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[Background]: Saccharomyces cerevisiae is generally considered safe, and is involved in the production of many types of foods and dietary supplements. However, some isolates, which are genetically related to strains used in brewing and baking, have shown virulent traits, being able to produce infections in humans, mainly in immunodeficient patients. This can lead to systemic infections in humans. [Methods]: In this work, we studied S. cerevisiae isolates in an in vitro human endothelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans. [Results]: The results showed that this food related yeast is able to cross the endothelial barrier in vitro. However, in contrast to C. glabrata and C. albicans, S. cerevisiae showed very low levels of traversal. [Conclusions]: We conclude that using an in vitro human endothelial barrier model with S. cerevisiae can be useful to evaluate the safety of S. cerevisiae strains isolated from foods. ; This work was supported by grants AGL2012-39937-C02-01 and AGL2015-67504-C3-1-R from the Spanish Government and ERDF (European Regional Development Fund) and by grant PROMETEO (project PROMETEOII/2014/042) from Generalitat Valenciana to AQ. ; We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI). ; Peer reviewed

[Background] Aroma is one of the most important attributes defining wine quality in which yeasts play a crucial role, synthesizing aromatic compounds or releasing odourless conjugates. A present-day trend in winemaking consists of lowering fermentation temperature to achieve higher aroma production and retention. S. cerevisiae × S. kudriavzevii hybrids seem to have inherited beneficial traits from their parental species, like fermenting efficiently at low temperature or producing higher amounts of certain aromatic compounds. In this study, allelic composition and gene expression of the genes related to aroma synthesis in two genetically and phenotypically different S. cerevisiae × S. kudriavzevii hybrids, Lalvin W27 and VIN7, were compared and related to aroma production in microvinifications at 12 and 28 °C. In addition, the contribution of the allele coming from each parental to the overall expression was explored by RT-PCR. ; [Results] The results indicated large differences in allele composition, gene expression and the contribution of each parental to the overall expression at the fermentation temperatures tested. Results obtained by RT-PCR showed that in ARO1 and ATF2 genes the S. kudriavzevii allele was more expressed than that of S. cerevisiae particularly at 12 °C. ; [Conclusions] This study revealed high differences regarding allele composition and gene expression in two S. cerevisiae × S. kudriavzevii hybrids, which may have led to different aroma profiles in winemaking conditions. The contribution of the alleles coming from each parental to the overall expression has proved to differently influence aroma synthesis. Besides, the quantitative contribution to the overall gene expression of the alleles coming from one parental strain or the other was clearly determined by the fermentation temperature for some genes. ; This work has been supported by the projects AGL2012-39937-C02-01 from the Spanish Government and FEDER to A. Querol. AG acknowledge to her PhD contract from I3P program. ; 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

Semi-synthetic must containing standard nutrients, a phenolic and aromatic fraction extracted from Albariño grapes and synthetic precursors of 3-mercaptohexanol (MOH) and 4-mercapto-4-pentan-2-one (MMP) were fermented with three different selected commercial S. cerevisiae yeasts. Wines were subjected to anoxic aging at 50 °C for 1, 2, 5 and 8 weeks, and their volatile composition was comprehensively determined by the analysis of 86 different aroma compounds using five different GC methods. Yeasts exert a strong influence on wine aroma throughout the whole aging period. Their effects extend beyond the well-known actions on yeast secondary metabolites, including the formation of little amounts of Strecker aldehydes or the formation of acids precursors of fruity esters, and on the enzymatic actions on the different grape aroma precursors. Additionally, yeasts influence wine aroma; first, by producing SO2 which reacts with β-damascenone and increases Strecker aldehyde production in fermentation; second, by inducing the differential accumulation of Strecker aldehydes during anoxic aging; and third, by determining the stability of MOH, likely because of its reaction with vinylphenols or other yeast-related electrophiles. ; The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement number 764364. The authors declare that no conflict of interest exists. ; Peer reviewed

