Insights into intraspecific diversity of central carbon metabolites in Saccharomyces cerevisiae during wine fermentation.

Saccharomyces cerevisiae is a major actor in winemaking that converts sugars from the grape must into ethanol and CO2 with outstanding efficiency. Primary metabolites produced during fermentation have a great importance in wine. While ethanol content contributes to the overall profile, other metabolites like glycerol, succinate, acetate or lactate also have significant impacts, even when present in lower concentrations. S. cerevisiae is known for its great genetic diversity that is related to its natural or technological environment. However, the variation range of metabolic diversity which can be exploited to enhance wine quality depends on the pathway considered. Our experiment assessed the diversity of primary metabolites production in a set of 51 S. cerevisiae strains from various genetic backgrounds. Results pointed out great yield differences depending on the metabolite considered, with ethanol having the lowest variation. A negative correlation between ethanol and glycerol was observed, confirming glycerol synthesis as a suitable lever to reduce ethanol yield. Genetic groups were linked to specific yields, such as the wine group and high α-ketoglutarate and low acetate yields. This research highlights the potential of using natural yeast diversity in winemaking. It also provides a detailed data set on production of well known (ethanol, glycerol, acetate) or little-known (lactate) primary metabolites.

Comparing the hierarchy of inter- and intra-species interactions with population dynamics of wine yeast cocultures.

In winemaking, the development of new fermentation strategies, such as the use of mixed starter cultures with Saccharomyces cerevisiae (Sc) yeast and non-Saccharomyces (NS) species, requires a better understanding of how yeasts interact, especially at the beginning of fermentation. Despite the growing knowledge on interactions between Sc and NS, few data are available on the interactions between different species of NS. It is furthermore still unclear whether interactions are primarily driven by generic differences between yeast species or whether individual strains are the evolutionarily relevant unit for biotic interactions. This study aimed at acquiring knowledge of the relevance of species and strain in the population dynamics of cocultures between five yeast species: Hanseniaspora uvarum, Lachancea thermotolerans, Starmerella bacillaris, Torulaspora delbrueckii and Sc. We performed cocultures between 15 strains in synthetic grape must and monitored growth in microplates. Both positive and negative interactions were identified. Based on an interaction index, our results showed that the population dynamics seemed mainly driven by the two species involved. Strain level was more relevant in modulating the strength of the interactions. This study provides fundamental insights into the microbial dynamics in early fermentation and contribute to the understanding of more complex consortia encompassing multiple yeasts trains.

Influence of ergosterol and phytosterols on wine alcoholic fermentation with Saccharomyces cerevisiae strains.

Sterols are a fraction of the eukaryotic lipidome that is essential for the maintenance of cell membrane integrity and its good functionality. During alcoholic fermentation, they enhance yeast growth, metabolism and viability, as well as resistance to high sugar content and ethanol stress. Grape musts clarified in excess lead to the loss of solid particles rich in sterols, resulting in sluggish and stuck fermentations. Two sterol sources can help Saccharomyces cerevisiae yeasts to adapt to fermentation stress conditions: ergosterol (synthesized by yeast under aerobic conditions) and phytosterols (plant sterols imported by yeast cells from grape musts under anaerobiosis). Little is known about the physiological impact of phytosterols assimilation in comparison with ergosterol and the influence of sterol type on fermentation kinetics parameters. Moreover, studies to date have analyzed a limited number of yeast strains. Thus, the aim of this work was to compare the performances of a set of Saccharomyces cerevisiae wine strains that represent the diversity of industrial wine yeast, fermenting with phytosterols or ergosterol under two conditions: sterol limitation (sterol starvation) and high sugar content (the most common stress during fermentation). Results indicated that yeast cell viability was negatively impacted by both stressful conditions, resulting in sluggish and stuck fermentations. This study revealed the huge phenotype diversity of the S. cerevisiae strains tested, in particular in terms of cell viability. Indeed, strains with better viability maintenance completed fermentation earlier. Interestingly, we showed for the first time that sterol type differently affects a wide variety of phenotype, such as viability, biomass, fermentation kinetics parameters and biosynthesis of carbon central metabolism (CCM) metabolites. Ergosterol allowed preserving more viable cells at the end of fermentation and, as a consequence, a better completion of fermentation in both conditions tested, even if phytosterols also enabled the completion of alcoholic fermentation for almost all strains. These results highlighted the essential role of sterols during wine alcoholic fermentation to ensure yeast growth and avoid sluggish or stuck fermentations. Finally, this study emphasizes the importance of taking into account sterol types available during wine fermentation.

