Genome-wide effect of non-optimal temperatures under anaerobic conditions on gene expression in Saccharomyces cerevisiae.

Understanding of thermal adaptation mechanisms in yeast is crucial to develop better-adapted strains to industrial processes, providing more economical and sustainable products. We have analyzed the transcriptomic responses of three Saccharomyces cerevisiae strains, a commercial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and a commercial bioethanol strain, Ethanol Red, grown at non-optimal temperatures under anaerobic chemostat conditions. Transcriptomic analysis of the three strains revealed a huge complexity of cellular mechanisms and responses. Overall, cold exerted a stronger transcriptional response in the three strains comparing with heat conditions, with a higher number of down-regulating genes than of up-regulating genes regardless the strain analyzed. The comparison of the transcriptome at both sub- and supra-optimal temperatures showed the presence of common genes up- or down-regulated in both conditions, but also the presence of common genes up- or down-regulated in the three studied strains. More specifically, we have identified and validated three up-regulated genes at sub-optimal temperature in the three strains, OPI3, EFM6 and YOL014W. Finally, the comparison of the transcriptomic data with a previous proteomic study with the same strains revealed a good correlation between gene activity and protein abundance, mainly at low temperature. Our work provides a global insight into the specific mechanisms involved in temperature adaptation regarding both transcriptome and proteome, which can be a step forward in the comprehension and improvement of yeast thermotolerance.

The potential role of yeasts in the mitigation of health issues related to beer consumption.

Food consumption of healthier products has become an essential trend in the food sector. This is also the case in beer, a biochemical process of transformation performed by yeast cells. More and more studies proclaim the need to reduce ethanol content in alcoholic drinks, certainly the most important health issue of beer consumption. In this review we gather key health issues related to beer consumption and the last advances regarding the use of yeast to attenuate those health problems. Furthermore, we have included the latest findings about the general positive impact of yeast in health as a consequence of its ability to biotransform polyphenolic compounds present in the wort, producing healthy compounds as hydroxytyrosol or melatonin, and its ability to perform as a probiotic driver. Besides, a group of population with chronic diseases as diabetes or celiac disease could take advantage of low carbohydrate or gluten-free beers, respectively. The role of yeast in beer production has been traditionally associated to its fermentative power. But here we have found a change in this dogma in the last years toward yeasts being a main driver to enhance healthy aspects of beer. The key findings are discussed and possible future directions are proposed.

Modulation of aroma and chemical composition of Albariño semi-synthetic wines by non-wine Saccharomyces yeasts and bottle aging.

Saccharomyces yeasts from different origins and species fermented in a semi-synthetic must containing aroma precursor of cv. Albariño and polyfunctional mercaptans precursors. The resulting wines were subjected to accelerate anoxic aging. Afterward, aroma profiles were analyzed by distinct gas chromatography methodologies. Cryotolerant strains showed better fermentation performances with significant differences in volatile and non-volatile fermentation products than Saccharomyces cerevisiae (S. cerevisiae). We suggested that the highest levels γ-butyrolactone and diethyl succinate in Saccharomyces uvarum (S. uvarum) strains, together with their substantial succinic acid yields, could be related to greater flux through the GABA shunt. These strains also had the highest production of ß-phenylethyl acetate, geraniol, and branched-chain ethyl esters. The latter compounds were highly increased by aging, while acetates and some terpenes decreased. S. kudriavzevii strains showed a remarkable ability to release polyfunctional mercaptans, with SK1 strain yielding up to 47-fold and 8-fold more 4-methyl-4-mercaptopentan-2-one (4MMP) than S. cerevisiae and S. uvarum strains, respectively. The wild S. cerevisiae beer isolate showed a particular aroma profile due to the highest production of ethyl 4-methylvalerate (lactic and fruity notes), γ-octalactone (coconut), and furfurylthiol (roasted coffee). The latter compound is possibly produced from the pentose phosphate pathway (PPP). Since erythritol, another PPP intermediate was largely produced by this strain.

Screening of Saccharomyces strains for the capacity to produce desirable fermentative compounds under the influence of different nitrogen sources in synthetic wine fermentations.