The toxic effect of ethanol is one of the most important handicaps for many biotechnological applications of yeasts, such as bioethanol production. Elucidation of ethanol stress response will help to improve yeast performance in biotechnological processes. In the yeast Saccharomyces cerevisiae, ethanol stress has been recently described as an activator of the unfolded protein response (UPR), a conserved intracellular signalling pathway that regulates the transcription of ER homoeostasis-related genes. However, the signal and activation mechanism has not yet been unravelled. Here, we studied UPR's activation after ethanol stress and observed the upregulation of the key target genes, like INO1, involved in lipid metabolism. We found that inositol content influenced UPR activation after ethanol stress and we observed significant changes in lipid composition, which correlate with a major membrane fluidity alteration by this amphipathic molecule. Then, we explored the hypothesis that membrane fluidity changes cause UPR activation upon ethanol stress by studying UPR response against fluidification or rigidification agents and by studying a mutant, erg2, with altered membrane fluidity. The results suggest that the membrane fluidification effects of ethanol and other agents are the signal for UPR activation, a mechanism that has been proposed in higher eukaryotes. ; E. Navarro‐Tapia was supported by an FPU grant from the Ministerio de Educación y Ciencia (ref. AP2009‐0787). This work was supported by grants AGL2012‐39937‐C02‐01 and AGL2015‐67504‐C3‐1‐R from the Spanish Government and FEDER and by grant PROMETEO (Project PROMETEO/2009/019) from the Generalitat Valenciana to A. Querol. ; Peer Reviewed

The unfolded protein response (UPR) is a conserved intracellular signaling pathway that controls transcription of endoplasmic reticulum (ER) homeostasis related genes. Ethanol stress has been recently described as an activator of the UPR response in yeast Saccharomyces cerevisiae, but very little is known about the causes of this activation. Although some authors ensure that the UPR is triggered by the unfolded proteins generated by ethanol in the cell, there are studies which demonstrate that protein denaturation occurs at higher ethanol concentrations than those used to trigger the UPR. Here, we studied UPR after ethanol stress by three different approaches and we concluded that unfolded proteins do not accumulate in the ER under. We also ruled out inositol depletion as an alternative mechanism to activate the UPR under ethanol stress discarding that ethanol effects on the cell decreased inositol levels by different methods. All these data suggest that ethanol, at relatively low concentrations, does not cause unfolded proteins in the yeasts and UPR activation is likely due to other unknown mechanism related with a restructuring of ER membrane due to the effect of ethanol. ; EN was supported by a FPU grant from the Ministerio de Educación y Ciencia (ref. AP2009-0787). This work was supported by grants AGL2012-39937-C02-01 and AGL2015-67504-C3-1-R from the Spanish Government and FEDER and by grant PROMETEO (project PROMETEOII/2014/042) from Generalitat Valenciana to AQ. ; Peer reviewed ; Peer Reviewed

The unfolded protein response (UPR) is a conserved intracellular signaling pathway that controls transcription of endoplasmic reticulum (ER) homeostasis related genes. Ethanol stress has been recently described as an activator of the UPR response in yeast Saccharomyces cerevisiae, but very little is known about the causes of this activation. Although some authors ensure that the UPR is triggered by the unfolded proteins generated by ethanol in the cell, there are studies which demonstrate that protein denaturation occurs at higher ethanol concentrations than those used to trigger the UPR. Here, we studied UPR after ethanol stress by three different approaches and we concluded that unfolded proteins do not accumulate in the ER under. We also ruled out inositol depletion as an alternative mechanism to activate the UPR under ethanol stress discarding that ethanol effects on the cell decreased inositol levels by different methods. All these data suggest that ethanol, at relatively low concentrations, does not cause unfolded proteins in the yeasts and UPR activation is likely due to other unknown mechanism related with a restructuring of ER membrane due to the effect of ethanol. ; EN was supported by a FPU grant from the Ministerio de Educación y Ciencia (ref. AP2009-0787). This work was supported by grants AGL2012-39937-C02-01 and AGL2015-67504-C3-1-R from the Spanish Government and FEDER and by grant PROMETEO (project PROMETEOII/2014/042) from Generalitat Valenciana to AQ. ; Peer reviewed

Temperature is one of the most important parameters to affect the duration and rate of alcoholic fermentation and final wine quality. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae, which was the case of cryotolerant yeasts Saccharomyces uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the proteomic landscape of these cryotolerant species grown at 12 °C and 28 °C, which we compared with the proteome of S. cerevisiae (poorly adapted at low temperature). Our results showed that the main differences among the proteomic profiling of the three Saccharomyces strains grown at 12 °C and 28 °C lay in translation, glycolysis and amino acid metabolism. Our data corroborate previous transcriptomic results, which suggest that S. kudriavzevii is better adapted to grow at low temperature as a result of enhanced more efficient translation. Fitter amino acid biosynthetic pathways can also be mechanisms that better explain biomass yield in cryotolerant strains. Yet even at low temperature, S. cerevisiae is the most fermentative competitive species. A higher concentration of glycolytic and alcoholic fermentation enzymes in the S. cerevisiae strain might explain such greater fermentation activity. ; This work has been financially supported by grants PCIN-2015-143, AGL2012-39937-C02-01 from the Spanish government, FEDER and PROMETEOII/2014/042 from the Spanish government and the Generalitat Valenciana, respectively, awarded to JMG and AQ. EGR also thanks the Spanish government for an FPI grant BES-2011-044498. ; Peer reviewed

