Cerevisiae Populations Research Articles

Fermentative and Enological Features of Saccharomyces cerevisiae Populations Generated Through Adaptive Laboratory Evolution

Adaptive laboratory evolution (ALE) is a non-GMO technique utilized for the amelioration of wine yeast strains. Employing two-step ALE strategies, we recently acquired six evolved Saccharomyces cerevisiae populations with improved fermentative abilities compared to their parental strains in synthetic broths. Herein, we evaluated the qualities of the abovementioned evolved populations under real winemaking conditions, using the grape musts Assyrtiko and Roditis. The ethanol-tolerant populations evolved solely with glucose delayed to complete the fermentation due to slow fructose assimilation, albeit showing improved ethanol yields, compared to their parental strains. The volatile compounds of the evolved populations were significantly different from those of parental strains. Statistically significant differences were observed in the organoleptic profiles between the evolved populations’ and parental strains’ wines. Notably, wine from one evolved population (BLR200) was rated higher in overall aroma and quality. This study supports the magnitude of ALE strategies for the generation of novel wine yeasts.

Empirical evidence of resource dependent evolution of payoff matrices in Saccharomyces cerevisiae populations.

In evolutionary game theory, a relative comparison of the cost and benefit associated with obtaining a resource, called payoff, is used as an indicator of fitness of an organism. Payoffs of different strategies, quantitatively represented as payoff matrices, are used to understand complex inter-species and intra-species interactions like cooperation, mutualism, and altruism. Payoff matrices, however, are usually treated as invariant with time - largely due to the absence of any empirical data quantifying their evolution. In this paper, we present empirical evidence of three types of resource-dependent changes in the payoff matrices of evolving Saccharomyces cerevisiae populations. We show that depending on the carbon source and participating genotypes, N-player games could collapse, be born, or be maintained. Our results highlight the need to consider the dynamic nature of payoff matrices while making even short-term predictions about population interactions and dynamics.

Asynchronous abundance fluctuations can drive giant genotype frequency fluctuations.

Large stochastic population abundance fluctuations are ubiquitous across the tree of life1-7, impacting the predictability of population dynamics and influencing eco-evolutionary outcomes. It has generally been thought that these large abundance fluctuations do not strongly impact evolution, as the relative frequencies of alleles in the population will be unaffected if the abundance of all alleles fluctuate in unison. However, we argue that large abundance fluctuations can lead to significant genotype frequency fluctuations if different genotypes within a population experience these fluctuations asynchronously. By serially diluting mixtures of two closely related E. coli strains, we show that such asynchrony can occur, leading to giant frequency fluctuations that far exceed expectations from models of genetic drift. We develop a flexible, effective model that explains the abundance fluctuations as arising from correlated offspring numbers between individuals, and the large frequency fluctuations result from even slight decoupling in offspring numbers between genotypes. This model accurately describes the observed abundance and frequency fluctuation scaling behaviors. Our findings suggest chaotic dynamics underpin these giant fluctuations, causing initially similar trajectories to diverge exponentially; subtle environmental changes can be magnified, leading to batch correlations in identical growth conditions. Furthermore, we present evidence that such decoupling noise is also present in mixed-genotype S. cerevisiae populations. We demonstrate that such decoupling noise can strongly influence evolutionary outcomes, in a manner distinct from genetic drift. Given the generic nature of asynchronous fluctuations, we anticipate that they are widespread in biological populations, significantly affecting evolutionary and ecological dynamics.

Occurrence and Persistence of Saccharomyces cerevisiae Population in Spontaneous Fermentation and the Relation with "Winery Effect".

The yeast Saccharomyces cerevisiae ensures successful fermentation in winemaking, although the persistent use of commercial strains lead to the loss of aroma complexity of wines. Hence, the research of indigenous S. cerevisiae with proper oenological features and well adapted to specific wine-growing areas become of great interest for winemakers. Here, 206 pure cultures of S. cerevisiae were isolated from two wineries during a two-year sampling campaign and bio-typed through interdelta sequences analyses with the aim to evaluate the occurrence and persistence of the S. cerevisiae wild population linked to each winery. Both wineries belong to the same Verdicchio DOC wine area (Castelli di Jesi), and never used commercial yeasts during fermentation. Results showed 19 different biotypes with a specific population of S. cerevisiae in each winery, without cross-contamination with each other and with commercial starter strains. Moreover, inside each winery a persistence of some dominant biotypes was observed over time (three biotypes in winery 1; 95% of isolates in the two years and one biotype in winery 2; 20% of isolates in the two years), indicating a sort of "winery-effect". The evaluation of S. cerevisiae populations for the oenological characters by microfermentations showed a proper and well distinct aromatic imprinting on the resulted wines supporting the concept of "winery effect".

