Differences in the management of intracellular redox state between wine yeast species dictate their fermentation performances and metabolite production.

The maintenance of the balance between oxidised and reduced redox cofactors is essential for the functioning of many cellular processes in all living organisms. While the electron transport chain plays a key role in maintaining this balance under respiratory conditions, its inactivity in the absence of oxygen poses a challenge that yeasts such as Saccharomyces cerevisiae overcome through the production of various metabolic end-products during alcoholic fermentation. In this study, we investigated the diversity occurring between wine yeast species in their management of redox balance and its consequences on the fermentation performances and the formation of metabolites. To this aim, we quantified the changes in NAD(H) and NADP(H) concentrations and redox status throughout the fermentation of 6 wine yeast species. While the availability of NADP and NADPH remained balanced and stable throughout the process for all the strains, important differences between species were observed in the dynamics of NAD and NADH intracellular pools. A comparative analysis of these data with the fermentation capacity and metabolic profiles of the strains revealed that Saccharomyces cerevisiae, Torulaspora delbrueckii and Lachancea thermotolerans strains were able to reoxidise NADH to NAD throughout the fermentation, mainly by the formation of glycerol. These species exhibited good fermentation capacities. Conversely, Starmerella bacillaris and Metschnikowia pulcherrima species were unable to regenerate NAD as early as one third of sugars were consumed, explaining at least in part their poor growth and fermentation performances. The Kluyveromyces marxianus strain exhibited a specific behaviour, by maintaining similar levels of NAD and NADH throughout the process. This balance between oxidised and reduced redox cofactors ensured the consumption of a large part of sugars by this species, despite a low fermentation rate. In addition, the dynamics of redox cofactors affected the production of by-products by the various strains either directly or indirectly, through the formation of precursors. Major examples are the increased formation of glycerol by S. bacillaris and M. pulcherrima strains, as a way of trying to reoxidise NADH, and the greater capacity to produce acetate and derived metabolites of yeasts capable of maintaining their redox balance. Overall, this study provided new insight into the contribution of the management of redox status to the orientation of yeast metabolism during fermentation. This information should be taken into account when developing strategies for more efficient and effective fermentation.

Beyond S. cerevisiae for winemaking: Fermentation-related trait diversity in the genus Saccharomyces.

Saccharomyces cerevisiae is the yeast of choice for most inoculated wine fermentations worldwide. However, many other yeast species and genera display phenotypes of interest that may help address the environmental and commercial challenges the wine industry has been facing in recent years. This work aimed to provide, for the first time, a systematic phenotyping of all Saccharomyces species under winemaking conditions. For this purpose, we characterized the fermentative and metabolic properties of 92 Saccharomyces strains in synthetic grape must at two different temperatures. The fermentative potential of alternative yeasts was higher than expected, as nearly all strains were able to complete fermentation, in some cases more efficiently than commercial S. cerevisiae strains. Various species showed interesting metabolic traits, such as high glycerol, succinate and odour-active compound production, or low acetic acid production, compared to S. cerevisiae. Altogether, these results reveal that non-cerevisiae Saccharomyces yeasts are especially interesting for wine fermentation, as they may offer advantages over both S. cerevisiae and non-Saccharomyces strains. This study highlights the potential of alternative Saccharomyces species for winemaking, paving the way for further research and, potentially, for their industrial exploitation.

The growth and metabolome of Saccharomyces uvarum in wine fermentations are strongly influenced by the route of nitrogen assimilation.

Nitrogen is a critical nutrient in beverage fermentations, influencing fermentation performance and formation of compounds that affect organoleptic properties of the product. Traditionally, most commercial wine fermentations rely on Saccharomyces cerevisiae but the potential of alternative yeasts is increasingly recognised because of the possibility to deliver innovative products and process improvements. In this regard, Saccharomyces uvarum is an attractive non-traditional yeast that, while quite closely related to S. cerevisiae, displays a different fermentative and aromatic profile. Although S. uvarum is used in cider-making and in some winemaking, better knowledge of its physiology and metabolism is required if its full potential is to be realised. To address this gap, we performed a comparative analysis of the response of S. uvarum and S. cerevisiae to 13 different sources of nitrogen, assessing key parameters such as growth, fermentation performance, the production of central carbon metabolites and aroma volatile compounds. We observed that the two species differ in the production of acetate, succinate, medium-chain fatty acids, phenylethanol, phenylethyl acetate, and fusel/branched acids in ways that reflect different distribution of fluxes in the metabolic network. The integrated analysis revealed different patterns of yeast performance and activity linked to whether growth was on amino acids metabolised via the Ehrlich pathway or on amino acids and compounds assimilated through the central nitrogen core. This study highlights differences between the two yeasts and the importance that nitrogen metabolism can play in modulating the sensory profile of wine when using S. uvarum as the fermentative yeast.

