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Additional file of Expression variability of co-regulated genes differentiates Saccharomyces cerevisiae strains

Authors’ original file for figure 7

Additional file 3: Relevant aCGH data. Summary of gene copy number differences observed between the Saccharomyces cerevisiae strains included in this study. Relative gene copy number values (fold change relatively to strain S288C) were obtained by comparative genome hybridization on array (Carreto et al. 2008. BMC Genomics 9: 524). Only genes relevant for discussion in the present manuscript were represented. (PDF 89 KB)

Additional file 2: Co-expression cluster line graphs. Gene co-expression was investigated for strains 06L3FF02, 06L6FF20, AEB Fermol Rouge, Lalvin ICV D254, Lalvin EC-1118, J940047 and S288C from the measurements of relative transcript abundance during fermentation. Graphs represent the average relative transcript abundance (log2 ratio) determined for groups of genes having highly correlated expression profiles across samples. (PDF 125 KB)

Additional file 6: Correlation between gene expression and aCGH. Correlation between relative transcript abundance and putative differences in gene load was found for some genes. The relative transcript abundance values determined for each strain relatively to those of strain S288C were plotted against the relative gene copy number differences for the same strains. The analysis was performed using the datasets from T2 (Panel A) and T4 (Panel B) stages of fermentation. (PDF 60 KB)

Additional file 4: Variability in TATA box genes. Variability in gene expression was biased towards TATA box genes. The graphics show the frequency of TATA box genes as a function of gene expression variability. The average deviation from the mean of the relative gene expression value was taken as a measure of expression variability. Panel A was obtained considering all the investigated strains while Panel B represents the distribution obtained for the environmental and commercial strains. (PDF 55 KB)

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Authors’ original file for figure 1

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

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

The main component of the volatile acidity of wines is acetic acid. The maximum acceptable limit for volatile acidity in most wines is 1.2 g/L of acetic acid due to the associated unpleasant vinegar aroma and acrid taste. Acetic acid is a by-product of alcoholic fermentation by Saccharomyces cerevisiae under winemaking conditions. However, this acid may also appear in wine due to spoilage agents, such as the acetic acid bacteria and spoilage yeasts. Winemakers have been using a refermentation process to lower the concentration of acetic acid of wines with high volatile acidity, which consists in mixing the acidic wine with freshly crushed grapes or marcs in a proportion of no more than 20-30% (v/v). Though this process implies low costs it harbors the risk of unexpected and detrimental effects on refermented wines. Thus, one challenge to find new solutions for the reduction of excessive volatile acidity is the selection of yeast from refermentation processes of acidic wines to use as starters in a controlled biological process. To this end we set up an isolation protocol with Wallerstein Laboratory Nutrient Agar (WL) to select yeast strains from refermentation processes of acidic wines carried at the winery scale. Among the isolates obtained, 135 were then randomly selected, based on the different colony color pattern and size, and tested for their ability to consume acetic acid in the presence of glucose. For this purpose we used a modified version of a Zygosaccharomyces bailii differential medium containing acetic acid and glucose. Characterization of the isolates obtained in this medium by fingerprinting with primer T3B confirmed three Saccharomyces strains and one non-Saccharomyces strain as predicted by WL and L-Lysine media. Our previous studies revealed that the yeast strains selected by this approach are adequate for the correction of acidic musts and wines with excessive levels of volatile acidity.

The level of acetic acid, the main component of volatile acidity, is critical for wine quality. Winemakers have been using a refermentation process to lower the concentration of acetic acid of wines with high volatile acidity, whichconsistsinmixingtheacidicwinewithfreshlycrushedgrapesormarcsinaproportionofnomorethan 20-30% (v/v). Though this process implies low costs it harbors the risk of unexpected and detrimental effects on refermented wines. Thus, one challenge to find new solutions for the reduction of excessive volatile acidity is the selection of yeast from refermentation processes of acidic wines to use as starters in a controlled biological process.To this end we setup an isolation protocolwithWallerstein Laboratory Nutrient Agar(WL)to select yeast strains from refermentation processes of acidic wines carried atthe winery scale. Among the isolates obtained, 135 were then randomly selected, based on the different colony color pattern and size, and tested for their ability to consume acetic acid in the presence of glucose. For this purpose we used a modified version of a Zygosaccharomycesbailiidifferentialmediumcontainingaceticacidandglucose.Characterizationoftheisolates obtained in this medium by fingerprinting with primer T3B confirmed three Saccharomyces strains and one nonSaccharomyces strain as predicted by WL and L-Lysine media. Our previous studies revealed that the yeast strains selected by this approach are adequate forthe correction of acidic musts and wines with excessive levels of volatile acidity., This work received financial support from the IBB/CGB – UTAD and was supported by FEDER throughPOFC – COMPETEand byPortuguese funds from FCTthrough the projectPEst-OE/BIA/UI4050/2014., info:eu-repo/semantics/publishedVersion

