Convergent adaptation of Saccharomyces uvarum to sulfite, an antimicrobial preservative widely used in human-driven fermentations.

Different species can find convergent solutions to adapt their genome to the same evolutionary constraints, although functional convergence promoted by chromosomal rearrangements in different species has not previously been found. In this work, we discovered that two domesticated yeast species, Saccharomyces cerevisiae, and Saccharomyces uvarum, acquired chromosomal rearrangements to convergently adapt to the presence of sulfite in fermentation environments. We found two new heterologous chromosomal translocations in fermentative strains of S. uvarum at the SSU1 locus, involved in sulfite resistance, an antimicrobial additive widely used in food production. These are convergent events that share similarities with other SSU1 locus chromosomal translocations previously described in domesticated S. cerevisiae strains. In S. uvarum, the newly described VIIXVI and XIXVI chromosomal translocations generate an overexpression of the SSU1 gene and confer increased sulfite resistance. This study highlights the relevance of chromosomal rearrangements to promote the adaptation of yeast to anthropic environments.

A novel aminotransferase gene and its regulator acquired in Saccharomyces by a horizontal gene transfer event.

BACKGROUND: Horizontal gene transfer (HGT) is an evolutionary mechanism of adaptive importance, which has been deeply studied in wine S. cerevisiae strains, where those acquired genes conferred improved traits related to both transport and metabolism of the nutrients present in the grape must. However, little is known about HGT events that occurred in wild Saccharomyces yeasts and how they determine their phenotypes. RESULTS: Through a comparative genomic approach among Saccharomyces species, we detected a subtelomeric segment present in the S. uvarum, S. kudriavzevii, and S. eubayanus species, belonging to the first species to diverge in the Saccharomyces genus, but absent in the other Saccharomyces species. The segment contains three genes, two of which were characterized, named DGD1 and DGD2. DGD1 encodes dialkylglicine decarboxylase, whose specific substrate is the non-proteinogenic amino acid 2-aminoisobutyric acid (AIB), a rare amino acid present in some antimicrobial peptides of fungal origin. DGD2 encodes putative zinc finger transcription factor, which is essential to induce the AIB-dependent expression of DGD1. Phylogenetic analysis showed that DGD1 and DGD2 are closely related to two adjacent genes present in Zygosaccharomyces. CONCLUSIONS: The presented results show evidence of an early HGT event conferring new traits to the ancestor of the Saccharomyces genus that could be lost in the evolutionary more recent Saccharomyces species, perhaps due to loss of function during the colonization of new habitats.

Saccharomyces cerevisiae wine strains show a wide range of competitive abilities and differential nutrient uptake behavior in co-culture with S. kudriavzevii.

The wine industry has implemented complex starters with multiple yeast species as an efficient method to improve certain wine properties. Strains' competitive fitness becomes essential for its use in such cases. In the present work, we studied this trait in 60 S. cerevisiae strains from different origins, co-inoculated with a S. kudriavzevii strain, and confirmed it to be associated with the strains' origin. To gather deeper knowledge about the characteristics of strains with highly competitive ability versus the rest, microfermentations using representative strains from each group were performed and the carbon and nitrogen sources uptake was analysed. Our results demonstrate that despite wine strains being the subclade with the highest competitive ability, they present a wide range of behaviors as well as nutrient uptake dynamics, which points to a heterogeneous nature of domestication processes. An interesting strategy was observed in the highly competitive strains (GRE and QA23), the nitrogen sources uptake in the competition was accelerated and the sugar fermentation was slowing despite the fermentation finish at the same time. Therefore, this competition study, using particular combinations of strains, expands the knowledge in the field of the usage of mixed starters in wine manufactured products.

Metabolic differences between a wild and a wine strain of Saccharomyces cerevisiae during fermentation unveiled by multi-omic analysis.

