that unstable clones could change during the industrial process to obtain active dry yeast. When no changes in molecular markers and ploidy were observed after this process, no changes in genetic composition were confirmed by comparative genome hybridization, considering the clone as a stable hybrid. According to our results, under these conditions, fermentation steps 3 and 5 (30–50 generations) would suffice to obtain genetically stable interspecific and intraspecific hybrids, respectively. ; This work has been supported by grants AGL2009-12673-CO2-01 and AGL2009-12673-CO2-02 and AGL2012-39937-C02-01 and AGL2012-39937-C02-02 from the Spanish Government to A. Querol and E. Barrio, respectively. L. P-T. and C. L. wish to acknowledge the CSIC and the Spanish Ministry of Education and Science for an I3P fellowship and a postdoctoral contract, respectively ; Peer Reviewed

Although the genus Saccharomyces has been thoroughly studied, some species in the genus has not yet been accurately resolved; an example is S. bayanus, a taxon that includes genetically diverse lineages of pure and hybrid strains. This diversity makes the assignation and classification of strains belonging to this species unclear and controversial. They have been subdivided by some authors into two varieties (bayanus and uvarum), which have been raised to the species level by others. In this work, we evaluate the complexity of 46 different strains included in the S. bayanus taxon by means of PCR-RFLP analysis and by sequencing of 34 gene regions and one mitochondrial gene. Using the sequence data, and based on the S. bayanus var. bayanus reference strain NBRC 1948, a hypothetical pure S. bayanus was reconstructed for these genes that showed alleles with similarity values lower than 97% with the S. bayanus var. uvarum strain CBS 7001, and of 99-100% with the non S. cerevisiae portion in S. pastorianus Weihenstephan 34/70 and with the new species S. eubayanus . Among the S. bayanus strains under study, different levels of homozygosity, hybridization and introgression were found; however, no pure S. bayanus var. bayanus strain was identified. These S. bayanus hybrids can be classified into two types: homozygous (type I) and heterozygous hybrids (type II), indicating that they have been originated by different hybridization processes. Therefore, a putative evolutionary scenario involving two different hybridization events between a S. bayanus var. uvarum and unknown European S. eubayanus-like strains can be postulated to explain the genomic diversity observed in our S. bayanus var. bayanus strains. © 2014 Pérez-Través et al. ; This work was supported by grants AGL2009-12673-CO2 (01 and 02) and AGL2012-39937-CO2 (01 and 02) from the Spanish Government to AQ and EB, respectively. LP-T and CAL acknowledge to CSIC and Fundación Carolina for the I3P and Formación Permanente fellowships respectively. ; Peer Reviewed

Abstract Background Interspecific hybrids between S. cerevisiae × S. kudriavzevii have frequently been detected in wine and beer fermentations. Significant physiological differences among parental and hybrid strains under different stress conditions have been evidenced. In this study, we used comparative genome hybridization analysis to evaluate the genome composition of different S. cerevisiae × S. kudriavzevii natural hybrids isolated from wine and beer fermentations to infer their evolutionary origins and to figure out the potential role of common S. kudriavzevii gene fraction present in these hybrids. Results Comparative genomic hybridization (CGH) and ploidy analyses carried out in this study confirmed the presence of individual and differential chromosomal composition patterns for most S. cerevisiae × S. kudriavzevii hybrids from beer and wine. All hybrids share a common set of depleted S. cerevisiae genes, which also are depleted or absent in the wine strains studied so far, and the presence a common set of S. kudriavzevii genes, which may be associated with their capability to grow at low temperatures. Finally, a maximum parsimony analysis of chromosomal rearrangement events, occurred in the hybrid genomes, indicated the presence of two main groups of wine hybrids and different divergent lineages of brewing strains. Conclusion Our data suggest that wine and beer S. cerevisiae × S. kudriavzevii hybrids have been originated by different rare-mating events involving a diploid wine S. cerevisiae and a haploid or diploid European S. kudriavzevii strains. Hybrids maintain several S. kudriavzevii genes involved in cold adaptation as well as those related to S. kudriavzevii mitochondrial functions. ; This work was supported by Spanish Governments projects AGL2009-12673-CO2-01 and AGL2009-12673-CO2-02 to AQ and EB respectively, and by grant PROMETEO/2009/019 from Generalitat Valenciana to AQ, EB and CB. DP and CL acknowledges to the Spanish Government for a FPI fellowship and a postdoctoral contract, respectively. We also thank Ksenija Lopandić, José Manuel Álvarez-Pérez, Sandi Orlić, Lallemand Bio and Anchor Wine Yeasts for providing yeast strains. ; Peer Reviewed