The evolution and role of the periplasmic asparaginase Asp3 in yeast.

The study of nitrogen assimilation in yeast is of interest from genetic, evolutionary, and biotechnological perspectives. Over the course of evolution, yeasts have developed sophisticated control mechanisms to regulate nitrogen metabolism, with domesticated lineages sometimes displaying particular specialisation. The focus of this study was on assimilation of asparagine, which is a significant nutritional source for some alcoholic fermentations. We were particularly interested in ASP3, which encodes a periplasmic asparaginase and that was proposed to have been acquired relatively recently in S. cerevisiae by horizontal gene transfer. We examined 1680 S. cerevisiae genome assemblies to evaluate the distribution and evolutionary trajectory of ASP3. Our findings suggest an alternative hypothesis that ASP3 is an ancient Saccharomyces gene that has generally been lost over the course of evolution but has been retained in certain fermentative environments. As asparagine is the major nitrogen source in apple juice, we explored whether the presence of ASP3 would confer a growth advantage. Interestingly, we found that although ASP3 enhances growth when asparagine is the sole nitrogen source, the same effect is not seen in apple juice. These data indicate that growth in pure culture may not reflect the original selective environment for ASP3+ strains and highlight the role that complex regulation may play in optimising nitrogen assimilation in yeasts.

Species-Dependent Metabolic Response to Lipid Mixtures in Wine Yeasts.

Lipids are essential energy storage compounds and are the core structural elements of all biological membranes. During wine alcoholic fermentation, the ability of yeasts to adjust the lipid composition of the plasma membrane partly determines their ability to cope with various fermentation-related stresses, including elevated levels of ethanol and the presence of weak acids. In addition, the lipid composition of grape juice also impacts the production of many wine-relevant aromatic compounds. Several studies have evaluated the impact of lipids and of their metabolism on fermentation performance and aroma production in the dominant wine yeast Saccharomyces cerevisiae, but limited information is available on other yeast species. Thus, the aim of this study was to evaluate the influence of specific fatty acid and sterol mixtures on various non-Saccharomyces yeast fermentation rates and the production of primary fermentation metabolites. The data show that the response to different lipid mixtures is species-dependent. For Metschnikowia pulcherrima, a slight increase in carbon dioxide production was observed in media enriched with unsaturated fatty acids whereas Kluyveromyces marxianus fermented significantly better in synthetic media containing a higher concentration of polyunsaturated fatty acids than monounsaturated fatty acids. Torulaspora delbrueckii fermentation rate increased in media supplemented with lipids present at an equimolar concentration. The data indicate that these different responses may be linked to variations in the lipid profile of these yeasts and divergent metabolic activities, in particular the regulation of acetyl-CoA metabolism. Finally, the results suggest that the yeast metabolic footprint and ultimately the wine organoleptic properties could be optimized via species-specific lipid adjustments.

Genetic bases for the metabolism of the DMS precursor S-methylmethionine by Saccharomyces cerevisiae.

Dimethyl sulfide (DMS) is a sulfur containing volatile that enhances general fruity aroma and imparts aromatic notes in wine. The most important precursor of DMS is S-methylmethionine (SMM), which is synthesized by grapes and can be metabolized by the yeast S. cerevisiae during wine fermentation. Precursor molecules left after fermentation are chemically converted to DMS during wine maturation, meaning that wine DMS levels are determined by the amount of remaining precursors at bottling. To elucidate SMM metabolism in yeast we performed quantitative trait locus (QTL) mapping using a population of 130 F2-segregants obtained from a cross between two wine yeast strains, and we detected one major QTL explaining almost 30% of trait variation. Within the QTL, gene YLL058W and SMM transporter gene MMP1 were found to influence SMM metabolism, from which MMP1 has the bigger impact. We identified and characterized a variant coding for a truncated transporter with superior SMM preserving attributes. A population analysis with 85 yeast strains from different origins revealed a significant association of the variant to flor strains and minor occurrence in cheese and wine strains. These results will help selecting and improving S. cerevisiae strains for the production of wine and other fermented foods containing DMS such as cheese or beer.

