QTL mapping reveals novel genes and mechanisms underlying variations in H2S production during alcoholic fermentation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae requirement for reduced sulfur to synthesize methionine and cysteine during alcoholic fermentation, is mainly fulfilled through the sulfur assimilation pathway. Saccharomyces cerevisiae reduces sulfate into sulfur dioxide (SO2) and sulfide (H2S), whose overproduction is a major issue in winemaking, due to its negative impact on wine aroma. The amount of H2S produced is highly strain-specific and also depends on SO2 concentration, often added to grape must. Applying a bulk segregant analysis to a 96-strain-progeny derived from two strains with different abilities to produce H2S, and comparing allelic frequencies along the genome of pools of segregants producing contrasting H2S quantities, we identified two causative regions involved in H2S production in the presence of SO2. A functional genetic analysis allowed the identification of variants in four genes able to impact H2S formation, viz; ZWF1, ZRT2, SNR2, and YLR125W, and involved in functions and pathways not associated with sulfur metabolism until now. These data point out that, in wine fermentation conditions, redox status, and zinc homeostasis are linked to H2S formation while providing new insights into the regulation of H2S production, and a new vision of the interplay between the sulfur assimilation pathway and cell metabolism.

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.

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.

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.

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.

Oxygen availability and strain combination modulate yeast growth dynamics in mixed culture fermentations of grape must with Starmerella bacillaris and Saccharomyces cerevisiae.

Starmerella bacillaris (synonym Candida zemplinina) is a non-Saccharomyces yeast that has been proposed as a co-inoculant of selected Saccharomyces cerevisiae strains in mixed culture fermentations to enhance the analytical composition of the wines. In order to acquire further knowledge on the metabolic interactions between these two species, in this study we investigated the impact of oxygen addition and combination of Starm. bacillaris with S. cerevisiae strains on the microbial growth and metabolite production. Fermentations were carried out under two different conditions of oxygen availability. Oxygen availability and strain combination clearly influenced the population dynamics throughout the fermentation. Oxygen concentration increased the survival time of Starm. bacillaris and decreased the growth rate of S. cerevisiae strains in mixed culture fermentations, whereas it did not affect the growth of the latter in pure culture fermentations. This study reveals new knowledge about the influence of oxygen availability on the successional evolution of yeast species during wine fermentation.

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.

Yeast multistress resistance and lag-phase characterisation during wine fermentation.

Saccharomyces cerevisiae has been used to perform wine fermentation for several millennia due to its endurance and unmatched qualities. Nevertheless, at the moment of inoculation, wine yeasts must cope with specific stress factors that still challenge wine makers by slowing down or compromising the fermentation process. To better assess the role of genetic and environmental factors that govern multistress resistance during the wine fermentation lag phase, we used a factorial plan to characterise the individual and combined impact of relevant stress factors on eight S. cerevisiae and two non-S. cerevisiae wine yeast strains that are currently commercialised. The S. cerevisiae strains are very genetically diverse, belonging to the wine and flor groups, and frequently contain a previously described XVIVIII translocation that confers increased resistance to sulphites. We found that low temperature and osmotic stress substantially affected all strains, promoting considerably extended lag phases. SO2 addition had a partially temperature-dependent effect, whereas low phytosterol and thiamine concentrations impacted the lag phase in a strain-dependent manner. No major synergic effects of multistress were detected. The diversity of lag-phase durations and stress responses observed among wine strains offer new insights to better control this critical step of fermentation.

Exploring the potential of Saccharomyces eubayanus as a parent for new interspecies hybrid strains in winemaking.

Yeast cryotolerance brings some advantages for wine fermentations, including the improved aromatic complexity of white wines. Naturally cold-tolerant strains are generally less adept at wine fermentation but fermentative fitness can potentially be improved through hybridization. Here we studied the potential of using hybrids involving Saccharomyces eubayanus and a S. cerevisiae wine strain for low-temperature winemaking. Through screening the performance in response to variable concentrations of sugar, nitrogen and temperature, we isolated one hybrid strain that exhibited the superior performance. This hybrid strain was propagated and dried in pilot scale and tested for the fermentation of Macabeu and Sauvignon blanc grape musts. We obtained highly viable active dry yeast, which was able to efficiently ferment the grape musts with superior production of aroma active volatiles, in particular, 2-phenylethanol. The genome sequences of the hybrid strains revealed variable chromosome inheritance among hybrids, particularly within the S. cerevisiae subgenome. With the present paper, we expand the knowledge on the potentialities of using S. eubayanus hybrids in industrial fermentation at beverages other than lager beer.