Adaptation of Saccharomyces to High Glucose Concentrations and Its Impact on Growth Kinetics of Alcoholic Fermentations.

Prior adaptation of Saccharomyces cerevisiae to the fermentation medium ensures its implantation and success in alcoholic fermentations. Fermentation kinetics can be characterized with mathematical models to objectively measure the success of adaptation and growth. The study aims at assessing and comparing two pre-culture procedures using, respectively, one or two adaptation steps, analyzing the impact of different initial glucose concentrations on the fermentation profiles of S. cerevisiae cultures, and assessing the performance of three predictive growth models (Buchanan's, modified Gompertz, and Baranyi and Roberts models) under varied initial glucose concentrations. We concluded that both protocols produced S. cerevisiae pre-cultures with similar viability and biomass increase, which suggests that short protocols may be more cost-effective. Furthermore, the study highlights the need of inoculating a high S. cerevisiae population to minimize the depletion of dissolved oxygen in the medium and to ensure that glucose is predominantly directed toward the ethanol formation at early fermentative steps. This study shows that the relationship between kinetic parameters is model-dependent, which hinders inter-study comparisons and stresses the need for standardized growth models. We advocate for the generalized use of confidence intervals of the kinetic parameters to facilitate objective inter-study comparisons.

Genomic sequencing reveals convergent adaptation during experimental evolution in two budding yeast species

Convergent evolution is central in the origins of multicellularity. Identifying the basis for convergent multicellular evolution is challenging because of the diverse evolutionary origins and environments involved. Haploid Kluyveromyces lactis populations evolve multicellularity during selection for increased settling in liquid media. Strong genomic and phenotypic convergence is observed between K. lactis and previously selected S. cerevisiae populations under similar selection, despite their >100-million-year divergence. We find K. lactis multicellularity is conferred by mutations in genes ACE2 or AIM44, with ACE2 being predominant. They are a subset of the six genes involved in the S. cerevisiae multicellularity. Both ACE2 and AIM44 regulate cell division, indicating that the genetic convergence is likely due to conserved cellular replication mechanisms. Complex population dynamics involving multiple ACE2/AIM44 genotypes are found in most K. lactis lineages. The results show common ancestry and natural selection shape convergence while chance and contingency determine the degree of divergence.

The loci of environmental adaptation in a model eukaryote

While the underlying genetic changes have been uncovered in some cases of adaptive evolution, the lack of a systematic study prevents a general understanding of the genomic basis of adaptation. For example, it is unclear whether protein-coding or noncoding mutations are more important to adaptive evolution and whether adaptations to different environments are brought by genetic changes distributed in diverse genes and biological processes or concentrated in a core set. We here perform laboratory evolution of 3360 Saccharomyces cerevisiae populations in 252 environments of varying levels of stress. We find the yeast adaptations to be primarily fueled by large-effect coding mutations overrepresented in a relatively small gene set, despite prevalent antagonistic pleiotropy across environments. Populations generally adapt faster in more stressful environments, partly because of greater benefits of the same mutations in more stressful environments. These and other findings from this model eukaryote help unravel the genomic principles of environmental adaptation.

Identification of Key Parameters Inducing Microbial Modulation during Backslopped Kombucha Fermentation.