Impact of Leavening Agent and Wheat Variety on Bread Organoleptic and Nutritional Quality.

Leavened bread can be made with different wheat varieties and leavening agents. Several studies have now demonstrated that each of these factors can play a role in bread quality. However, their relative impact in artisanal bread making remains to be elucidated. Here, we assessed the impact of two wheat varieties as well as the impact of sourdoughs and yeasts on multiple components of bread organoleptic and nutritional quality. Using a participatory research approach including scientists and bakers, we compared breads leavened with three different sourdoughs and three different commercial yeasts as well as a mix of sourdough and yeast. Breads were made from two wheat varieties commonly used in organic farming: the variety "Renan" and the landrace "Barbu". Except for bread minerals contents that mostly depended on wheat variety, bread quality was mostly driven by the fermenting agent. Sourdough breads had lower sugar and organic acids contents. These differences were mostly attributable to lower amounts of maltose and malate. They also had a higher proportion of soluble proteins than yeast breads, with specific aroma profiles. Finally, their aroma profiles were specific and more diverse compared to yeast breads. Interestingly, we also found significant nutritional and organoleptic quality differences between sourdough breads. These results highlight the value of sourdough bread and the role of sourdough microbial diversity in bread nutritional and organoleptic quality.

Consideration of Maintenance in Wine Fermentation Modeling.

We show that a simple model with a maintenance term can satisfactorily reproduce the simulations of several existing models of wine fermentation from the literature, as well as experimental data. The maintenance describes a consumption of the nitrogen that is not entirely converted into biomass. We show also that considering a maintenance term in the model is equivalent to writing a model with a variable yield that can be estimated from data.

Data integration uncovers the metabolic bases of phenotypic variation in yeast.

The relationship between different levels of integration is a key feature for understanding the genotype-phenotype map. Here, we describe a novel method of integrated data analysis that incorporates protein abundance data into constraint-based modeling to elucidate the biological mechanisms underlying phenotypic variation. Specifically, we studied yeast genetic diversity at three levels of phenotypic complexity in a population of yeast obtained by pairwise crosses of eleven strains belonging to two species, Saccharomyces cerevisiae and S. uvarum. The data included protein abundances, integrated traits (life-history/fermentation) and computational estimates of metabolic fluxes. Results highlighted that the negative correlation between production traits such as population carrying capacity (K) and traits associated with growth and fermentation rates (Jmax) is explained by a differential usage of energy production pathways: a high K was associated with high TCA fluxes, while a high Jmax was associated with high glycolytic fluxes. Enrichment analysis of protein sets confirmed our results. This powerful approach allowed us to identify the molecular and metabolic bases of integrated trait variation, and therefore has a broad applicability domain.

Correction: Investigations of the mechanisms of interactions between four non-conventional species with Saccharomyces cerevisiae in oenological conditions.

[This corrects the article DOI: 10.1371/journal.pone.0233285.].

Evidence for Two Main Domestication Trajectories in Saccharomyces cerevisiae Linked to Distinct Bread-Making Processes.