Saccharomyces cerevisiae, a widespread yeast present both in the wild and in fermentative processes, like winemaking. During the colonization of these human-associated fermentative environments, certain strains of S. cerevisiae acquired differential adaptive traits that enhanced their physiological properties to cope with the challenges imposed by these new ecological niches. The advent of omics technologies allowed unveiling some details of the molecular bases responsible for the peculiar traits of S. cerevisiae wine strains. However, the metabolic diversity within yeasts remained poorly explored, in particular that existing between wine and wild strains of S. cerevisiae. For this purpose, we performed a dual transcriptomic and metabolomic comparative analysis between a wild and a wine S. cerevisiae strains during wine fermentations performed at high and low temperatures. By using this approach, we could correlate the differential expression of genes involved in metabolic pathways, such as sulfur, arginine and thiamine metabolisms, with differences in the amounts of key metabolites that can explain some important differences in the fermentation performance between the wine and wild strains.

Kluyveromyces osmophilus is not a synonym of Zygosaccharomyces mellis; reinstatement as Zygosaccharomyces osmophilus comb. nov.

Kluyveromyces osmophilus, a single-strain species isolated from Mozambique sugar, has been treated a synonym of Zygosaccharomyces mellis. Analyses of D1/D2 LSU rRNA gene sequences confirmed that the species belongs to the genus Zygosaccharomyces but showed it to be distinct from strains of Z. mellis. During studies of yeasts associated with stingless bees in Brazil, nine additional isolates of the species were obtained from unripe and ripe honey and pollen of Scaptotrigona cfr. bipunctata, as well as ripe honey of Tetragonisca angustula. The D1/D2 sequences of the Brazilian isolates were identical to those of the type strain of K. osmophilus CBS 5499 (=ATCC 22027), indicating that they represent the same species. Phylogenomic analyses using 4038 orthologous genes support the reinstatement of K. osmophilus as a member of the genus Zygosaccharomyces. We, therefore, propose the name Zygosaccharomyces osmophilus comb. nov. (lectotype ATCC 22027; MycoBank no. MB 833739).

A new chromosomal rearrangement improves the adaptation of wine yeasts to sulfite.

Sulfite-generating compounds are widely used during winemaking as preservatives because of its antimicrobial and antioxidant properties. Thus, wine yeast strains have developed different genetic strategies to increase its sulfite resistance. The most efficient sulfite detoxification mechanism in Saccharomyces cerevisiae uses a plasma membrane protein called Ssu1 to efflux sulfite. In wine yeast strains, two chromosomal translocations (VIIItXVI and XVtXVI) involving the SSU1 promoter region have been shown to upregulate SSU1 expression and, as a result, increase sulfite tolerance. In this study, we have identified a novel chromosomal rearrangement that triggers wine yeast sulfite adaptation. An inversion in chromosome XVI (inv-XVI) probably due to sequence microhomology, which involves SSU1 and GCR1 regulatory regions, increases the expression of SSU1 and the sulfite resistance of a commercial wine yeast strain. A detailed dissection of this chimeric SSU1 promoter indicates that both the removed SSU1 promoter sequence and the relocated GCR1 sequence contribute to SSU1 upregulation and sulfite tolerance. However, no relevant function has been attributed to the SSU1-promoter-binding transcription factor Fzf1. These results unveil a new genomic event that confers an evolutive advantage to wine yeast strains.

Dominance of wine Saccharomyces cerevisiae strains over S. kudriavzevii in industrial fermentation competitions is related to an acceleration of nutrient uptake and utilization.

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.

On the origins and industrial applications of Saccharomyces cerevisiae × Saccharomyces kudriavzevii hybrids.

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. Copyright © 2017 John Wiley & Sons, Ltd.

Alternative yeasts for winemaking: Saccharomyces non-cerevisiae and its hybrids.

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.

Mitochondrial introgression suggests extensive ancestral hybridization events among Saccharomyces species.