Key role of lipid management in nitrogen and aroma metabolism in an evolved wine yeast strain.

BACKGROUND: Fermentative aromas play a key role in the organoleptic profile of young wines. Their production depends both on yeast strain and fermentation conditions. A present-day trend in the wine industry consists in developing new strains with aromatic properties using adaptive evolution approaches. An evolved strain, Affinity™ ECA5, overproducing esters, was recently obtained. In this study, dynamics of nitrogen consumption and of the fermentative aroma synthesis of the evolved and its ancestral strains were compared and coupled with a transcriptomic analysis approach to better understand the metabolic reshaping of Affinity™ ECA5. RESULTS: Nitrogen assimilation was different between the two strains, particularly amino acids transported by carriers regulated by nitrogen catabolite repression. We also observed differences in the kinetics of fermentative aroma production, especially in the bioconversion of higher alcohols into acetate esters. Finally, transcriptomic data showed that the enhanced bioconversion into acetate esters by the evolved strain was associated with the repression of genes involved in sterol biosynthesis rather than an enhanced expression of ATF1 and ATF2 (genes coding for the enzymes responsible for the synthesis of acetate esters from higher alcohols). CONCLUSIONS: An integrated approach to yeast metabolism-combining transcriptomic analyses and online monitoring data-showed differences between the two strains at different levels. Differences in nitrogen source consumption were observed suggesting modifications of NCR in the evolved strain. Moreover, the evolved strain showed a different way of managing the lipid source, which notably affected the production of acetate esters, likely because of a greater availability of acetyl-CoA for the evolved strain.

Starmerella bacillaris and Saccharomyces cerevisiae mixed fermentations to reduce ethanol content in wine.

Decreasing the ethanol content in wine is a current challenge, mainly due to the global climate change and to the consumer preference for wines from grapes with increased maturity. In this study, a central composite design (CCD) and response surface methodology (RSM) approach was used to investigate the potential application of Starmerella bacillaris (synonym Candida zemplinina) in combination with Saccharomyces cerevisiae, in mixed (co-inoculated and sequential) cultures, to understand better the mechanism of co-habitation and achieve the objective of reducing the ethanol in wines. Laboratory scale fermentations demonstrated a decrease up to 0.7 % (v/v) of ethanol and an increase of about 4.2 g/L of glycerol when S. cerevisiae was inoculated with a delay of 48 h with respect to the inoculation of S. bacillaris. Pilot-scale fermentations, carried out in winemaking conditions, confirmed the laboratory results. This study demonstrates that the combination of strains and inoculation protocol could help to reduce the ethanol content in wines.

Combined effects of nutrients and temperature on the production of fermentative aromas by Saccharomyces cerevisiae during wine fermentation.

Volatile compounds produced by yeast during fermentation greatly influence the organoleptic qualities of wine. We developed a model to predict the combined effects of initial nitrogen and phytosterol content and fermentation temperature on the production of volatile compounds. We used a Box-Behnken design and response surface modeling to study the response of Lalvin EC1118® to these environmental conditions. Initial nitrogen content had the greatest influence on most compounds; however, there were differences in the value of fermentation parameters required for the maximal production of the various compounds. Fermentation parameters affected differently the production of isobutanol and isoamyl alcohol, although their synthesis involve the same enzymes and intermediate. We found differences in regulation of the synthesis of acetates of higher alcohols and ethyl esters, suggesting that fatty acid availability is the main factor influencing the synthesis of ethyl esters whereas the production of acetates depends on the activity of alcohol acetyltransferases. We also evaluated the effect of temperature on the total production of three esters by determining gas-liquid balances. Evaporation largely accounted for the effect of temperature on the accumulation of esters in liquid. Nonetheless, the metabolism of isoamyl acetate and ethyl octanoate was significantly affected by this parameter. We extended this study to other strains. Environmental parameters had a similar effect on aroma production in most strains. Nevertheless, the regulation of the synthesis of fermentative aromas was atypical in two strains: Lalvin K1M® and Affinity™ ECA5, which produces a high amount of aromatic compounds and was obtained by experimental evolution.

Reduction of ethanol yield and improvement of glycerol formation by adaptive evolution of the wine yeast Saccharomyces cerevisiae under hyperosmotic conditions.