The aim of this study was to assess the impact of production parameters on the reproducibility of kombucha fermentation over several production cycles based on backslopping. Six conditions with varying oxygen accessibility (specific interface surface) and initial acidity (through the inoculation rate) of the cultures were carried out and compared to an original kombucha consortium and a synthetic consortium assembled from yeasts and bacteria isolated from the original culture. Output parameters monitored were microbial populations, biofilm weight, key physico-chemical parameters and metabolites. Results highlighted the existence of phases in microbial dynamics as backslopping cycles progressed. The transitions between phases occurred faster for the synthetic consortium compared to the original kombucha. This led to microbial dynamics and fermentative kinetics that were reproducible over several cycles but that could also deviate and shift abruptly to different behaviors. These changes were mainly induced by an increase in the Saccharomyces cerevisiae population, associated with an intensification of sucrose hydrolysis, sugar consumption and an increase in ethanol content, without any significant acceleration in the rate of acidification. The study suggests that the reproducibility of kombucha fermentations relies on high biodiversity to slow down the modulations of microbial dynamics induced by the sustained rhythm of backslopping cycles.

The rate of adaptive molecular evolution in wild and domesticated Saccharomyces cerevisiae populations.

Through its fermentative capacities, Saccharomyces cerevisiae was central in the development of civilisation during the Neolithic period, and the yeast remains of importance in industry and biotechnology, giving rise to bona fide domesticated populations. Here, we conduct a population genomic study of domesticated and wild populations of S. cerevisiae. Using coalescent analyses, we report that the effective population size of yeast populations decreased since the divergence with S. paradoxus. We fitted models of distributions of fitness effects to infer the rate of adaptive ( ) and non-adaptive ( ) non-synonymous substitutions in protein-coding genes. We report an overall limited contribution of positive selection to S. cerevisiae protein evolution, albeit with higher rates of adaptive evolution in wild compared to domesticated populations. Our analyses revealed the signature of background selection and possibly Hill-Robertson interference, as recombination was found to be negatively correlated with and positively correlated with . However, the effect of recombination on was found to be labile, as it is only apparent after removing the impact of codon usage bias on the synonymous site frequency spectrum and disappears if we control for the correlation with , suggesting that it could be an artefact of the decreasing population size. Furthermore, the rate of adaptive non-synonymous substitutions is significantly correlated with the residue solvent exposure, a relation that cannot be explained by the population's demography. Together, our results provide a detailed characterisation of adaptive mutations in protein-coding genes across S. cerevisiae populations.

Populations of Saccharomyces cerevisiae in Vineyards: Biodiversity and Persistence Associated with Terroir

The origin terroir provides distinctive characteristics for wines, in relation to soil, climate, oenological practices, etc. Hence, the characterization of each wine region by multiple aspects would allow differentiation of its wines. Several approaches at different scales have studied terroir microbiological fingerprints: from global microbiome analysis up to intraspecific Saccharomyces biodiversity. Mature grapes are the primary source of yeasts, and S. cerevisiae is a key wine fermentative species. Malbec is the emblematic Argentinean variety and is mainly cultivated in the “Zona Alta del Rio Mendoza” (ZARM). In this work, the diversity of S. cerevisiae grape populations was studied at three vintages in two Malbec vineyards of the ZARM, to evaluate their annual diversity and behavior in different vintages. Rarefaction of classical ecological indices was applied for a statistically adequate biodiversity analysis. A total of 654 S. cerevisiae isolates were differentiated by Interdelta-PCR. Each yeast grape population showed a unique composition of S. cerevisiae strains; however, a narrow genetic relationship was found in each vineyard. A slight increase in the initial diversity and a stabilization in the diversity of S. cerevisiae populations were confirmed. These results add to the discussion about the contribution of yeasts to the terroir microbiological concept, and its limitations and stability over the time.

Discovery of a rapidly evolving yeast defense factor, KTD1, against the secreted killer toxin K28

Secreted protein toxins are widely used weapons in conflicts between organisms. Elucidating how organisms genetically adapt to defend themselves against these toxins is fundamental to understanding the coevolutionary dynamics of competing organisms. Within yeast communities, "killer" toxins are secreted to kill nearby sensitive yeast, providing a fitness advantage in competitive growth environments. Natural yeast isolates vary in their sensitivity to these toxins, but to date, no polymorphic genetic factors contributing to defense have been identified. We investigated the variation in resistance to the killer toxin K28 across diverse natural isolates of the Saccharomyces cerevisiae population. Using large-scale linkage mapping, we discovered a novel defense factor, which we named KTD1. We identified many KTD1 alleles, which provided different levels of K28 resistance. KTD1 is a member of the DUP240 gene family of unknown function, which is rapidly evolving in a region spanning its two encoded transmembrane helices. We found that this domain is critical to KTD1's protective ability. Our findings implicate KTD1 as a key polymorphic factor in the defense against K28 toxin.