A collection of 33 Saccharomyces yeasts were used for wine fermentation with a sole nitrogen source: ammonium and four individual aroma-inducing amino acids. The fermentation performance and chemical wine composition were evaluated. The most valuable nitrogen sources were valine as a fermentation promoter on non-cerevisiae strains, phenylalanine as fruity aromas enhancer whereas the ethanol yield was lessened by leucine and isoleucine. S. cerevisiae SC03 and S. kudriavzevii SK02 strains showed to be the greatest producers of fruity ethyl esters while S. kudriavzevii strains SK06 and SK07 by shortening the fermentation duration. S. uvarum strains produced the greatest succinic acid amounts and, together with S. eubayanus, they reached the highest production of 2-phenylethanol and its acetate ester; whereas S. kudriavzevii strains were found to be positively related to high glycerol production.

Phenotypic and genomic differences among S. cerevisiae strains in nitrogen requirements during wine fermentations.

Nitrogen requirements by S. cerevisiae during wine fermentation are highly strain-dependent. Different approaches were applied to explore the nitrogen requirements of 28 wine yeast strains. Based on the growth and fermentation behaviour displayed at different nitrogen concentrations, high and low nitrogen-demanding strains were selected and further verified by competition fermentation. Biomass production with increasing nitrogen concentrations in the exponential fermentation phase was analysed by chemostat cultures. Low nitrogen-demanding (LND) strains produced a larger amount of biomass in nitrogen-limited synthetic grape musts, whereas high nitrogen-demanding (HND) strains achieved a bigger biomass yield when the YAN concentration was above 100 mg/L. Constant rate fermentation was carried out with both strains to determine the amount of nitrogen required to maintain the highest fermentation rate. Large differences appeared in the analysis of the genomes of low and high-nitrogen demanding strains showed for heterozygosity and the amino acid substitutions between orthologous proteins, with nitrogen recycling system genes showing the widest amino acid divergences. The CRISPR/Cas9-mediated genome modification method was used to validate the involvement of GCN1 in the yeast strain nitrogen needs. However, the allele swapping of gene GCN1 from low nitrogen-demanding strains to high nitrogen-demanding strains did not significantly influence the fermentation rate.

Nitrogen sources preferences of non-Saccharomyces yeasts to sustain growth and fermentation under winemaking conditions.

Wine-related non-Saccharomyces yeasts are becoming more widely used in oenological practice for their ability to confer wine a more complex satisfying aroma, but their metabolism remains unknown. Our study explored the nitrogen utilisation profile of three popular non-Saccharomyces species, Torulaspora delbrueckii, Metschnikowia pulcherrima and Metschnikowia fructicola. The nitrogen source preferences to support growth and fermentation as well as the uptake order of different nitrogen sources during wine fermentation were investigated. While T. delbrueckii and S. cerevisiae strains shared the same nitrogen source preferences, Metschnikowia sp. Displayed a lower capacity to efficiently use the preferred nitrogen compounds, but were able to assimilate a wider range of amino acids. During alcoholic fermentation, the non-Saccharomyces strains consumed different nitrogen sources in a similar order as S. cerevisiae, but not as quickly. Furthermore, when all the nitrogen sources were supplied in the same amount, their assimilation order was similarly affected for both S. cerevisiae and non-Saccharomyces strains. Under this condition, the rate of nitrogen source consumption of non-Saccharomyces strains and S. cerevisiae was comparable. Overall, this study expands our understanding about the preferences and consumption rates of individual nitrogen sources by the investigated non-Saccharomyces yeasts in a wine environment. This knowledge provides useful information for a more efficient exploitation of non-Saccharomyces strains that improves the management of the wine fermentation.

Mechanisms of Yeast Adaptation to Wine Fermentations.

Cells face genetic and/or environmental changes in order to outlast and proliferate. Characterization of changes after stress at different "omics" levels is crucial to understand the adaptation of yeast to changing conditions. Wine fermentation is a stressful situation which yeast cells have to cope with. Genome-wide analyses extend our cellular physiology knowledge by pointing out the mechanisms that contribute to sense the stress caused by these perturbations (temperature, ethanol, sulfites, nitrogen, etc.) and related signaling pathways. The model organism, Saccharomyces cerevisiae, was studied in response to industrial stresses and changes at different cellular levels (transcriptomic, proteomic, and metabolomics), which were followed statically and/or dynamically in the short and long terms. This chapter focuses on the response of yeast cells to the diverse stress situations that occur during wine fermentations, which induce perturbations, including nutritional changes, ethanol stress, temperature stress, oxidative stress, etc.