Torulaspora delbrueckii Phenotypic and Metabolic Profiling towards Its Biotechnological Exploitation.

Wine is a particularly complex beverage resulting from the combination of several factors, with yeasts being highlighted due to their fundamental role in its development. For many years, non-Saccharomyces yeasts were believed to be sources of spoilage and contamination, but this idea was challenged, and many of these yeasts are starting to be explored for their beneficial input to wine character. Among this group, Torulaspora delbrueckii is gaining relevance within the wine industry, owing to its low volatile acidity production, increased release of aromatic compounds and enhanced color intensity. In addition, this yeast was also attracting interest in other biotechnological areas, such as bread and beer fermentation. In this work, a set of 40 T. delbrueckii strains, of varied geographical and technological origins, was gathered in order to characterize the phenotypic behavior of this species, focusing on different parameters of biotechnological interest. The fermentative performance of the strains was also evaluated through individual fermentations in synthetic grape must with the isolates' metabolic profile being assessed by HPLC. Data analysis revealed that T. delbrueckii growth is significantly affected by high temperature (37 °C) and ethanol concentrations (up to 18%), alongside 1.5 mM SO2, showing variable fermentative power and yields. Our computation models suggest that the technological origin of the strains seems to prevail over the geographical origin as regards the influence on yeast properties. The inter-strain variability and profile of the products through the fermentative processes reinforce the potential of T. delbrueckii from a biotechnological point of view.

How to modulate the formation of negative volatile sulfur compounds during wine fermentation?

Beyond the production of positive aromas during alcoholic fermentation, Saccharomyces cerevisiae metabolism also results in the formation of volatile compounds detrimental to wine quality, including a wide range of volatile sulfur compounds (VSCs). The formation of these VSCs during wine fermentation is strongly variable and depends on biological and environmental factors. First, the comparison of the VSCs profile of 22 S. cerevisiae strains provided a comprehensive overview of the intra-species diversity in VSCs production: according to their genetic background, strains synthetized from 1 to 6 different sulfur molecules, in a 1- to 30-fold concentration range. The impact of fermentation parameters on VSCs production was then investigated. We identified yeast assimilable nitrogen, cysteine, methionine and pantothenic acid contents - but not SO2 content - as the main factors modulating VSCs production. In particular, ethylthioacetate and all the VSCs deriving from methionine catabolism displayed a maximal production at yeast assimilable nitrogen concentrations around 250 mg/L; pantothenic acid had a positive impact on compounds deriving from methionine catabolism through the Ehrlich pathway but a negative one on the production of thioesters. Overall, these results highlight those factors to be taken into account to modulate the formation of negative VSCs and limit their content in wines.

Influence of single nitrogen compounds on growth and fermentation performance of Starmerella bacillaris and Saccharomyces cerevisiae during alcoholic fermentation.

Nitrogen is among the essential nutriments that govern interactions between yeast species in the wine environment. A thorough knowledge of how these yeasts assimilate the nitrogen compounds of grape juice is an important prerequisite for a successful co- or sequential fermentation. In the present study, we investigated the efficiency of 18 nitrogen sources for sustaining the growth and fermentation of two Starm. bacillaris strains displaying metabolic properties, compared to the reference yeast S. cerevisiae The analysis of growth and fermentation parameters provided a comprehensive picture of Starm. bacillaris preferences with respect to nitrogen sources for sustained growth and fermentation. Important differences were observed in S. cerevisiae regarding rates, final population and CO2 production. In particular, Lys and His supported substantial Starm. bacillaris growth and fermentation contrary to S. cerevisiae, while only 3 nitrogen sources, Arg, NH4+ and Ser, promoted S. cerevisiae growth more efficiently than that of Starm. bacillaris strains. Furthermore, Starm. bacillaris strains displayed a higher fermentative activity than S. cerevisiae during the first phase of culture with Gly or Thr, when the former species consumed solely fructose. Finally, no correlation has been shown between the ability of nitrogen sources to support growth and their fermentation efficiency. The specificities of Starm. bacillaris regarding nitrogen sources preferences are related to its genetic background, but further investigations are needed to elucidate the molecular mechanisms involved. These data are essential elements to be taken into account in order to make the best use of the potential of the two species.IMPORTANCE Mixed fermentations combining non-Saccharomyces and S. cerevisiae strains are increasingly implemented in the wine sector as they offer promising opportunities to diversify the flavour profile of end-products. However, competition for nutrients between species can cause fermentation problems, which is a severe hindrance to the development of these approaches. With the knowledge provided in this study on the nitrogen preferences of Starm. bacillaris, winemakers will be able to set up a nitrogen nutrition scheme adapted to the requirement of each species during mixed fermentation, through must supplementation with relevant nitrogen compounds. This will prevent nitrogen depletion or competition between yeasts for nitrogen sources, and consequently potential issues during fermentation. The data of this study highlight the importance of an appropriate nitrogen resource management during co- or sequential fermentation for fully exploiting the phenotypic potential of non-Saccharomyces yeasts.