Hotspot of de novo telomere addition stabilizes linear amplicons in yeast grown in sulfate-limiting conditions.

Evolution is driven by the accumulation of competing mutations that influence survival. A broad form of genetic variation is the amplification or deletion of DNA (≥50 bp) referred to as copy number variation (CNV). In humans, CNV may be inconsequential, contribute to minor phenotypic differences, or cause conditions such as birth defects, neurodevelopmental disorders, and cancers. To identify mechanisms that drive CNV, we monitored the experimental evolution of Saccharomyces cerevisiae populations grown under sulfate-limiting conditions. Cells with increased copy number of the gene SUL1, which encodes a primary sulfate transporter, exhibit a fitness advantage. Previously, we reported interstitial inverted triplications of SUL1 as the dominant rearrangement in a haploid population. Here, in a diploid population, we find instead that small linear fragments containing SUL1 form and are sustained over several generations. Many of the linear fragments are stabilized by de novo telomere addition within a telomere-like sequence near SUL1 (within the SNF5 gene). Using an assay that monitors telomerase action following an induced chromosome break, we show that this region acts as a hotspot of de novo telomere addition and that required sequences map to a region of <250 base pairs. Consistent with previous work showing that association of the telomere-binding protein Cdc13 with internal sequences stimulates telomerase recruitment, mutation of a four-nucleotide motif predicted to associate with Cdc13 abolishes de novo telomere addition. Our study suggests that internal telomere-like sequences that stimulate de novo telomere addition can contribute to adaptation by promoting genomic plasticity.

Improving ethanol tolerance of Saccharomyces cerevisiae through adaptive laboratory evolution using high ethanol concentrations as a selective pressure

Saccharomyces cerevisiae is a microorganism of remarkable biotechnological interest, owing to its involvement in bioethanol production, as well as in beverages and bread production. High ethanol concentration during alcoholic fermentation constitutes a major stress for yeast cells, leading to decreased fermentation rate and reduced ethanol production. Herein we describe an innovative Adaptive Laboratory Evolution (ALE) strategy for the acquisition of S. cerevisiae populations more tolerant to ethanol toxicity. In this strategy, a weak selective pressure environment (encouraging diversity) is alternated by a strong selective pressure environment (eliminating low-tolerant clones). Two different parental strains, named S. cerevisiae CFB and S. cerevisiae BLR, were used and the resulting evolved populations obtained after 100 generations of evolution were proven capable of surviving at 23% v/v ethanol for 1 h and at 25% v/v ethanol for 2 h, respectively. Two evolved populations originated from S. cerevisiae CFB and S. cerevisiae BLR after 150 and 200 generations of evolution respectively, exhibited higher fermentation rates than their parental strains in synthetic broth with 200 g/l glucose, completing the fermentation 166 h and 130 h earlier, respectively. The evolved populations derived after 100 generations of evolution produced 94.77 and 98.67 g/l ethanol, for S. cerevisiae CFB and S. cerevisiae BLR, respectively, in comparison with 84.91 and 78.09 g/l of their parental strains. Contrary, the maximum specific growth rate (μmax) was improved solely in S. cerevisiae BLR, reaching the value of 0.25 1/h after 100 generations of evolution in comparison with 0.12 1/h of the parental strain. It is concluded that the fermentation ability of the two S. cerevisiae strains was significantly improved through evolution using ethanol as a selective factor.

Long-Term Adaptation to Galactose as a Sole Carbon Source Selects for Mutations Outside the Canonical GAL Pathway.