Replenishment and mobilization of intracellular nitrogen pools decouples wine yeast nitrogen uptake from growth.

Wine yeast capacity to take up nitrogen from the environment and catabolize it to support population growth, fermentation, and aroma production is critical to wine production. Under nitrogen restriction, yeast nitrogen uptake is believed to be intimately coupled to reproduction with nitrogen catabolite repression (NCR) suggested mediating this link. We provide a time- and strain-resolved view of nitrogen uptake, population growth, and NCR activity in wine yeasts. Nitrogen uptake was found to be decoupled from growth due to early assimilated nitrogen being used to replenish intracellular nitrogen pools rather than being channeled directly into reproduction. Internally accumulated nitrogen was later mobilized to support substantial population expansion after external nitrogen was depleted. On good nitrogen sources, the decoupling between nitrogen uptake and growth correlated well with relaxation of NCR repression, raising the potential that the latter may be triggered by intracellular build-up of nitrogen. No link between NCR activity and nitrogen assimilation or growth on poor nitrogen sources was found. The decoupling between nitrogen uptake and growth and its influence on NCR activity is of relevance for both wine production and our general understanding of nitrogen use.

Evolutionary engineering of a wine yeast strain revealed a key role of inositol and mannoprotein metabolism during low-temperature fermentation.

BACKGROUND: Wine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes. RESULTS: We used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 °C. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1 (Thr108)) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins. CONCLUSIONS: In this study we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms.

Global phenotypic and genomic comparison of two Saccharomyces cerevisiae wine strains reveals a novel role of the sulfur assimilation pathway in adaptation at low temperature fermentations.

BACKGROUND: The wine industry needs better-adapted yeasts to grow at low temperature because it is interested in fermenting at low temperature to improve wine aroma. Elucidating the response to cold in Saccharomyces cerevisiae is of paramount importance for the selection or genetic improvement of wine strains. RESULTS: We followed a global approach by comparing transcriptomic, proteomic and genomic changes in two commercial wine strains, which showed clear differences in their growth and fermentation capacity at low temperature. These strains were selected according to the maximum growth rate in a synthetic grape must during miniaturized batch cultures at different temperatures. The fitness differences of the selected strains were corroborated by directly competing during fermentations at optimum and low temperatures. The up-regulation of the genes of the sulfur assimilation pathway and glutathione biosynthesis suggested a crucial role in better performance at low temperature. The presence of some metabolites of these pathways, such as S-Adenosilmethionine (SAM) and glutathione, counteracted the differences in growth rate at low temperature in both strains. Generally, the proteomic and genomic changes observed in both strains also supported the importance of these metabolic pathways in adaptation at low temperature. CONCLUSIONS: This work reveals a novel role of the sulfur assimilation pathway in adaptation at low temperature. We propose that a greater activation of this metabolic route enhances the synthesis of key metabolites, such as glutathione, whose protective effects can contribute to improve the fermentation process.

Transcriptomics of cryophilic Saccharomyces kudriavzevii reveals the key role of gene translation efficiency in cold stress adaptations.

BACKGROUND: Comparative transcriptomics and functional studies of different Saccharomyces species have opened up the possibility of studying and understanding new yeast abilities. This is the case of yeast adaptation to stress, in particular the cold stress response, which is especially relevant for the food industry. Since the species Saccharomyces kudriavzevii is adapted to grow at low temperatures, it has been suggested that it contains physiological adaptations that allow it to rapidly and efficiently acclimatise after cold shock. RESULTS: In this work, we aimed to provide new insights into the molecular basis determining this better cold adaptation of S. kudriavzevii strains. To this end, we have compared S. cerevisiae and S. kudriavzevii transcriptome after yeast adapted to cold shock. The results showed that both yeast mainly activated the genes related to translation machinery by comparing 12°C with 28°C, but the S. kudriavzevii response was stronger, showing an increased expression of dozens of genes involved in protein synthesis. This suggested enhanced translation efficiency at low temperatures, which was confirmed when we observed increased resistance to translation inhibitor paromomycin. Finally, 35S-methionine incorporation assays confirmed the increased S. kudriavzevii translation rate after cold shock. CONCLUSIONS: This work confirms that S. kudriavzevii is able to grow at low temperatures, an interesting ability for different industrial applications. We propose that this adaptation is based on its enhanced ability to initiate a quick, efficient translation of crucial genes in cold adaptation among others, a mechanism that has been suggested for other microorganisms.