Production of leavened bread dates to the second millennium BCE. Since then, the art of bread making has developed, yet the evolution of bread-associated microbial species remains largely unknown. Nowadays, leavened bread is made either by using a pure commercial culture of the yeast Saccharomyces cerevisiae or by propagating a sourdough-a mix of flour and water spontaneously fermented by yeasts and bacteria. We studied the domestication of S. cerevisiae originating from industrial sources and artisanal sourdoughs and tested whether different bread-making processes led to population divergence. We found that S. cerevisiae bakery strains are polyphyletic with 67% of strains clustering into two main clades: most industrial strains were tetraploid and clustered with strains having diverse origins, including beer. By contrast, most sourdough strains were diploid and grouped in a second clade of strains having mosaic genomes and diverse origins, including fruits and natural environments. They harbored a higher copy number of genes involved in maltose utilization, and a high level of gene flow from multiple contributors was detected. Bakery strains displayed higher CO2 production than do strains from other domesticated lineages (such as beer and wine), revealing a specific phenotypic signature of domestication. Interestingly, industrial strains had a shorter fermentation onset than sourdough strains, which were better adapted to a sourdough-like environment, suggesting divergent selection by industrial and artisanal processes. Our results reveal that the domestication of bakery yeast has been accompanied by dispersion, hybridization, and divergent selection through industrial and artisanal processes.

Understanding the Mechanisms of Positive Microbial Interactions That Benefit Lactic Acid Bacteria Co-cultures.

Microorganisms grow in concert, both in natural communities and in artificial or synthetic co-cultures. Positive interactions between associated microbes are paramount to achieve improved substrate conversion and process performance in biotransformation and fermented food production. The mechanisms underlying such positive interactions have been the focus of numerous studies in recent decades and are now starting to be well characterized. Lactic acid bacteria (LAB) contribute to the final organoleptic, nutritional, and health properties of fermented food products. However, interactions in LAB co-cultures have been little studied, apart from the well-characterized LAB co-culture used for yogurt manufacture. LAB are, however, multifunctional microorganisms that display considerable potential to create positive interactions between them. This review describes why LAB co-cultures are of such interest, particularly in foods, and how their extensive nutritional requirements can be used to favor positive interactions. In that respect, our review highlights the benefits of co-cultures in different areas of application, details the mechanisms underlying positive interactions and aims to show how mechanisms based on nutritional interactions can be exploited to create efficient LAB co-cultures.

Investigations of the mechanisms of interactions between four non-conventional species with Saccharomyces cerevisiae in oenological conditions.

Fermentation by microorganisms is a key step in the production of traditional food products such as bread, cheese, beer and wine. In these fermentative ecosystems, microorganisms interact in various ways, namely competition, predation, commensalism and mutualism. Traditional wine fermentation is a complex microbial process performed by Saccharomyces and non-Saccharomyces (NS) yeast species. To better understand the different interactions occurring within wine fermentation, isolated yeast cultures were compared with mixed co-cultures of one reference strain of S. cerevisiae with one strain of four NS yeast species (Metschnikowia pulcherrima, M. fructicola, Hanseniaspora opuntiae and H. uvarum). In each case, we studied population dynamics, resource consumed and metabolites produced from central carbon metabolism. This phenotyping of competition kinetics allowed us to confirm the main mechanisms of interaction between strains of four NS species. S. cerevisiae competed with H. uvarum and H. opuntiae for resources although both Hanseniaspora species were characterized by a strong mortality either in mono or mixed fermentations. M. pulcherrima and M. fructicola displayed a negative interaction with the S. cerevisiae strain tested, with a decrease in viability in co-culture. Overall, this work highlights the importance of measuring specific cell populations in mixed cultures and their metabolite kinetics to understand yeast-yeast interactions. These results are a first step towards ecological engineering and the rational design of optimal multi-species starter consortia using modeling tools. In particular the originality of this paper is for the first times to highlight the joint-effect of different species population dynamics on glycerol production and also to discuss on the putative role of lipid uptake on the limitation of some non-conventional species growth although interaction processes.

Interactions between Kazachstania humilis Yeast Species and Lactic Acid Bacteria in Sourdough.