Galactose is a secondary fermentable sugar that requires specific regulatory and structural genes for its assimilation, which are under catabolite repression by glucose. When glucose is absent, the catabolic repression is attenuated, and the structural GAL genes are fully activated. In Saccharomyces cerevisiae, the GAL pathway is under selection in environments where galactose is present. However, it is unclear the adaptive strategies in response to long-term propagation in galactose as a sole carbon source in laboratory evolution experiments. Here, we performed a 4,000-generation evolution experiment using 48 diploid Saccharomyces cerevisiae populations to study adaptation in galactose. We show that fitness gains were greater in the galactose-evolved population than in identically evolved populations with glucose as a sole carbon source. Whole-genome sequencing of 96 evolved clones revealed recurrent de novo single nucleotide mutations in candidate targets of selection, copy number variations, and ploidy changes. We find that most mutations that improve fitness in galactose lie outside of the canonical GAL pathway. Reconstruction of specific evolved alleles in candidate target of selection, SEC23 and IRA1, showed a significant increase in fitness in galactose compared to glucose. In addition, most of our evolved populations (28/46; 61%) fixed aneuploidies on Chromosome VIII, suggesting a parallel adaptive amplification. Finally, we show greater loss of extrachromosomal elements in our glucose-evolved lineages compared with previous glucose evolution. Broadly, these data further our understanding of the evolutionary pressures that drive adaptation to less-preferred carbon sources.

Acetaldehyde accumulation during wine micro oxygenation: The influence of microbial metabolism

Acetaldehyde is a key oxidation product during red wine micro oxygenation (Mox) and it appears to accumulate by the metabolism of residual yeast Saccharomyces cerevisiae (S. cerevisiae), which may be difficult to detect by plating due to low numbers. The aim of this study was to investigate the roles and interaction of S. cerevisiae and lactic acid bacterial (LAB) with regard to acetaldehyde accumulation during red wine Mox treatment. Immediately after alcohol fermentation using three different yeast S. cerevisiae strains, some wine was sterile filtered, and of that, some was inoculated with the same three yeasts. All were treated with Mox; acetaldehyde and malolactic fermentation (MLF) were monitored. Besides, the capacity of LAB to metabolize acetaldehyde was also evaluated in model wine. The results showed that Mox appeared to maintain S. cerevisiae populations, and the survival of S. cerevisiae postponed spontaneous MLF. S. cerevisiae did produce acetaldehyde during Mox, while levels varied by strains, production rates declined during treatment, and levels dropped after treatment ended. The appearance of LAB coincided with an increase in production, but levels later dropped more quickly when LAB were present. Residual sugar and amino acid levels did not change significantly during microbial metabolism of Mox wines. The acetaldehyde generated from chemical oxidation continued to accumulate during Mox. In the experiment of model wine, LAB was shown to produce acetaldehyde in the inception phase and a certain amount of acetaldehyde did not influence the start of MLF. The results confirmed the variation character of acetaldehyde with microbial action and can serve as a theoretical underpinning for better control of Mox applications in enology.

Effect of Combined Treatment with Cinnamon Oil and petit-High Pressure CO2 against Saccharomyces cerevisiae.

This study investigated the effects of the combined treatment with cinnamon oil (CIN) and petit-high pressure CO2 (p-HPCO2) against Saccharomyces cerevisiae. The results showed that CIN and p-HPCO2 exhibited a synergistic antifungal effect against S. cerevisiae. After being treated by CIN at a final concentration of 0.02% and p-HPCO2 under 1.3 MPa at 25 °C for 2 h, the S. cerevisiae population decreased by 3.35 log10 CFU/mL, which was significantly (p &lt; 0.05) higher than that of CIN (1.11 log10 CFU/mL) or p-HPCO2 (0.31 log10 CFU/mL). Through scanning electron microscopy, fluorescence staining, and other approaches, a disorder of the structure and function of the cell membrane was observed after the CIN + p-HPCO2 treatment, such as severe morphological changes, increased membrane permeability, decreased cell membrane potential, and loss of membrane integrity. CIN + p-HPCO2 also induced mitochondrial membrane depolarization in S. cerevisiae cells, which could be associated with the decrease in intracellular ATP observed in this study. Moreover, the expression of genes involved in ergosterol synthesis in S. cerevisiae was up-regulated after exposure to CIN + p-HPCO2, which might be an adaptive response to membrane damage. This work demonstrates the potential of CIN and p-HPCO2 in combination as an alternative pasteurization technique for use in the food industry.

Screening and Enzymatic Evaluation of Saccharomyces cerevisiae Populations from Spontaneous Fermentation of Organic Verdejo Wines.