Horizontal gene transfer (HGT) in eukaryotic plastids and mitochondrial genomes is common, and plays an important role in organism evolution. In yeasts, recent mitochondrial HGT has been suggested between S. cerevisiae and S. paradoxus. However, few strains have been explored given the lack of accurate mitochondrial genome annotations. Mitochondrial genome sequences are important to understand how frequent these introgressions occur, and their role in cytonuclear incompatibilities and fitness. Indeed, most of the Bateson-Dobzhansky-Muller genetic incompatibilities described in yeasts are driven by cytonuclear incompatibilities. We herein explored the mitochondrial inheritance of several worldwide distributed wild Saccharomyces species and their hybrids isolated from different sources and geographic origins. We demonstrated the existence of several recombination points in mitochondrial region COX2-ORF1, likely mediated by either the activity of the protein encoded by the ORF1 (F-SceIII) gene, a free-standing homing endonuclease, or mostly facilitated by A+T tandem repeats and regions of integration of GC clusters. These introgressions were shown to occur among strains of the same species and among strains of different species, which suggests a complex model of Saccharomyces evolution that involves several ancestral hybridization events in wild environments.

Saccharomyces uvarum is responsible for the traditional fermentation of apple chicha in Patagonia.

Apple chicha is a fresh low alcoholic beverage elaborated by aboriginal communities of Andean Patagonia (Argentina and Chile). In the present work, we identified the yeast microbiota associated with this fermentation, and characterized genetically those belonging to the genus Saccharomyces. Both Saccharomyces cerevisiae and S. uvarum were found in the analyzed fermentations. Phylogenetic and population structure analyses based on genes sequence analysis were carried out for both S. cerevisiae and S. uvarum strains obtained in this study and a set of additional strains from diverse origins. The results demonstrate that S. cerevisiae strains from apple chicha belong to the big group of wine/European strains of this species, while S. uvarum strains were included in the Holartic population of this species. Additionally, some S. uvarum strains from chichas evidenced as an admixture of both pure Holartic and pure South American populations. Our results suggest that Holartic strains could have been introduced in South America together with the domestication of apple trees by Mapuche communities. This Holartic population suffered admixis with the naturally present South American population of this species, originating strains bearing genetic features from the two populations, detectable in both chichas and natural habitats.

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.

Two interbreeding populations of Saccharomyces cerevisiae strains coexist in cachaça fermentations from Brazil.

In this study, the phylogenetic relationships between cachaça strains of Saccharomyces cerevisiae isolated from different geographical areas in Brazil were obtained on the basis of sequences of one mitochondrial (COX2) and three nuclear (EGT2, CAT8, and BRE5) genes. This analysis allowed us to demonstrate that different types of strains coexist in cachaça fermentations: wine strains, exhibiting alleles related or identical to those present in European wine strains; native strains, containing alleles similar to those found in strains isolated from traditional fermentations from Latin America, North America, Malaysian, Japan, or West Africa; and their intraspecific hybrids or 'mestizo' strains, heterozygous for both types of alleles. Wine strains and hybrids with high proportions of wine-type alleles predominate in southern and southeastern Brazil, where cachaça production coexists with winemaking. The high frequency of 'wine-type' alleles in these regions is probably due to the arrival of wine immigrant strains introduced from Europe in the nearby wineries due to the winemaking practices. However, in north and northeastern states, regions less suited or not suited for vine growing and winemaking, wine-type alleles are much less frequent because 'mestizo' strains with intermediate or higher proportions of 'native-type' alleles are predominant.

Saccharomyces eubayanus and Saccharomyces uvarum associated with the fermentation of Araucaria araucana seeds in Patagonia.

Mudai is a traditional fermented beverage, made from the seeds of the Araucaria araucana tree by Mapuche communities. The main goal of the present study was to identify and characterize the yeast microbiota responsible of Mudai fermentation as well as from A. araucana seeds and bark from different locations in Northern Patagonia. Only Hanseniaspora uvarum and a commercial bakery strain of Saccharomyces cerevisiae were isolated from Mudai and all Saccharomyces isolates recovered from A. araucana seed and bark samples belonged to the cryotolerant species Saccharomyces eubayanus and Saccharomyces uvarum. These two species were already reported in Nothofagus trees from Patagonia; however, this is the first time that they were isolated from A. araucana, which extends their ecological distribution. The presence of these species in A. araucana seeds and bark samples, led us to postulate a potential role for them as the original yeasts responsible for the elaboration of Mudai before the introduction of commercial S. cerevisiae cultures. The molecular and genetic characterization of the S. uvarum and S. eubayanus isolates and their comparison with European S. uvarum strains and S. eubayanus hybrids (S. bayanus and S. pastorianus), allowed their ecology and evolution us to be examined.