Sourdoughs harbor simple microbial communities usually composed of a few prevailing lactic acid bacteria species (LAB) and yeast species. However, yeast and LAB found in sourdough have been described as highly diverse. Even if LAB and yeast associations have been widely documented, the nature of the interactions between them has been poorly described. These interactions define the composition and structure of sourdough communities, and therefore, the characteristics of the final bread product. In this study, the nature of the interactions between strains of two commonly found sourdough yeast species, Kazachstania humilis and Saccharomyces cerevisiae, and lactic acid bacteria isolated from sourdoughs has been analyzed. Population density analysis showed no evidence of positive interactions, but instead revealed neutral or negative asymmetric interaction outcomes. When in coculture, the yeasts´ population size decreased in the presence of LAB regardless of the strain, while the LAB´s population size was rarely influenced by the presence of yeasts. However, a higher maltose depletion was shown in maltose-negative K. humilis and maltose-positive obligately heterofermentative LAB cocultures compared to monocultures. In addition, tested pairs of obligately heterofermentative LAB and K. humilis strains leavened dough as much as couples of LAB and S. cerevisiae strains, while K. humilis strains never leavened dough as much as S. cerevisiae when in monoculture. Taken together, our results demonstrate that even if higher fermentation levels with increased maltose depletion were detected for K. humilis and obligately heterofermentative LAB pairs, these interactions cannot be ecologically classified as positive, leading us to rethink the established hypothesis of coexistence by facilitation in sourdoughs.

QTL mapping of modelled metabolic fluxes reveals gene variants impacting yeast central carbon metabolism.

The yeast Saccharomyces cerevisiae is an attractive industrial microorganism for the production of foods and beverages as well as for various bulk and fine chemicals, such as biofuels or fragrances. Building blocks for these biosyntheses are intermediates of yeast central carbon metabolism (CCM), whose intracellular availability depends on balanced single reactions that form metabolic fluxes. Therefore, efficient product biosynthesis is influenced by the distribution of these fluxes. We recently demonstrated great variations in CCM fluxes between yeast strains of different origins. However, we have limited understanding of flux modulation and the genetic basis of flux variations. In this study, we investigated the potential of quantitative trait locus (QTL) mapping to elucidate genetic variations responsible for differences in metabolic flux distributions (fQTL). Intracellular metabolic fluxes were estimated by constraint-based modelling and used as quantitative phenotypes, and differences in fluxes were linked to genomic variations. Using this approach, we detected four fQTLs that influence metabolic pathways. The molecular dissection of these QTLs revealed two allelic gene variants, PDB1 and VID30, contributing to flux distribution. The elucidation of genetic determinants influencing metabolic fluxes, as reported here for the first time, creates new opportunities for the development of strains with optimized metabolite profiles for various applications.

Nonlinear phenotypic variation uncovers the emergence of heterosis in Arabidopsis thaliana.

Heterosis describes the phenotypic superiority of hybrids over their parents in traits related to agronomic performance and fitness. Understanding and predicting nonadditive inheritance such as heterosis is crucial for evolutionary biology as well as for plant and animal breeding. However, the physiological bases of heterosis remain debated. Moreover, empirical data in various species have shown that diverse genetic and molecular mechanisms are likely to explain heterosis, making it difficult to predict its emergence and amplitude from parental genotypes alone. In this study, we examined a model of physiological dominance initially proposed by Sewall Wright to explain the nonadditive inheritance of traits like metabolic fluxes at the cellular level. We evaluated Wright's model for two fitness-related traits at the whole-plant level, growth rate and fruit number, using 450 hybrids derived from crosses among natural accessions of A. thaliana. We found that allometric relationships between traits constrain phenotypic variation in a nonlinear and similar manner in hybrids and accessions. These allometric relationships behave predictably, explaining up to 75% of heterosis amplitude, while genetic distance among parents at best explains 7%. Thus, our findings are consistent with Wright's model of physiological dominance and suggest that the emergence of heterosis on plant performance is an intrinsic property of nonlinear relationships between traits. Furthermore, our study highlights the potential of a geometric approach of phenotypic relationships for predicting heterosis of major components of crop productivity and yield.

Bakery yeasts, a new model for studies in ecology and evolution.