New insights into the adaptation of yeast to wine making using comparative genomics. 10. Symposium International d’Œnologie de Bordeaux, Œno2015

This study aims to identify the fermentative yeasts communities of the vineyards from the mid-Atlantic islands ecosystems of the Azores Archipelago and to evaluate whether the populations vary according to the islands geography, grape variety and type of vineyard. From an ecological point of view, vineyards of these isolated volcanic islands terroirs correspond to very particular ecosystems, whose yeast flora is completely unknown. During the harvests of 2009 and 2010, 88 and 75 grape samples were collected from eight islands of the archipelago, respectively. They were obtained from traditional grape varieties (Arinto, Verdelho and Terrantez) and hybrid varieties. The sampling plan covered 36 locations, including vineyards in appellations of origin and vineyards that were abandoned for at least 5 years. A total of 4890 yeast isolates was obtained from the crushed grapes. Species identification was performed by restriction fragment length polymorphism analysis (RFLP) and sequencing of ITS regions. Twenty-seven yeast species were identified, being Hanseniaspora uvarum, Issatchenkia terricola, Candida zemplinina and Metschnikowia pulcherrima the most representative ones. Three putative new species were also found. In 2009, differences in the microflora composition were apparent between islands, grape varieties and type of vineyard. A high percentage of C. zemplinina differentiated the yeast microflora of the eastern islands Santa Maria and São Miguel (25% and 36%, respectively). The species M. pulcherrima appeared associated with the traditional grape varieties (20% and 2% in traditional and hybrid varieties, respectively). H. uvarum was found in higher proportions in abandoned vineyards (53% and 43% in abandoned and non-abandoned vineyards, respectively). In the following sampling year, characterized by unfavorable climatic conditions and very low grape production, the yeast species richness decreased considerably from 25 species in 2009 to 14 species in 2010 and H. uvarum represented 86% of the total isolates, 40% more than in the previous year., João Drumonde-Neves is recipient of a fellowship supported by the program PRO-EMPREGO and the Azores Government. This study was financially supported by FP7 (nº 232454). Financial support was also obtained from FEDER funds through the program COMPETE and by national funds through FCT by the project FCOMP-01-0124-008775.

Within our previous work [1] we developed computational models to predict strains with specific phenotypes (e.g. low ethanol resistance, growth at 30ºC and growth in media containing galactose, raffinose or urea) from microsatellite allelic patterns. The objective of the present work was to gain deeper understanding of the phenotypic diversity of a heterogeneous Saccharomyces cerevisiae strain collection, using a large battery of tests with biotechnological relevance, and apply computational data mining algorithms to predict a strain´s potential to be used as a winemaking strain from a few selected phenotypic data. A S. cerevisiae collection was constituted, comprising 172 strains of different geographical origins and technological uses (winemaking, brewing, bakery, distillery, etc.). Phenotypic screening was performed considering 30 physiological traits that are important from an oenological point of view, such as ethanol tolerance, growth in synthetic must media at various temperatures or resistance to fungicides. Data was analyzed using Principal Component Analysis and some phenotypes were identified (growth in the presence of potassium bisulfite, growth at 40˚C, and resistance to ethanol) as being responsible for the highest strain variability. Statistical analysis revealed relevant associations between several phenotypes and the strains technological use. Based on the phenotypic data, naїve Bayesian classifier, as implemented in the software Orange [2], correctly assigned (AUC=0.70) most of strains from vineyards (73%) and commercial strains (77%) to the respective group. Data mining approaches identified, for the group of commercial strains, 18 phenotypic tests with the highest weight. Globally, the growth patterns of this group of strains in must containing iprodion (0,05mg/mL) or cycloheximide (0,1µg/mL) revealed to have the highest predictive score for the assignment of a strain as a commercial strain. The results obtained herein demonstrate the potential of computational approaches to explore phenotypic variability and to predict the probability of a S. cerevisiae strain to be used as a commercial strain., Fundação para a Ciência e a Tecnologia (FCT)