Grape must is a sugar‐rich habitat for a complex microbiota which is replaced by Saccharomyces cerevisiae strains during the first fermentation stages. Interest on yeast competitive interactions has recently been propelled due to the use of alternative yeasts in the wine industry to respond to new market demands. The main issue resides in the persistence of these yeasts due to the specific competitive activity of S. cerevisiae. To gather deeper knowledge of the molecular mechanisms involved, we performed a comparative transcriptomic analysis during fermentation carried out by a wine S. cerevisiae strain and a strain representative of the cryophilic S. kudriavzevii, which exhibits high genetic and physiological similarities to S. cerevisiae, but also differences of biotechnological interest. In this study, we report that transcriptomic response to the presence of a competitor is stronger in S. cerevisiae than in S. kudriavzevii. Our results demonstrate that a wine S. cerevisiae industrial strain accelerates nutrient uptake and utilization to outcompete the co‐inoculated yeast, and that this process requires cell‐to‐cell contact to occur. Finally, we propose that this competitive phenotype evolved recently, during the adaptation of S. cerevisiae to man‐manipulated fermentative environments, since a non‐wine S. cerevisiae strain, isolated from a North American oak, showed a remarkable low response to competition. ; JAR was supported by a FPI grant from the Ministerio de Economia y Competitividad, Spain (ref. BES‐2013‐066434). This work was funded by grants AGL2015‐67504‐C3‐1‐R and AGL2015‐67504‐C3‐3‐R from the Spanish Government and European Regional Development Fund (ERDF) to AQ and EB respectively. RPT is supported by the aforementioned grant associated to AQ. ; Peer reviewed

Saccharomyces cerevisiae is the main microorganism responsible for the fermentation of wine. Nevertheless, in the last years wineries are facing new challenges due to current market demands and climate change effects on the wine quality. New yeast starters formed by non-conventional Saccharomyces species (such as S. uvarum or S. kudriavzevii) or their hybrids (S. cerevisiae x S. uvarum and S. cerevisiae x S. kudriavzevii) can contribute to solve some of these challenges. They exhibit good fermentative capabilities at low temperatures, producing wines with lower alcohol and higher glycerol amounts. However, S. cerevisiae can competitively displace other yeast species from wine fermentations, therefore the use of these new starters requires an analysis of their behavior during competition with S. cerevisiae during wine fermentation. In the present study we analyzed the survival capacity of non-cerevisiae strains in competition with S. cerevisiae during fermentation of synthetic wine must at different temperatures. First, we developed a new method, based on QPCR, to quantify the proportion of different Saccharomyces yeasts in mixed cultures. This method was used to assess the effect of competition on the growth fitness. In addition, fermentation kinetics parameters and final wine compositions were also analyzed. We observed that some cryotolerant Saccharomyces yeasts, particularly S. uvarum, seriously compromised S. cerevisiae fitness during competences at lower temperatures, which explains why S. uvarum can replace S. cerevisiae during wine fermentations in European regions with oceanic and continental climates. From an enological point of view, mixed co-cultures between S. cerevisiae and S. paradoxus or S. eubayanus, deteriorated fermentation parameters and the final product composition compared to single S. cerevisiae inoculation. However, in co-inoculated synthetic must in which S. kudriavzevii or S. uvarum coexisted with S. cerevisiae, there were fermentation performance improvements and the final wines contained less ethanol and higher amounts of glycerol. Finally, it is interesting to note that in co-inoculated fermentations, wine strains of S. cerevisiae and S. uvarum performed better than non-wine strains of the same species. ; JA was supported by a FPI grant from the Ministerio de Economía y Competitividad (ref. BES-2013-066434). This work was supported by grants AGL2012-39937-C02-01 and AGL2015-67504-C3-1-R from the Spanish Government and FEDER to AQ, AGL2012-39937-C02-02 and AGL2015-67504-C3-3-R from the Spanish Government and FEDER to EB, and PROMETEO (project PROMETEOII/2014/042) from Generalitat Valenciana to AQ. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). ; Peer reviewed ; Peer Reviewed

