Nicotinic acid availability impacts redox cofactor metabolism in Saccharomyces cerevisiae during alcoholic fermentation.

Anaerobic alcoholic fermentation, particularly in high-sugar environments, presents metabolic challenges for yeasts. Crabtree-positive yeasts, including Saccharomyces cerevisiae, prefer fermentation even in the presence of oxygen. These yeasts rely on internal NAD+ recycling and extracellular assimilation of its precursor, nicotinic acid (vitamin B3), rather than de novo NAD+ production. Surprisingly, nicotinic acid assimilation is poorly characterized, even in S. cerevisiae. This study elucidated the timing of nicotinic acid uptake during grape juice-like fermentation and its impact on NAD(H) levels, the NAD+/NADH ratio, and metabolites produced. Complete uptake of extracellular nicotinic acid occurred premid-exponential phase, thereafter small amounts of vitamin B3 were exported back into the medium. Suboptimal levels of nicotinic acid were correlated with slower fermentation and reduced biomass, disrupting redox balance and impeding NAD+ regeneration, thereby affecting metabolite production. Metabolic outcomes varied with nicotinic acid concentrations, linking NAD+ availability to fermentation efficiency. A model was proposed encompassing rapid nicotinic acid uptake, accumulation during cell proliferation, and recycling with limited vitamin B3 export. This research enhances the understanding of nicotinic acid uptake dynamics during grape juice-like fermentation. These insights contribute to advancing yeast metabolism research and have profound implications for the enhancement of biotechnological practices and the wine-making industry.

Exploring fermentative metabolic response to varying exogenous supplies of redox cofactor precursors in selected wine yeast species.

The use of non-Saccharomyces yeasts in winemaking is gaining traction due to their specific phenotypes of technological interest, including their unique profile of central carbon metabolites and volatile compounds. However, the lack of knowledge about their physiology hinders their industrial exploitation. The intracellular redox status, involving NAD/NADH and NADP/NADPH cofactors, is a key driver of yeast activity during fermentation, notably directing the formation of metabolites that contribute to the wine bouquet. The biosynthesis of these cofactors can be modulated by the availability of their precursors, nicotinic acid and tryptophan, and their ratio by that of thiamine. In this study, a multifactorial experiment was designed to assess the effects of these three nutrients and their interactions on the metabolic response of various wine yeast species. The data indicated that limiting concentrations of nicotinic acid led to a species-dependent decrease in intracellular NAD(H) concentrations, resulting in variations of fermentation performance and production of metabolic sinks. Thiamine limitation did not directly affect redox cofactor concentrations or balance, but influenced redox management and subsequently the production of metabolites. Overall, this study identified nicotinic acid and thiamine as key factors to consider for species-specific modulation of the metabolic footprint of wine yeasts.

Oxygen alters redox cofactor dynamics and induces metabolic shifts in Saccharomyces cerevisiae during alcoholic fermentation.

Environmental conditions significantly impact the metabolism of Saccharomyces cerevisiae, a Crabtree-positive yeast that maintains a fermentative metabolism in high-sugar environments even in the presence of oxygen. Although the introduction of oxygen has been reported to induce alterations in yeast metabolism, knowledge of the mechanisms behind these metabolic adaptations in relation to redox cofactor metabolism and their implications in the context of wine fermentation remains limited. This study aimed to compare the intracellular redox cofactor levels, the cofactor ratios, and primary metabolite production in S. cerevisiae under aerobic and anaerobic conditions in synthetic grape juice. The molecular mechanisms underlying these metabolic differences were explored using a transcriptomic approach. Aerobic conditions resulted in an enhanced fermentation rate and biomass yield. Total NADP(H) levels were threefold higher during aerobiosis, while a decline in the total levels of NAD(H) was observed. However, there were stark differences in the ratio of NAD+/NADH between the treatments. Despite few changes in the differential expression of genes involved in redox cofactor metabolism, anaerobiosis resulted in an increased expression of genes involved in lipid biosynthesis pathways, while the presence of oxygen increased the expression of genes associated with thiamine, methionine, and sulfur metabolism. The production of fermentation by-products was linked with differences in the redox metabolism in each treatment. This study provides valuable insights that may help steer the production of metabolites of industrial interest during alcoholic fermentation (including winemaking) by using oxygen as a lever of redox metabolism.