The fitness advantage of commercial wine yeasts in relation to the nitrogen concentration, temperature, and ethanol content under microvinification conditions.

The effect of the main environmental factors governing wine fermentation on the fitness of industrial yeast strains has barely received attention. In this study, we used the concept of fitness advantage to measure how increasing nitrogen concentrations (0 to 200 mg N/liter), ethanol (0 to 20%), and temperature (4 to 45°C) affects competition among four commercial wine yeast strains (PDM, ARM, RVA, and TTA). We used a mathematical approach to model the hypothetical time needed for the control strain (PDM) to out-compete the other three strains in a theoretical mixed population. The theoretical values obtained were subsequently verified by competitive mixed fermentations in both synthetic and natural musts, which showed a good fit between the theoretical and experimental data. Specifically, the data show that the increase in nitrogen concentration and temperature values improved the fitness advantage of the PDM strain, whereas the presence of ethanol significantly reduced its competitiveness. However, the RVA strain proved to be the most competitive yeast for the three enological parameters assayed. The study of the fitness of these industrial strains is of paramount interest for the wine industry, which uses them as starters of their fermentations. Here, we propose a very simple method to model the fitness advantage, which allows the prediction of the competitiveness of one strain with respect to different abiotic factors.

Biomarkers for detecting nitrogen deficiency during alcoholic fermentation in different commercial wine yeast strains.

Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. Several putative biomarkers were tested in order to analyze their appropriateness to detect nitrogen stress in the yeast. To this aim, four commercial wine strains (PDM, ARM, RVA and TTA) were grown in a synthetic grape must with different nitrogen concentrations. Trehalose accumulation, arginase activity and the expression of eleven genes were tested in these wine strains, known to have different nitrogen requirements. The overall response of the four strains was similar, with differences in response intensity (PDM and RVA with higher intensity) and response time (which was also related with nitrogen consumption time). Trehalose response was mostly related to entry into the stationary phase, whereas arginase activity was responsive to nitrogen depletion, although its measurement is too complicated to be used for routine monitoring during winemaking. The expression of the genes DAL4, DAL5, DUR3 and GAP1 was clearly related to nitrogen depletion and thus, GAP1 and DAL4 were selected as markers of nitrogen deficiency. In order to adapt expression analysis to winemaking conditions, the original strains were transformed into reporter strains based on the expression of green fluorescent protein (GFP) under control of the promoters for GAP1 and DAL4. The transformants had a similar fermentative capacity to the parental strains and were able to detect alterations in yeast physiological status due to nitrogen limitations.

The Role of the PAA1 Gene on Melatonin Biosynthesis in Saccharomyces cerevisiae: A Search of New Arylalkylamine N-Acetyltransferases.

Recently, the presence of melatonin in fermented beverages has been correlated with yeast metabolism during alcoholic fermentation. Melatonin, originally considered a unique product of the pineal gland of vertebrates, has been also identified in a wide range of invertebrates, plants, bacteria, and fungi in the last two decades. These findings bring the challenge of studying the function of melatonin in yeasts and the mechanisms underlying its synthesis. However, the necessary information to improve the selection and production of this interesting molecule in fermented beverages is to disclose the genes involved in the metabolic pathway. So far, only one gene has been proposed as involved in melatonin production in Saccharomyces cerevisiae, PAA1, a polyamine acetyltransferase, a homolog of the vertebrate's aralkylamine N-acetyltransferase (AANAT). In this study, we assessed the in vivo function of PAA1 by evaluating the bioconversion of the different possible substrates, such as 5-methoxytryptamine, tryptamine, and serotonin, using different protein expression platforms. Moreover, we expanded the search for new N-acetyltransferase candidates by combining a global transcriptome analysis and the use of powerful bioinformatic tools to predict similar domains to AANAT in S. cerevisiae. The AANAT activity of the candidate genes was validated by their overexpression in E. coli because, curiously, this system evidenced higher differences than the overexpression in their own host S. cerevisiae. Our results confirm that PAA1 possesses the ability to acetylate different aralkylamines, but AANAT activity does not seem to be the main acetylation activity. Moreover, we also prove that Paa1p is not the only enzyme with this AANAT activity. Our search of new genes detected HPA2 as a new arylalkylamine N-acetyltransferase in S. cerevisiae. This is the first report that clearly proves the involvement of this enzyme in AANAT activity.