There is a strong demand from the wine industry for methodologies to reduce the alcohol content of wine without compromising wine's sensory characteristics. We assessed the potential of adaptive laboratory evolution strategies under hyperosmotic stress for generation of Saccharomyces cerevisiae wine yeast strains with enhanced glycerol and reduced ethanol yields. Experimental evolution on KCl resulted, after 200 generations, in strains that had higher glycerol and lower ethanol production than the ancestral strain. This major metabolic shift was accompanied by reduced fermentative capacities, suggesting a trade-off between high glycerol production and fermentation rate. Several evolved strains retaining good fermentation performance were selected. These strains produced more succinate and 2,3-butanediol than the ancestral strain and did not accumulate undesirable organoleptic compounds, such as acetate, acetaldehyde, or acetoin. They survived better under osmotic stress and glucose starvation conditions than the ancestral strain, suggesting that the forces that drove the redirection of carbon fluxes involved a combination of osmotic and salt stresses and carbon limitation. To further decrease the ethanol yield, a breeding strategy was used, generating intrastrain hybrids that produced more glycerol than the evolved strain. Pilot-scale fermentation on Syrah using evolved and hybrid strains produced wine with 0.6% (vol/vol) and 1.3% (vol/vol) less ethanol, more glycerol and 2,3-butanediol, and less acetate than the ancestral strain. This work demonstrates that the combination of adaptive evolution and breeding is a valuable alternative to rational design for remodeling the yeast metabolic network.

Investigating Key Volatile Compound Diffusion in Cocoa Beans during Yeast Fermentation-like Incubation.

An experimental setup was devised to investigate the permeability of cocoa bean seed coat and pulp to key volatile compounds during fermentation. Four labeled compounds (ethyl acetate-d3, ethyl octanoate-d15, 2-phenylethanol-d5, linalool-d5) and 2 unlabeled (beta-damascenone, delta-decalactone) were chosen for the investigation. The beans (cotyledons), depulped beans, or pulped beans were immersed separately in a concentrated solution of these volatile compounds at 36 or 46 °C for durations ranging from 3 to 120 h. The imbibed beans were dissected, and the cotyledons were analyzed by SPME-GC/MS. The diffusion of volatile compounds from the external solution to the seed was categorized into three groups: (1) not diffusible (ethyl octanoate-d15); (2) semidiffusible (ethyl acetate); and (3) totally diffusible (2-phenylethanol-d5, linalool-d5, beta-damascenone, delta-decalactone). The impact of the yeast on volatile compound diffusion was also investigated by immerging the pulped beans into the same concentrated solution with a yeast starter. Results highlighted the positive role of yeast in the diffusion of volatile compounds. The starter positively contributed to volatile compound diffusion after a transition phase occurring at approximately 48 h of fermentation, enriching the cocoa beans with key aromatic volatile compounds.

Pilot-scale evaluation the enological traits of a novel, aromatic wine yeast strain obtained by adaptive evolution.

In the competitive context of the wine market, there is a growing interest for novel wine yeast strains that have an overall good fermentation capacity and that contribute favorably to the organoleptic quality of wine. Using an adaptive evolution strategy based on growth on gluconate as sole carbon source, we recently obtained wine yeasts with improved characteristics in laboratory-scale fermentations. The characteristics included enhanced fermentation rate, decreased formation of acetate and greater production of fermentative aroma. We report an evaluation of the potential value of the evolved strain ECA5™ for winemaking, by comparing its fermentation performance and metabolite production to those of the parental strain in pilot-scale fermentation trials, with various grape cultivars and winemaking conditions. We show that the evolved strain has outstanding attributes relative to the parental wine yeast strain, and in particular the production of less volatile acidity and greater production of desirable volatile esters, important for the fruity/flowery character of wines. This study highlights the potential of evolutionary engineering for the generation of strains with a broad range of novel properties, appropriate for rapid application in the wine industry.

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.

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.

Evolutionary engineered Saccharomyces cerevisiae wine yeast strains with increased in vivo flux through the pentose phosphate pathway.

Amplification of the flux toward the pentose phosphate (PP) pathway might be of interest for various S. cerevisiae based industrial applications. We report an evolutionary engineering strategy based on a long-term batch culture on gluconate, a substrate that is poorly assimilated by S. cerevisiae cells and is metabolized by the PP pathway. After adaptation for various periods of time, we selected strains that had evolved a greater consumption capacity for gluconate. (13)C metabolic flux analysis on glucose revealed a redirection of carbon flux from glycolysis towards the PP pathway and a greater synthesis of lipids. The relative flux into the PP pathway was 17% for the evolved strain (ECA5) versus 11% for the parental strain (EC1118). During wine fermentation, the evolved strains displayed major metabolic changes, such as lower levels of acetate production, higher fermentation rates and enhanced production of aroma compounds. These represent a combination of novel traits, which are of great interest in the context of modern winemaking.