The Saccharomyces genus is the main yeast involved in wine fermentations to play a crucial role in the production and release of aromatic compounds. Despite the several studies done into the genome-wide expression analysis using DNA microarray technology in wine S. cerevisiae strains, this is the first to investigate other species of the Saccharomyces genus. This research work investigates the expression of the genes involved in flavor compound production in three different Saccharomyces species (S. cerevisiae, S. bayanus var. uvarum and S. kudriavzevii ) under low (12°C) and moderate fermentation temperatures (28°C). The global genes analysis showed that 30% of genes appeared to be differently expressed in the three cryophilic strains if compared to the reference strain (mesophilic S. cerevisiae), suggesting a very close cold adaptation response. Remarkable differences in the gene expression level were observed when comparing the three species, S. cerevisiae, S. bayanus var. uvarum and S. kudriavzevii, which will result in different aroma profiles. Knowledge of these differences in the transcriptome can be a tool to help modulate aroma to create wines with the desired aromatic traits. © 2014 Gamero et al. ; AGL2009-12673-CO2-01, AGL2012-39937-C02-01 and from the Spanish Government and PROMETEO grant (Project PROMETEO/2009/019) from the Generalitat Valenciana to A. Querol. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ; Peer Reviewed

Temperature is one of the most important parameters affecting the length and rate of alcoholic fermentation and final wine quality. Wine produced at low temperature is often considered to have improved sensory qualities. However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase, and sluggish or stuck fermentations. To investigate the effects of temperature on commercial wine yeast, we compared its metabolome growing at 12°C and 28°C in a synthetic must. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae. This is the case of the cryotolerant yeasts Saccharomyces bayanus var. uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the metabolome of these species growing at 12°C, which we compared with the metabolome of S. cerevisiae (not well adapted at low temperature) at the same temperature. Our results show that the main differences between the metabolic profiling of S. cerevisiae growing at 12°C and 28°C were observed in lipid metabolism and redox homeostasis. Moreover, the global metabolic comparison among the three species revealed that the main differences between the two cryotolerant species and S. cerevisiae were in carbohydrate metabolism, mainly fructose metabolism. However, these two species have developed different strategies for cold resistance. S. bayanus var. uvarum presented elevated shikimate pathway activity, while S. kudriavzevii displayed increased NAD+ synthesis. © 2013 López-Malo et al. ; This work has been financially supported by the grants AGL2010-22001-C02-01 and AGL2009-12673-C02-01, from the Spanish government, awarded to JMG and AQ, respectively; PROMETEO/2009/019 and ACOMP/2012/014 from Generalitat Valenciana, awarded to AQ and JMG, respectively. MLM also wishes to thank the Spanish government for her FPI grant. ; 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

© 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

Wine fermentation and innovation have focused mostly on Saccharomyces cerevisiae strains. However, recent studies have shown that other Saccharomyces species can also be involved in wine fermentation or are useful for wine bouquet, such as Saccharomyces uvarum and Saccharomyces paradoxus. Many interspecies hybrids have also been isolated from wine fermentation, such as S. cerevisiae × Saccharomyces kudriavzevii hybrids. In this study, we explored the genetic diversity and fermentation performance of Spanish S. kudriavzevii strains, which we compared to other S. kudriavzevii strains. Fermentations of red and white grape musts were performed, and the phenotypic differences between Spanish S. kudriavzevii strains under different temperature conditions were examined. An ANOVA analysis suggested striking similarity between strains for glycerol and ethanol production, although a high diversity of aromatic profiles among fermentations was found. The sources of these phenotypic differences are not well understood and require further investigation. Although the Spanish S. kudriavzevii strains showed desirable properties, particularly must fermentations, the quality of their wines was no better than those produced with a commercial S. cerevisiae. We suggest hybridization or directed evolution as methods to improve and innovate wine. ; D.P. acknowledges the Spanish Government for its Ministry of Science and Innovation (FPI) fellowship. L.P. acknowledges the CSIC and the Spanish Ministry of Education and Science (MEC) for an I3P fellowship. This work has been supported by grants AGL2012-39937-C02-01 from the Spanish Government, FEDER and Generalitat ValencianaPROMETEOII/2014/042 to AQ. ; 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