This work aims to evaluate diversity of Saccharomyces cerevisiae strains from vineyards of the Azores Archipelago and to estimate the degree of phenotypic and genetic differentiation among geographically isolated islands. During two consecutive years, 163 grape musts were obtained from grape samples collected in eight islands of the Archipelago, that belonged to traditional (Arinto, Verdelho and Terrantez) and hybrid grape varieties. The sampling plan included vineyards in appellations of origin and abandoned vineyards. Ninety-four spontaneous fermentations were achieved. From the final fermentative stages, 2670 yeast isolates were obtained. The species identification was performed by restriction fragment length polymorphism analysis (RFLP) and sequencing of ITS regions. S. cerevisiae strains delimitation was performed by interdelta sequences analysis. S. cerevisiae strains were phenotypically evaluated regarding the combined resistance to ethanol/SO2, ß-glucosidade activity, H2S production and the killer/sensitive phenotype. From all isolates collected, 22.2% belonged to non-Saccharomyces species and 77.8% to S. cerevisiae, which were classified in 284 strains. We found phenotypic variations among strains according to their origin (islands, grape variety or vineyard management). Fifty percent of S. cerevisiae isolates from Graciosa, the island with the largest area of contiguous vineyards, showed killer activity, whereas this value was considerably lower in the other six islands (0 – 12 %). On this island we also found a high genetic diversity, since the average number of S. cerevisiae strains per fermentation was of 6.4 and 6.7 on the islands Graciosa and Santa Maria, respectively. Lower values (ranging between 1.2 and 4.9) were observed in each of the other five islands. On each island of the Azores Archipelago, S. cerevisiae isolates from abandoned vineyards tended to be less resistant to SO2 (100 ppm) in comparison with strains obtained from cultivated vineyards. Sequencing of genetically most distinctive strains is currently underway., João Drumonde-Neves is recipient of a fellowship supported by the program PRO-EMPREGO and the Azores Government. This study was financially supported by FP7 (nº 232454). Financial support was also obtained from FEDER funds through the program COMPETE and by national funds through FCT by the project FCOMP-01-0124-008775.

Genome sequencing is essential to understand individual variation and to study the relationship between genotype and phenotype. Recently, large-scale sequencing projects of Saccharomyces cerevisiae revealed the existence of a few well defined lineages and some mosaics of that lineages, and suggested the occurrence of two domestication events during the history of association to human activities, one for sake strains and one for wine yeasts. Although the diversity of S. cerevisiae strains in winemaking environments is rather high, suggesting the occurrence of specific natural strains associated with particular terroirs, scarce information is available regarding phenotypic variability among strains used for different biotechnological applications. The objective of the present work was to undertake high-throughput approaches for a genetic evaluation of 172 S. cerevisiae strains from different geographical origins and technological uses (winemaking, brewing, bakery, distillery, laboratory, natural, etc.) and computationally relate the results with 30 phenotypic tests that were previously obtained. Genetic characterization was performed using eleven polymorphic S. cerevisiae specific microsatellite loci. More than 200 different alleles were obtained, being around 30 responsible for the highest strain variability. 8944 data points were generated and Principal Component Analysis (PCA) revealed the microsatellites C4 and ScYOR267c, as well as the alleles AAT5-256 and AAT6-256 as the most contributing to intra-strain variability. Based on microsatellite allelic information, the software Orange [1] was used to find sub-groups of strains with similar values of phenotypic features and microsatellite allelic patterns. Globally, our study demonstrates that computational approaches can be successfully used to estimate a strain’s biotechnological potential from genotypic data, simplifying laborious strain selection programs by partially replacing phenotypic screens through a preliminary selection based on a strain’s microsatellite allelic combinations., Fundação para a Ciência e a Tecnologia (FCT)