Differences in metabolism among Saccharomyces species and their hybrids during wine fermentation.

This study aimed to investigate how parental genomes contribute to yeast hybrid metabolism using a metabolomic approach. Previous studies have explored central carbon and nitrogen metabolism in Saccharomyces species during wine fermentation, but this study analyses the metabolomes of Saccharomyces hybrids for the first time. We evaluated the oenological performance and intra- and extracellular metabolomes, and we compared the strains according to nutrient consumption and production of the main fermentative by-products. Surprisingly, no common pattern was observed for hybrid genome influence; each strain behaved differently during wine fermentation. However, this study suggests that the genome of the S. cerevisiae species may play a more relevant role in fermentative metabolism. Variations in biomass/nitrogen ratios were also noted, potentially linked to S. kudriavzevii and S. uvarum genome contributions. These results open up possibilities for further research using different "omics" approaches to comprehend better metabolic regulation in hybrid strains with genomes from different species.

Understanding the role of GRE3 in the erythritol biosynthesis pathway in Saccharomyces uvarum and its implication in osmoregulation and redox homeostasis.

Erythritol is produced in yeasts via the reduction of erythrose into erythritol by erythrose reductases (ERs). However, the genes codifying for the ERs involved in this reaction have not been described in any Saccharomyces species yet. In our laboratory, we recently showed that, during alcoholic fermentation, erythritol is differentially produced by Saccharomyces cerevisiae and S. uvarum species, the latter being the largest producer. In this study, by using BLAST analysis and phylogenetic approaches the genes GRE3, GCY1, YPR1, ARA1 and YJR096W were identified as putative ERs in Saccharomyces cerevisiae Then, these genes were knocked out in our S. uvarum strain (BMV58) with higher erythritol biosynthesis compared to control S. cerevisiae wine strain, to evaluate their impact on erythritol synthesis and global metabolism. Among the mutants, the single deletion of GRE3 markedly impacts erythritol production, although ΔYPR1ΔGCY1ΔGRE3 was the combination that most decreased erythritol synthesis. Consistent with the increased production of fermentative by-products involved in redox balance in the Saccharomyces uvarum strain BMV58, erythritol synthesis increases at higher sugar concentrations, hinting it might be a response to osmotic stress. However, the expression of GRE3 in the S. uvarum strain was found to peak just before the start of the stationary phase, being consistent with the observation that erythritol increases at the start of the stationary phase, when there is low sugar in the medium and nitrogen sources are depleted. This suggests that GRE3 plays its primary function to help the yeast cells to maintain the redox balance during the last phases of fermentation.

A Dynamic Genome-Scale Model Identifies Metabolic Pathways Associated with Cold Tolerance in Saccharomyces kudriavzevii.