Effect of low temperature upon vitality of Saccharomyces cerevisiae phospholipid mutants.

The phospholipid metabolism of Saccharomyces cerevisiae plays a central role in its adaptation to low temperatures. In order to detect the key genes in this adaptation, various phospholipid mutants from the EUROSCARF collection of Saccharomyces cerevisiae BY4742 were tested to ascertain whether the suppression of some genes could improve the fermentation vitality of the cells at low temperature. The cell vitality and phospholipid composition of these mutants were analysed. Some knockouts improved (hmn1Δ) or impaired (cho2Δ and psd1Δ) their vitality at low temperature (13 ° C) but were not affected at optimum temperature (25 ° C). A common trait of the mutants that had some defect in vitality was a lower concentration of phosphatidylcholine and/or phosphatidylethanolamine. The supplementation with choline allowed them to recover viability, probably by synthesis through the Kennedy pathway. Hmn1Δ showed a lower concentration of phosphatidylcholine, which explains the dominant role of the de novo pathway in cellular phosphatidylethanolamine and phosphatidylcholine vs the Kennedy pathway. The absence of such genes as CRD1 or OPI3 produced important changes in phospholipid composition. Cardiolipin was not detected in crd1Δ but phosphatidylglycerol circumvents most of the functions assigned to CL. The considerable reduction in PC diminished the cell vitality of opi3Δ at both temperatures, although the decrease at 13 ° C was more marked.

Analysis of low temperature-induced genes (LTIG) in wine yeast during alcoholic fermentation.

Fermentations carried out at low temperatures, that is, 10-15 °C, not only enhance the production and retention of flavor volatiles, but also increase the chances of slowing or arresting the process. In this study, we determined the transcriptional activity of 10 genes that were previously reported as induced by low temperatures and involved in cold adaptation, during fermentation with the commercial wine yeast strain QA23. Mutant and overexpressing strains of these genes were constructed in a haploid derivative of this strain to determine the importance of these genes in growth and fermentation at low temperature. In general, the deletion and overexpression of these genes did affect fermentation performance at low temperature. Most of the mutants were unable to complete fermentation, while overexpression of CSF1, HSP104, and TIR2 decreased the lag phase, increased the fermentation rate, and reached higher populations than that of the control strain. Another set of overexpressing strains were constructed by integrating copies of these genes in the delta regions of the commercial wine strain QA23. These new stable overexpressing strains again showed improved fermentation performance at low temperature, especially during the lag and exponential phases. Our results demonstrate the convenience of carrying out functional analysis in commercial strains and in an experimental set-up close to industrial conditions.

Nitrogen requirements of commercial wine yeast strains during fermentation of a synthetic grape must.

Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. Currently, the most common method for dealing with nitrogen-deficient fermentations is adding supplementary nitrogen (usually ammonium phosphate). However, it is important to know the specific nitrogen requirement of each strain, to avoid excessive addition that can lead to microbial instability and ethyl carbamate accumulation. In this study, we aimed to determine the effect of increasing nitrogen concentrations of three different nitrogen sources on growth and fermentation performance in four industrial wine yeast strains. This task was carried out using statistical modeling techniques. The strains PDM and RVA showed higher growth-rate and maximum population size and consumed nitrogen much more quickly than strains ARM and TTA. Likewise, the strains PDM and RVA were also the greatest nitrogen demanders. Thus, we can conclude that these differences in nitrogen demand positively correlated with higher growth rate and higher nitrogen uptake rate. The most direct effect of employing an adequate nitrogen concentration is the increase in biomass, which involves a higher fermentation rate. However, the impact of nitrogen on fermentation rate is not exclusively due to the increase in biomass because the strain TTA, which showed the worst growth behavior, had the best fermentation activity. Some strains may adapt a strategy whereby fewer cells with higher metabolic activity are produced. Regarding the nitrogen source used, all the strains showed the better and worse fermentation performance with arginine and ammonium, respectively.