One major issue for wine quality is volatile acidity. Above certain limits and depending on the wine, acetic acid may confer an undesirable sour taste and unpleasant vinegar aroma, which makes the wines unfit for consumption and translates into economic losses for the producer. In this context the removal of acetic acid from wine is an important issue for the wine industry. Biological removal of acetic acid from wines by Saccharomyces cerevisiae yeasts is an enological practice that has been studied [1] since the pioneer work of Peynaud in 1938 [2]. However, this deacidification process is inadequate for wines with high ethanol content and low pH [3]. Cell immobilization by entrapment in beads has been increasingly applied in the last years, due to numerous advantages such as continuous cell utilization and protection from inhibitory substances in the fermentative medium and from shear forces, as well as increased fermentation and production rates [3]. The aim of this work was to study the efficiency of acetic acid removal by immobilized cells of a previously characterized commercial strain of S. cerevisiae. In order to exploit its application in the reduction of volatile acidity of wines we evaluated two cell immobilization processes, one-layer Ca-alginate beads or double-layer alginate-chitosan beads, which have already been used in alcoholic fermentations by S. cerevisiae [4]. Furthermore, the effect of different parameters (cell concentration, initial pH and number of bead layers) on the deacidification process efficiency was also determined. The results showed that the outer chitosan layer improved the stability of the beads during fermentation, preventing cell leakage from the beads into the medium. In fact, immobilized cells in double layer alginate-chitosan beads (8.0x107 cell ml-1alginate) were able to reduce 61.8% of the initial volatile acidity of an acidic wine (1.1 g l-1 acetic acid) with 12.5% (v/v) ethanol and pH 3.12, after 72 h. Under these conditions beads maintained their integrity and no cell leakage occurred as confirmed by Scanning Electron Microscopy. Thus, immobilized S. cerevisiae cells in double-layer alginate-chitosan beads appear as an efficient alternative for the reduction of excessive volatile acidity of wines with low pH and high ethanol. UTAD

A ocorrência de vinhos com teores de acidez volátil excessiva é um problema grave e atual e as soluções enológicas disponíveis não são satisfatórias. A utilização de leveduras imobilizadas é uma alternativa promissora e eficiente para a correção da acidez volátil de vinhos e, consequentemente, para a melhoria da sua qualidade.

Volatile acidity (VA) is one of the major issues of wine quality. Its legal limit is 1.2 g L-1 and at concentrations above 0.8 g L-1 (1) confers to wine an undesirable acidic taste and unpleasant vinegar aroma. Excessive acetic acid, frequently associated with Botrytis-cinerea-infected grapes, is formed by yeasts during fermentation but can also be formed by lactic acid bacteria during malolactic fermentation or as a product of acetic acid bacteria. The commercial strain Saccharomyces cerevisiae S26 is able to decrease volatile acidity of acidic wines with a volatile acidity higher than 1.44 g L-1 acetic acid (2), with no detrimental impact on wine aroma (3). Herein we assessed the efficiency of acetic acid removal from acidic wines by immobilized cells of strain S26. The results obtained show that immobilized cells of the S. cerevisiae strain S26 in double layer alginate-chitosan (DL) beads (4.0x107 cell mL-1alginate) reduce 21.6% of the initial VA of an acidic wine (1.1 g L-1) with 12.5% (v/v) ethanol and pH 3.5, after 72 h. The level of deacidification did not change after 168 h and was associated with a slight decrease in ethanol concentration (1.1% v/v) and cell leakage from the beads (3.5x104 CFU mL-1). Duplication of the initial cell concentration and decrease of the pH to 3.12, increased VA removal up to 61.8%, depending on the initial acetic acid concentration. Moreover, no cell leakage occurred whereas ethanol concentration slightly decreased (0.7%, v/v). Scanning Electron Microscopy analysis of immobilized cells in DL beads confirmed that the initial pH value is critical for beads integrity maintenance. Thus, immobilized S. cerevisiae S26 cells in DL beads appear as an efficient alternative to improve wine quality with excessive VA.