Saccharomyces cerevisiae is the main microorganism responsible for the fermentation of wine. Nevertheless, in the last years wineries are facing new challenges due to current market demands and climate change effects on the wine quality. New yeast starters formed by non-conventional Saccharomyces species (such as S. uvarum or S. kudriavzevii) or their hybrids (S. cerevisiae x S. uvarum and S. cerevisiae x S. kudriavzevii) can contribute to solve some of these challenges. They exhibit good fermentative capabilities at low temperatures, producing wines with lower alcohol and higher glycerol amounts. However, S. cerevisiae can competitively displace other yeast species from wine fermentations, therefore the use of these new starters requires an analysis of their behavior during competition with S. cerevisiae during wine fermentation. In the present study we analyzed the survival capacity of non-cerevisiae strains in competition with S. cerevisiae during fermentation of synthetic wine must at different temperatures. First, we developed a new method, based on QPCR, to quantify the proportion of different Saccharomyces yeasts in mixed cultures. This method was used to assess the effect of competition on the growth fitness. In addition, fermentation kinetics parameters and final wine compositions were also analyzed. We observed that some cryotolerant Saccharomyces yeasts, particularly S. uvarum, seriously compromised S. cerevisiae fitness during competences at lower temperatures, which explains why S. uvarum can replace S. cerevisiae during wine fermentations in European regions with oceanic and continental climates. From an enological point of view, mixed co-cultures between S. cerevisiae and S. paradoxus or S. eubayanus, deteriorated fermentation parameters and the final product composition compared to single S. cerevisiae inoculation. However, in co-inoculated synthetic must in which S. kudriavzevii or S. uvarum coexisted with S. cerevisiae, there were fermentation performance improvements and the final wines contained less ethanol and higher amounts of glycerol. Finally, it is interesting to note that in co-inoculated fermentations, wine strains of S. cerevisiae and S. uvarum performed better than non-wine strains of the same species. ; JA was supported by a FPI grant from the Ministerio de Economía y Competitividad (ref. BES-2013-066434). This work was supported by grants AGL2012-39937-C02-01 and AGL2015-67504-C3-1-R from the Spanish Government and FEDER to AQ, AGL2012-39937-C02-02 and AGL2015-67504-C3-3-R from the Spanish Government and FEDER to EB, and PROMETEO (project PROMETEOII/2014/042) from Generalitat Valenciana to AQ. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). ; Peer reviewed

This work aims to describe the wine fermentation characteristics of 23 natural S. cerevisiae × S. kudriavzevii hybrid yeasts related to fermentative environments isolated from different regions and their significance for the aroma spectra of the produced wines. Fermentations were performed at 12 °C in artificial must, and S. cerevisiae and S. kudriavzevii pure species strains were used for comparison purposes. We determined the relevant kinetic parameters of fermentation, the concentration of the main metabolites and the main aroma-related compounds produced after fermentation. The results revealed that some strains that show well-rounded characteristics could be profitable yeast starters for low-temperature fermentation in winemaking, such as wine hybrid SPG172 but, surprisingly, also beer hybrid CECT11002, adding the efficient fermentative kinetics to the high production of aroma-related compounds. In addition, a novel metabolic correlation between fermentation performance and aroma production is described. ; G. Ortiz-Tovar was supported by CONACYT doctoral scholarship 176060. This work was supported by grants AGL2015-67504-C3-3-R from the Spanish Government and FEDER to EB. ; Peer reviewed

Available online 17 March 2015 ; Saccharomyces cerevisiae plays a main role in the winemaking process, although other species, like Saccharomyces uvarum or Saccharomyces paradoxus, have been associated with must fermentations. It has been reported in recent years, that yeast hybrids of different Saccharomyces species might be responsible for wine productions. Although S. cerevisiae × Saccharomyces kudriavzevii hybrids have been well studied, very little attention has been paid to S. cerevisiae × S. uvarum hybrids. In this work we characterized the genomic composition of S6U, a widely used commercial S. cerevisiae × S. uvarum yeast hybrid isolated in wine fermentations containing one copy of the genome of each parental species, which suggests a relatively recent hybridization event. We also studied its performance under diverse enological conditions. The results show enhanced performance under low temperature enological conditions, increased glycerol production, lower acetic acid production and increased production of interesting aroma compounds. We also examined the transcriptomic response of the S6U hybrid strain compared with the reference species under enological conditions. The results show that although the hybrid strain transcriptome is more similar to S. uvarum than to S. cerevisiae, it presents specifically regulated genes involved in stress response, lipids and amino acid metabolism. The enological performance and aroma profile of this S. cerevisiae × S. uvarum hybrid makes it a good candidate for participating in winemaking, especially at low temperatures. ; M. Combina is a member of the Scientific Research Career (CONICET) and was a beneficiary of a fellowship from the "Secretaría de Estado de Universidades e Investigación del Ministerio de Educación y Ciencia" from Spain. This work has been supported by grants AGL2012-39937-C02-01 and 02 from the Spanish Government and Generalitat Valenciana to A. Querol and E. Barrio, respectively, and to Project PROMETEOII/2014/042 to A. Querol. ; Peer reviewed