Yeasts have been involved in bread making since ancient times and have thus played an important role in the history and nutrition of humans. Bakery-associated yeasts have only recently attracted the attention of researchers outside of the bread industry. More than 30 yeast species are involved in bread making, and significant progress has been achieved in describing these species. Here, we present a review of bread-making processes and history, and we describe the diversity of yeast species and the genetic diversity of Saccharomyces cerevisiae isolated from bakeries. We then describe the metabolic functioning and diversity of these yeasts and their relevance to improvements in bread quality. Finally, we examine yeast and bacterial interactions in sourdoughs. The purpose of this review is to show that bakery yeast species are interesting models for studying domestication and other evolutionary and ecological processes. Studying these yeasts can contribute much to our fundamental understanding of speciation, evolutionary dynamics, and community assembly, and this knowledge could ultimately be used to adjust, modify, and improve the production of bread and the conservation of microbial diversity.

Heterosis Is a Systemic Property Emerging From Non-linear Genotype-Phenotype Relationships: Evidence From in Vitro Genetics and Computer Simulations.

Heterosis, the superiority of hybrids over their parents for quantitative traits, represents a crucial issue in plant and animal breeding as well as evolutionary biology. Heterosis has given rise to countless genetic, genomic and molecular studies, but has rarely been investigated from the point of view of systems biology. We hypothesized that heterosis is an emergent property of living systems resulting from frequent concave relationships between genotypic variables and phenotypes, or between different phenotypic levels. We chose the enzyme-flux relationship as a model of the concave genotype-phenotype (GP) relationship, and showed that heterosis can be easily created in the laboratory. First, we reconstituted in vitro the upper part of glycolysis. We simulated genetic variability of enzyme activity by varying enzyme concentrations in test tubes. Mixing the content of "parental" tubes resulted in "hybrids," whose fluxes were compared to the parental fluxes. Frequent heterotic fluxes were observed, under conditions that were determined analytically and confirmed by computer simulation. Second, to test this model in a more realistic situation, we modeled the glycolysis/fermentation network in yeast by considering one input flux, glucose, and two output fluxes, glycerol and acetaldehyde. We simulated genetic variability by randomly drawing parental enzyme concentrations under various conditions, and computed the parental and hybrid fluxes using a system of differential equations. Again we found that a majority of hybrids exhibited positive heterosis for metabolic fluxes. Cases of negative heterosis were due to local convexity between certain enzyme concentrations and fluxes. In both approaches, heterosis was maximized when the parents were phenotypically close and when the distributions of parental enzyme concentrations were contrasted and constrained. These conclusions are not restricted to metabolic systems: they only depend on the concavity of the GP relationship, which is commonly observed at various levels of the phenotypic hierarchy, and could account for the pervasiveness of heterosis.

Adaptation of S. cerevisiae to Fermented Food Environments Reveals Remarkable Genome Plasticity and the Footprints of Domestication.

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.

Feedback between environment and traits under selection in a seasonal environment: consequences for experimental evolution.

Batch cultures are frequently used in experimental evolution to study the dynamics of adaptation. Although they are generally considered to simply drive a growth rate increase, other fitness components can also be selected for. Indeed, recurrent batches form a seasonal environment where different phases repeat periodically and different traits can be under selection in the different seasons. Moreover, the system being closed, organisms may have a strong impact on the environment. Thus, the study of adaptation should take into account the environment and eco-evolutionary feedbacks. Using data from an experimental evolution on yeast Saccharomyces cerevisiae, we developed a mathematical model to understand which traits are under selection, and what is the impact of the environment for selection in a batch culture. We showed that two kinds of traits are under selection in seasonal environments: life-history traits, related to growth and mortality, but also transition traits, related to the ability to react to environmental changes. The impact of environmental conditions can be summarized by the length of the different seasons which weight selection on each trait: the longer a season is, the higher the selection on associated traits. Since phenotypes drive season length, eco-evolutionary feedbacks emerge. Our results show how evolution in successive batches can affect season lengths and strength of selection on different traits.

Diversity of flux distribution in central carbon metabolism of S. cerevisiae strains from diverse environments.