Microbial populations in spontaneous winemaking contribute to the distinctiveness and quality of the wines. In this study, molecular methods were applied to 484 isolated yeasts to survey the diversity of the Saccharomyces cerevisiae population in spontaneous fermentations of organic Verdejo grapes. Identification was carried out at strain level for samples from different vineyards correct.and stages of the winemaking process over the course of two vintages, establishing 54 different strains. The number of isolates belonging to each strain was not homogeneous, as two predominant strains represented more than half of the isolates independent of vineyard or vintage. Regarding the richness and abundance, differences among the stages of fermentation were confirmed, finding the highest diversity values in racked must and in the end of fermentation stages. Dissimilarity in S. cerevisiae communities was found among vineyards and vintages, distinguishing representative groups of isolates for each of the populations analysed. These results highlight the effect of vineyard and vintage on yeast communities as well as the presence of singular strains in populations of yeasts. Oenologically relevant enzymatic activities, β-lyase, protease and β-glucanase, were detected in 83.9%, 96.8% and 38.7% of the isolates, respectively, which may be of interest for potential future studies.

A new method for determining ribosomal DNA copy number shows differences between Saccharomyces cerevisiae populations

Ribosomal DNA genes (rDNA) encode the major ribosomal RNAs and in eukaryotes typically form tandem repeat arrays. Species have characteristic rDNA copy numbers, but there is substantial intra-species variation in copy number that results from frequent rDNA recombination. Copy number differences can have phenotypic consequences, however difficulties in quantifying copy number mean we lack a comprehensive understanding of how copy number evolves and the consequences. Here we present a genomic sequence read approach to estimate rDNA copy number based on modal coverage to help overcome limitations with existing mean coverage-based approaches. We validated our method using Saccharomyces cerevisiae strains with known rDNA copy numbers. Application of our pipeline to a global sample of S. cerevisiae isolates showed that different populations have different rDNA copy numbers. Our results demonstrate the utility of the modal coverage method, and highlight the high level of rDNA copy number variation within and between populations.

Start Codon Targeted (SCoT) markers for the assessment of genetic diversity in yeast isolated from Turkish sourdough

Molecular markers are valuable tools for assessing the genetic variation in yeast. Here, we investigated the utility of SCoT markers for the genetic characterization of yeast strains at inter and intraspecies levels. A total of 345 endogenous yeast strains were isolated from 65 Type I sourdough samples collected from six different regions of Turkey. The seven SCoT primers produced 221 bands, of which 95.47% were polymorphic. Each primer could successfully differentiate species, supported by PIC and RP values. The ITS sequencing of isolates selected from the UPGMA dendrogram revealed that Saccharomyces cerevisiae predominated the microflora, followed by Kazachstania servazzii, K. humilis, Wickerhamomyces anomalus, Torulaspora delbrueckii, and Pichia kudriavzevii, respectively. The AMOVA revealed a high genetic variation between (49%) and within populations (51%) for S. cerevisiae. The high gene flow observed among S. cerevisiae populations suggests that it may have contributed to the geographical evolution of S. cerevisiae via the transportation of the sourdough samples. The different geographical origins were most likely to group separately on the UPGMA and PCoA. Saccharomyces cerevisiae strains from more distant populations generally displayed more significant genetic variation. SCoT markers can successfully be used alone or with the other existing DNA markers for DNA fingerprinting and analyzing the genetic variation between and within species.

Effect of Saccharomyces cerevisiae on Probiotic Properties of Goat Milk Kefir

Goat milk kefir is a fermented milk product with kefir grain, as a probiotic agent and contains bioactive compounds. Kefir grains consist of bacteria and yeast. One of these yeasts are Saccharomyces cerevisiae, its role in the kefir system can be enhanced by restructure of kefir grain with increased Saccharomyces cerevisiae population. The objectives of this research were to analysis effect of the Saccharomyces cerevisiae concentration levels on kefir, as antibacterial against Escherichia coli, Salmonella typhi. Klebsiella pneumoniae, antioxidant activity, β-galactosidase synthesis. The research method was true experiment by Completely Randomized Design consist of 5 kind Saccharomyces cerevisiae concentration levels (S0: control; S1: 0.10%; S2: 0,25%; S3: 0.5% and S4: 1.0% v/w) of kefir grain. The results showed that kefir inhibited Escherichia coli, Salmonella typhi and Klebsiella pneumoniae, able to produce β-galactosidase and antioxidant activity. Kefir is relevant as food functional development.