Yeast mannoproteins contribute to several aspects of wine quality by protecting wine against protein haze, reducing astringency, retaining aroma compounds and stimulating lactic-acid bacteria growth. The selection of a yeast strain that simultaneously overproduces mannoproteins and presents good fermentative characteristics is a difficult task. In this work, a Saccharomyces cerevisiae×. S. cerevisiae hybrid bearing the two oenologically relevant features was constructed. According to the genomic characterisation of the hybrids, different copy numbers of some genes probably related with these physiological features were detected. The hybrid shared not only a similar copy number of genes SPR1, SWP1, MNN10 and YPS7 related to cell wall integrity with parental Sc1, but also a similar copy number of some glycolytic genes with parental Sc2, such as GPM1 and HXK1, as well as the genes involved in hexose transport, such as HXT9, HXT11 and HXT12. This work demonstrates that hybridisation and stabilisation under winemaking conditions constitute an effective approach to obtain yeast strains with desirable physiological features, like mannoprotein overproducing capacity and improved fermentation performance, which genetically depend of the expression of numerous genes (multigenic characters). ; This work has been supported by grants AGL2012-39937-CO2 (01 and 02), and AGL2009-07327 from the Spanish Government and FEDER to A. Querol, E. Barrio and R. Gonzalez, respectively and to PROMETEO grant (Project PROMETEOII/2014/042) from the Generalitat Valenciana to A.Q. L. P-T. and C. L. and wish to acknowledge the CSIC and the Spanish Ministry of Education and Science (MEC) for an I3P fellowship and a postdoctoral contract, respectively. ; Peer Reviewed

Fermentation performance at low temperature is a common approach to obtain wines with better aroma, and is critical in industrial applications. Natural hybrids S. cerevisiae × S. kudriavzevii, isolated from fermentations in cold-climate European countries, have provided an understanding of the mechanisms of adaptation to grow at low temperature. In this work, we studied the performance of 23 S. cerevisiae × S. kudriavzevii hybrids at low temperature (8, 12 and 24 °C) to characterize their phenotypes. Kinetic parameters and spot tests revealed a different ability to grow at low temperature. Interestingly, the genome content of the S. kudriavzevii in hybrids was moderately correlated with a shorter lag phase, and the genetic origin of hybrids influenced their performance at low temperature (8 °C). The parental expression of cold marker genes (NSR1, GUT2 and GPD1) showed that the relative expression of the S. kudriavzevii alleles was higher than the expression of the S. cerevisiae alleles in hybrids with a better growth at low-temperatures. These results suggest that the genomic contribution of S. kudriavzevii to hybrids is important for improving the fitness of these strains at low temperature. ; G. Ortiz-Tovar was supported by CONACYT doctoral scholarship 176060. This work was supported by grants AGL2015-67504-C3-1-R from the Spanish Government and FEDER; grant and PCIN-2015-143 to the European Project ERA-IB "YeastTempTation" and Spanish Government through MINECO and FEDER funds to AQ, grant AGL2015-67504-C3-3-R from the Spanish Government and FEDER to EB. ; Peer reviewed

Companies based on alcoholic fermentation products, such as wine, beer and biofuels, use yeasts to make their products. Each industrial process utilizes different media conditions, which differ in sugar content, the presence of inhibitors and fermentation temperature. Saccharomyces cerevisiae has traditionally been the main yeast responsible for most fermentation processes. However, the market is changing due to consumer demand and external factors such as climate change. Some processes, such as biofuel production or winemaking, require new yeasts to solve specific challenges, especially those associated with sustainability, novel flavours and altered alcohol content. One of the proposed solutions is the application of yeast hybrids. The lager beer market has been dominated by S. cerevisiae × S. eubayanus hybrids. However, several less thoroughly studied hybrids have been isolated from other diverse industrial processes. Here we focus on S. cerevisiae × S. kudriavzevii hybrids, which have been isolated from diverse industrial conditions that include wine, ale beer, cider and dietary supplements. Emerging data suggest an extended and complex story of adaptation of these hybrids to traditional industrial conditions. S. cerevisiae × S. kudriavzevii hybrids are also being explored for new industrial applications, such as biofuels. This review describes the past, present and future of S. cerevisiae × S. kudriavzevii hybrids. ; DP is a Marie Sklodowska-Curie fellow of the European Union's Horizon 2020 research and innovation programme, grant agreement No. 747775. DP and CTH: this material is based upon work supported by the National Science Foundation under Grant No. DEB-1253634, the Robert Draper Technology Innovation Fund from the Wisconsin Alumni Research Foundation, and funded in part by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494). CTH is supported by the USDA National Institute of Food and Agriculture (Hatch Project 1003258). CTH is a Pew Scholar in the Biomedical Sciences, supported by the Pew Charitable Trusts. AQ and EB acknowledge Generalitat Valenciana grant PROMETEOII/2014/042 and Spanish Government (MINECO) and European Union FEDER grants AGL2015-67504-C3-01R (to AQ) and AGL2015-67504-C3-03-R (to EB). RPT is supported by the aforementioned grants associated to AQ. ; Peer reviewed