BACKGROUND: S. cerevisiae has attracted considerable interest in recent years as a model for ecology and evolutionary biology, revealing a substantial genetic and phenotypic diversity. However, there is a lack of knowledge on the diversity of metabolic networks within this species. RESULTS: To identify the metabolic and evolutionary constraints that shape metabolic fluxes in S. cerevisiae, we used a dedicated constraint-based model to predict the central carbon metabolism flux distribution of 43 strains from different ecological origins, grown in wine fermentation conditions. In analyzing these distributions, we observed a highly contrasted situation in flux variability, with quasi-constancy of the glycolysis and ethanol synthesis yield yet high flexibility of other fluxes, such as the pentose phosphate pathway and acetaldehyde production. Furthermore, these fluxes with large variability showed multimodal distributions that could be linked to strain origin, indicating a convergence between genetic origin and flux phenotype. CONCLUSIONS: Flux variability is pathway-dependent and, for some flux, a strain origin effect can be found. These data highlight the constraints shaping the yeast operative central carbon network and provide clues for the design of strategies for strain improvement.

Efficient ammonium uptake and mobilization of vacuolar arginine by Saccharomyces cerevisiae wine strains during wine fermentation.

BACKGROUND: Under N-limiting conditions, Saccharomyces cerevisiae strains display a substantial variability in their biomass yield from consumed nitrogen -in particular wine yeasts exhibit high growth abilities- that is correlated with their capacity to complete alcoholic fermentation, a trait of interest for fermented beverages industries. The aim of the present work was to assess the contribution of nitrogen availability to the strain-specific differences in the ability to efficiently use N-resource for growth and to identify the underlying mechanisms. We compared the profiles of assimilation of several nitrogen sources (mostly ammonium, glutamine, and arginine) for high and low biomass-producing strains in various conditions of nitrogen availability. We also analyzed the intracellular fate of nitrogen compounds. RESULTS: Strains clustered into two groups at initial nitrogen concentrations between 85 and 385 mg N.L(-1): high biomass producers that included wine strains, were able to complete fermentation of 240 g.L(-1) glucose and quickly consume nitrogen, in contrast to low biomass producers. The two classes of strains exhibited distinctive characteristics that contributed to their differential capacity to produce biomass. The contribution of each characteristic varied according to nitrogen availability. In high biomass producers, the high rate of ammonium uptake resulted in an important consumption of this preferred nitrogen source that promoted the growth of these yeasts when nitrogen was provided in excess. Both classes of yeast accumulated poor nitrogen sources, mostly arginine, in vacuoles during the first stages of growth. However, at end of the growth phase when nitrogen had become limiting, high biomass producers more efficiently used this vacuolar nitrogen fraction for protein synthesis and further biomass formation than low biomass producers. CONCLUSIONS: Overall, we demonstrate that the efficient management of the nitrogen resource, including efficient ammonium uptake and efficient use of the amino acids stored in vacuoles during the late stages of growth, might lead to high biomass production by wine yeasts.

Phenotypic and genotypic convergences are influenced by historical contingency and environment in yeast.

Different organisms have independently and recurrently evolved similar phenotypic traits at different points throughout history. This phenotypic convergence may be caused by genotypic convergence and in addition, constrained by historical contingency. To investigate how convergence may be driven by selection in a particular environment and constrained by history, we analyzed nine life-history traits and four metabolic traits during an experimental evolution of six yeast strains in four different environments. In each of the environments, the population converged toward a different multivariate phenotype. However, the evolution of most traits, including fitness components, was constrained by history. Phenotypic convergence was partly associated with the selection of mutations in genes involved in the same pathway. By further investigating the convergence in one gene, BMH1, mutated in 20% of the evolved populations, we show that both the history and the environment influenced the types of mutations (missense/nonsense), their location within the gene itself, as well as their effects on multiple traits. However, these effects could not be easily predicted from ancestors' phylogeny or past selection. Combined, our data highlight the role of pleiotropy and epistasis in shaping a rugged fitness landscape.