Saccharomyces kudriavzevii is a cold-tolerant species identified as a good alternative for industrial winemaking. Although S. kudriavzevii has never been found in winemaking, its co-occurrence with Saccharomyces cerevisiae in Mediterranean oaks is well documented. This sympatric association is believed to be possible due to the different growth temperatures of the two yeast species. However, the mechanisms behind the cold tolerance of S. kudriavzevii are not well understood. In this work, we propose the use of a dynamic genome-scale model to compare the metabolic routes used by S. kudriavzevii at two temperatures, 25°C and 12°C, to decipher pathways relevant to cold tolerance. The model successfully recovered the dynamics of biomass and external metabolites and allowed us to link the observed phenotype with exact intracellular pathways. The model predicted fluxes that are consistent with previous findings, but it also led to novel results which we further confirmed with intracellular metabolomics and transcriptomic data. The proposed model (along with the corresponding code) provides a comprehensive picture of the mechanisms of cold tolerance that occur within S. kudriavzevii. The proposed strategy offers a systematic approach to explore microbial diversity from extracellular fermentation data at low temperatures. IMPORTANCE Nonconventional yeasts promise to provide new metabolic pathways for producing industrially relevant compounds and tolerating specific stressors such as cold temperatures. The mechanisms behind the cold tolerance of S. kudriavzevii or its sympatric relationship with S. cerevisiae in Mediterranean oaks are not well understood. This study proposes a dynamic genome-scale model to investigate metabolic pathways relevant to cold tolerance. The predictions of the model would indicate the ability of S. kudriavzevii to produce assimilable nitrogen sources from extracellular proteins present in its natural niche. These predictions were further confirmed with metabolomics and transcriptomic data. This finding suggests that not only the different growth temperature preferences but also this proteolytic activity may contribute to the sympatric association with S. cerevisiae. Further exploration of these natural adaptations could lead to novel engineering targets for the biotechnological industry.

Comparative genomics among Saccharomyces cerevisiae × Saccharomyces kudriavzevii natural hybrid strains isolated from wine and beer reveals different origins.

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.

The molecular characterization of new types of Saccharomyces cerevisiae×S. kudriavzevii hybrid yeasts unveils a high genetic diversity.

New double- and triple-hybrid Saccharomyces yeasts were characterized using PCR-restriction fragment length polymorphism of 35 nuclear genes, located on different chromosome arms, and the sequencing of one nuclear and one mitochondrial gene. Most of these new hybrids were originally isolated from fermentations; however, two of them correspond to clinical and dietary supplement isolates. This is the first time that the presence of double-hybrid S. cerevisiae×S. kudriavzevii in non-fermentative substrates has been reported and investigated. Phylogenetic analysis of the MET6 nuclear gene confirmed the double or triple parental origin of the new hybrids. Restriction analysis of gene regions in these hybrids revealed a high diversity of genome types. From these molecular characterizations, a reduction of the S. kudriavzevii fraction of the hybrid genomes is observed in most hybrids. Mitochondrial inheritance in hybrids was deduced from the analysis of mitochondrial COX2 gene sequences, which showed that most hybrids received the mitochondrial genome from the S. kudriavzevii parent. However, two strains inherited a S. cerevisiae COX2, being the first report of S. cerevisiae×S. kudriavzevii hybrids with S. cerevisiae mitochondrial genomes. These two strains are those showing a higher S. kudriavzevii nuclear genome reduction, especially in the wine hybrid AMH. This may be due to the release of selective pressures acting on the other hybrids to maintain kudriavzevii mitochondria-interacting genes.

Adaptive evolution in the Saccharomyces kudriavzevii Aro4p promoted a reduced production of higher alcohols.

The use of unconventional yeast species in human-driven fermentations has attracted a lot of attention in the last few years. This tool allows the alcoholic beverage industries to solve problems related to climate change or the consumer demand for newer high-quality products. In this sense, one of the most attractive species is Saccharomyces kudriavzevii, which shows interesting fermentative traits such as the increased and diverse aroma compound production in wines. Specifically, it has been observed that different isolates of this species can produce higher amounts of higher alcohols such as phenylethanol compared with Saccharomyces cerevisiae. In this work, we have shed light on this feature relating it to the S. kudriavzevii aromatic amino acid anabolic pathway in which the enzyme Aro4p plays an essential role. Unexpectedly, we observed that the presence of the S. kudriavzevii ARO4 variant reduces phenylethanol production compared with the S. cerevisiae ARO4 allele. Our experiments suggest that this can be explained by increased feedback inhibition, which might be a consequence of the changes detected in the Aro4p amino end such as L26 Q24 that have been under positive selection in the S. kudriavzevii specie.