Nitrogen metabolism in three non-conventional wine yeast species: A tool to modulate wine aroma profiles.

The positive impact of certain non-Saccharomyces yeasts on the aromatic profile of wines has been well documented in literature and their industrial use in association with S. cerevisiae is now recommended. Competition between non-Saccharomyces species and Saccharomyces cerevisiae for various nutrients, especially nitrogen sources, greatly impacts the production of aroma compounds. In this study, we further explored the impact of different nitrogen nutrition strategies on the production of carbon and sulphur volatile compounds of three non-Saccharomyces strains, namely Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae sequentially inoculated with S. cerevisiae in Sauvignon blanc and Shiraz grape musts. Nitrogen additions were implemented according the specific requirement of each species. At the end of fermentation, we observed specific metabolic signatures for each strain in response to the nature of the nitrogen source suggesting strain-specific metabolic fluxes present. Overall, these results confirmed and further explored the interconnection between nitrogen sources and aroma metabolism (including that of higher alcohols, fatty acids, esters and volatile sulphur compounds), and their variations according to species and the nature of the nitrogen source. The knowledge generated provides new insights to modulate the aroma profile of wines produced with non-Saccharomyces species.

Isotopic Tracers Unveil Distinct Fates for Nitrogen Sources during Wine Fermentation with Two Non-Saccharomyces Strains.

Non-Saccharomyces yeast strains have become increasingly prevalent in the food industry, particularly in winemaking, because of their properties of interest both in biological control and in complexifying flavour profiles in end-products. However, unleashing the full potential of these species would require solid knowledge of their physiology and metabolism, which is, however, very limited to date. In this study, a quantitative analysis using 15N-labelled NH4Cl, arginine, and glutamine, and 13C-labelled leucine and valine revealed the specificities of the nitrogen metabolism pattern of two non-Saccharomyces species, Torulaspora delbrueckii and Metschnikowia pulcherrima. In T. delbrueckii, consumed nitrogen sources were mainly directed towards the de novo synthesis of proteinogenic amino acids, at the expense of volatile compounds production. This redistribution pattern was in line with the high biomass-producer phenotype of this species. Conversely, in M. pulcherrima, which displayed weaker growth capacities, a larger proportion of consumed amino acids was catabolised for the production of higher alcohols through the Ehrlich pathway. Overall, this comprehensive overview of nitrogen redistribution in T. delbrueckii and M. pulcherrima provides valuable information for a better management of co- or sequential fermentation combining these species with Saccharomyces cerevisiae.

Impact of Nutrient Availability on the Fermentation and Production of Aroma Compounds Under Sequential Inoculation With M. pulcherrima and S. cerevisiae.