Genomic Adaptations of Saccharomyces Genus to Wine Niche.

Wine yeast have been exposed to harsh conditions for millennia, which have led to adaptive evolutionary strategies. Thus, wine yeasts from Saccharomyces genus are considered an interesting and highly valuable model to study human-drive domestication processes. The rise of whole-genome sequencing technologies together with new long reads platforms has provided new understanding about the population structure and the evolution of wine yeasts. Population genomics studies have indicated domestication fingerprints in wine yeast, including nucleotide variations, chromosomal rearrangements, horizontal gene transfer or hybridization, among others. These genetic changes contribute to genetically and phenotypically distinct strains. This review will summarize and discuss recent research on evolutionary trajectories of wine yeasts, highlighting the domestication hallmarks identified in this group of yeast.

Metabolic engineering of Saccharomyces cerevisiae for hydroxytyrosol overproduction directly from glucose.

Hydroxytyrosol (HT) is one of the most powerful dietary antioxidants with numerous applications in different areas, including cosmetics, nutraceuticals and food. In the present work, heterologous hydroxylase complex HpaBC from Escherichia coli was integrated into the Saccharomyces cerevisiae genome in multiple copies. HT productivity was increased by redirecting the metabolic flux towards tyrosol synthesis to avoid exogenous tyrosol or tyrosine supplementation. After evaluating the potential of our selected strain as an HT producer from glucose, we adjusted the medium composition for HT production. The combination of the selected modifications in our engineered strain, combined with culture conditions optimization, resulted in a titre of approximately 375 mg l-1 of HT obtained from shake-flask fermentation using a minimal synthetic-defined medium with 160 g l-1 glucose as the sole carbon source. To the best of our knowledge, this is the highest HT concentration produced by an engineered S. cerevisiae strain.

Effect of non-wine Saccharomyces yeasts and bottle aging on the release and generation of aromas in semi-synthetic Tempranillo wines.

Interest in the use of non-conventional yeasts in wine fermentation has been increased in the last years in the wine sector. The main objective of this manuscript was to explore the aromatic diversity produced by wild and non-wine strains of S. cerevisiae, S. eubayanus, S. kudriavzevii, and S. uvarum species in young and bottle-aged Tempranillo wines as well as evaluate their fermentation capacity and the yield on ethanol, glycerol, and organic acids, that can contribute to diminishing the effects of climate change on wines. S. uvarum strain U1 showed the highest ability to release or de novo produce monoterpenes, such as geraniol and citronellol, whose values were 1.5 and 3.5-fold higher than those of the wine S. cerevisiae strain. We found that compared to the normal values for red wines, ß-phenylethyl acetate was highly synthesized by U1 and E1 strains, achieving 1 mg/L. Additionally, after aging, wines of S. eubayanus strains contained the highest levels of this acetate. Malic acid was highly degraded by S. kudriavzevii yeasts, resulting in the highest yields of lactic acid (>5-fold) and ethyl lactate (>2.8-fold) in their wines. In aged wines, we observed that the modulating effects of yeast strain were very high in ß-ionone. S. uvarum strains U1 and BMV58 produced an important aging attribute, ethyl isobutyrate, which was highly enhanced during the aging. Also, the agave S. cerevisiae strain develops an essential aroma after aging, reaching the highest ethyl leucate contents. According to the results obtained, the use of wild non-wine strains of S. cerevisiae and strains of the cryotolerant species S. eubayanus, S. kudriavzevii, and S. uvarum in Tempranillo wine fermentation increase the aroma complexity. In addition, wines from S. kudriavzevii strains had twice additional glycerol, those from S. uvarum 4-fold more succinic acid, while wines from wild strains yielded 1% v/v less ethanol which may solve wine problems associated with climate change.