Non-Saccharomyces yeasts are currently widely used in winemaking to enhance aroma profile diversity among wines. The use of Metschnikowia pulcherrima in sequential inoculation with S. cerevisiae was compared to the inoculation of a pure culture of S. cerevisiae. Moreover, various concentrations of sugar, nitrogen and lipids were tested in synthetic must to assess their impact on fermentation and its outcomes using a Box-Behnken design. Due to its phenotypic specificities, early inoculation with M. pulcherrima led to important modifications, first altering the fermentation kinetics. This may relate, at least in part, to the depletion of some nitrogen sources by M. pulcherrima during the first part of fermentation. Beyond these negative interactions on fermentation performance, comparisons between pure cultures and sequentially inoculated cultures revealed changes in the distribution of carbon fluxes during fermentation in presence of M. pulcherrima, resulting in a positive impact on the production of central carbon metabolites and aromas. Furthermore, the expression of varietal thiols was strongly increased as a consequence of positive interactions between the two species. The mechanism of this release still needs to be investigated. Significant differences in the final concentrations of fermentative and varietal aromas depending on the initial must composition were obtained under both inoculation strategies. Interestingly, the response to changes in nutrient availability varied according to the inoculation modality. In particular, a greater incidence of lipids on the production of fatty acids and their ethyl esters derivatives was found during sequential fermentation compared with pure culture, to be viewed in combination with the metabolic characteristics of M. pulcherrima regarding the production of volatile compounds from acetyl-CoA. Overall, the importance of managing nutrient availability under M. pulcherrima/S. cerevisiae sequential inoculation in order to derive the maximum benefit from the potentialities of the non-Saccharomyces species while carrying out fermentation to dryness was highlighted.

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.

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

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

Analysing the impact of the nature of the nitrogen source on the formation of volatile compounds to unravel the aroma metabolism of two non-Saccharomyces strains.

Even though non-Saccharomyces yeasts were regarded as spoilage microorganisms for a long time, their abilities to improve and diversify the aromatic profile of wines are now well recognized. Consequently, their use in combination with S. cerevisiae strains during winemaking has attracted substantial attention over the last decade. However, our limited understanding of the metabolism and physiology of these species remains a barrier to promoting efficient exploitation of their full potential. In this study, we further explored the metabolism involved in the production of fermentative volatile compounds of two commercial non-Saccharomyces strains, T. delbrueckii Biodiva™ and M. pulcherrima Flavia®, in comparison with the reference wine yeast S. cerevisiae Lalvin EC1118®. After growing these strains in the presence of 24 different N-compounds, particular attention was paid to the influence of the nitrogen source on the profile of aroma compounds synthesized by these yeasts (higher alcohols and acids, medium-chain fatty acids and their acetate or ethyl esters derivatives). A comprehensive analysis of the dataset showed that these three species were able to produce all the fermentative aromas, regardless of the nitrogen source, demonstrating the key contribution of the central carbon metabolism to the formation of volatile molecules. Nevertheless, we also observed some specific phenotypic traits for each of the strains in their assimilation capacities for the various nitrogen nutrients as well as in their response to the nature of the nitrogen source in terms of the production of volatile molecules. These observations revealed the intricacy and interconnection between the networks involved in nitrogen consumption and aroma production. These differences are likely related to the genetic backgrounds of the strains. Overall, this study expands our understanding of the metabolic processes responsible for the formation of volatile compounds during wine fermentation and their variations according to species and the nature of the nitrogen source. This knowledge provides a new platform for the more efficient exploitation of non-Saccharomyces strains during winemaking, improving the management of the fermentation.

A comparison of the nitrogen metabolic networks of Kluyveromyces marxianus and Saccharomyces cerevisiae.

In grape must, nitrogen is available as a complex mixture of various compounds (ammonium and amino acids). Wine yeasts assimilate these multiple sources in order to suitably fulfil their anabolic requirements during alcoholic fermentation. Nevertheless, the order of uptake and the intracellular fate of these sources are likely to differ between strains and species. Using a two-pronged strategy of isotopic filiation and RNA sequencing, the metabolic network of nitrogen utilization and its regulation in Kluyveromyces marxianus were described, in comparison with those of Saccharomyces cerevisiae. The data highlighted differences in the assimilation of ammonium and arginine between the two species. The data also revealed that the metabolic fate of certain nitrogen sources differed, thereby resulting in the production of various amounts of key wine aroma compounds. These observations were corroborated by the gene expression analysis.

Impact of the timing and the nature of nitrogen additions on the production kinetics of fermentative aromas by Saccharomyces cerevisiae during winemaking fermentation in synthetic media.

During alcoholic fermentation, many parameters, including the nitrogen composition of the must, can affect aroma production. The aim of this study was to examine the impact of several types of nitrogen sources added at different times during fermentation. Nitrogen was added as ammonium or a mixture of amino acids at the beginning of fermentation or at the start of the stationary phase. These conditions were tested with two Saccharomyces cerevisiae strains that have different nitrogen requirements. The additions systematically reduced the fermentation duration. The aroma production was impacted by both the timing of the addition and the composition of the nitrogen source. Propanol appeared to be a metabolic marker of the presence of assimilable nitrogen in the must. The production of ethyl esters was slightly higher after the addition of any type of nitrogen; the production of higher alcohols other than propanol was unchanged, and acetate esters were overproduced due to the overexpression of the genes ATF1 and ATF2. Finally the parameter affecting the most the synthesis of beneficial aromas was the addition timing: The supply of organic nitrogen at the beginning of the stationary phase was more favorable for the synthesis of beneficial aromas.

Specific Phenotypic Traits of Starmerella bacillaris Related to Nitrogen Source Consumption and Central Carbon Metabolite Production during Wine Fermentation.

Over the last few years, the potential of non-Saccharomyces yeasts to improve the sensory quality of wine has been well recognized. In particular, the use of Starmerella bacillaris in mixed fermentations with Saccharomyces cerevisiae was reported as an appropriate way to enhance glycerol formation and reduce ethanol production. However, during sequential fermentation, many factors, such as the inoculation timing, strain combination, and physical and biochemical interactions, can affect yeast growth, the fermentation process, and/or metabolite synthesis. Among them, the availability of yeast-assimilable nitrogen (YAN), due to its role in the control of growth and fermentation, has been identified as a key parameter. Consequently, a comprehensive understanding of the metabolic specificities and the nitrogen requirements would be valuable to better exploit the potential of Starm. bacillaris during wine fermentation. In this study, marked differences in the consumption of the total and individual nitrogen sources were registered between the two species, while the two Starm. bacillaris strains generally behaved uniformly. Starm. bacillaris strains are differentiated by their preferential uptake of ammonium compared with amino acids that are poorly assimilated or even produced (alanine). Otherwise, the non-Saccharomyces yeast exhibits low activity through the acetaldehyde pathway, which triggers an important redistribution of fluxes through the central carbon metabolic network. In particular, the formation of metabolites deriving from the two glycolytic intermediates glyceraldehyde-3-phosphate and pyruvate is substantially increased during fermentations by Starm. bacillaris This knowledge will be useful to better control the fermentation process in mixed fermentation with Starm. bacillaris and S. cerevisiaeIMPORTANCE Mixed fermentations using a controlled inoculation of Starmerella bacillaris and Saccharomyces cerevisiae starter cultures represent a feasible way to modulate wine composition that takes advantage of both the phenotypic specificities of the non-Saccharomyces strain and the ability of S. cerevisiae to complete wine fermentation. However, according to the composition of grape juices, the consumption by Starm. bacillaris of nutrients, in particular of nitrogen sources, during the first stages of the process may result in depletions that further limit the growth of S. cerevisiae and lead to stuck or sluggish fermentations. Consequently, understanding the preferences of non-Saccharomyces yeasts for the nitrogen sources available in grape must together with their phenotypic specificities is essential for an efficient implementation of sequential wine fermentations with Starm. bacillaris and S. cerevisiae species. The results of our study demonstrate a clear preference for ammonium compared to amino acids for the non-Saccharomyces species. This finding underlines the importance of nitrogen sources, which modulate the functional characteristics of inoculated yeast strains to better control the fermentation process and product quality.

Fermentation performances and aroma production of non-conventional wine yeasts are influenced by nitrogen preferences.

Saccharomyces cerevisiae is currently the most important yeast involved in food fermentations, particularly in oenology. However, several other yeast species occur naturally in grape must that are highly promising for diversifying and improving the aromatic profile of wines. If the nitrogen requirement of S. cerevisiae has been described in detail, those of non-Saccharomyces yeasts remain poorly studied despite their increasingly widespread use in winemaking. With a view to improving the use of non-Saccharomyces yeasts in winemaking, we explored the fermentation performances, the utilisation of nitrogen sources and the volatile compound production of 10 strains of non-conventional yeasts in pure culture. Two different conditions were tested: one mimicking the grape juice's nitrogen composition and one with all the nitrogen sources at the same level. We highlighted the diversity in terms of nitrogen preference and amount consumed among the yeast strains. Some nitrogen sources (arginine, glutamate, glycine, tryptophan and γ-aminobutyric acid) displayed the largest variations between strains throughout the fermentation. Several non-Saccharomyces strains produced important aroma compounds such as higher alcohols, acetate and ethyl esters in significantly higher quantities than S. cerevisiae.

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.