The transcriptomic response of a wine strain of Lachancea thermotolerans to oxygen deprivation.

The yeast Lachancea thermotolerans is of significant biotechnological interest, and selected strains of this species have become commonly used starter cultures in wine fermentation. However, the impact of this species on wine is frequently limited by the rapid dominance of Saccharomyces cerevisiae strains which are better adapted to wine alcoholic fermentation conditions. Previous studies have shown that the major limiting factor for L. thermotolerans competitive performance in the wine ecosystem is oxygen availability, and not ethanol levels as had been previously suggested. Here we investigated the transcriptional response of L. thermotolerans to anaerobiosis in wine fermentation conditions. The data show that L. thermotolerans broadly redirects gene expression towards genes involved in central carbon metabolism, lipid metabolism, remodeling of the cell wall as well as autophagy. Furthermore, the induction of genes that are likely involved in the generation of lactate indicates a redirection of metabolic flux towards this metabolite. The data provide the first insight into the oxygen-dependent response of L. thermotolerans and suggest potential genetic targets to improve lactate production and/or anaerobic fermentation performance of this yeast.

RNA-seq based transcriptional analysis of Saccharomyces cerevisiae and Lachancea thermotolerans in mixed-culture fermentations under anaerobic conditions.

BACKGROUND: In wine fermentation starter cultures, the blending of non-Saccharomyces yeast with Saccharomyces cerevisiae to improve the complexity of wine has become common practice, but data regarding the impact of co-cultivation on yeast physiology and on genetic and metabolic regulation remain limited. Here we describe a transcriptomic analysis of mixed fermentations of Saccharomyces cerevisiae and Lachancea thermotolerans. The fermentations were carried out in carefully controlled environmental conditions in a bioreactor to reduce transcriptomic responses that would be due to factors other than the presence of the second species. RESULTS: The transcriptomic data revealed that both yeast species showed a clear response to the presence of the other. Affected genes primarily belonged to two groups: genes whose expression can be linked to the competition for certain trace elements such as copper and iron, as well as genes required for cell wall structure and integrity. Furthermore, the data revealed divergent transcriptional responses with regard to carbon metabolism in response to anoxic conditions. CONCLUSIONS: The results suggest that the mixed fermentation created a more competitive and stressful environment for the two species than single strain fermentations independently from total biomass, i.e. competition between cells of the same species is less stressful, or may present a different set of challenges, than interspecies competition. The changes in cell wall and adhesion properties encoding genes suggest that the adjustment of physical contact between cells may play a direct role in the response to the presence of competing species.

Impact of oxygenation on the performance of three non-Saccharomyces yeasts in co-fermentation with Saccharomyces cerevisiae.

The sequential or co-inoculation of grape must with non-Saccharomyces yeast species and Saccharomyces cerevisiae wine yeast strains has recently become a common practice in winemaking. The procedure intends to enhance unique aroma and flavor profiles of wine. The extent of the impact of non-Saccharomyces strains depends on their ability to produce biomass and to remain metabolically active for a sufficiently long period. However, mixed-culture wine fermentations tend to become rapidly dominated by S. cerevisiae, reducing or eliminating the non-Saccharomyces yeast contribution. For an efficient application of these yeasts, it is therefore essential to understand the environmental factors that modulate the population dynamics of such ecosystems. Several environmental parameters have been shown to influence population dynamics, but their specific effect remains largely uncharacterized. In this study, the population dynamics in co-fermentations of S. cerevisiae and three non-Saccharomyces yeast species: Torulaspora delbrueckii, Lachancea thermotolerans, and Metschnikowia pulcherrima, was investigated as a function of oxygen availability. In all cases, oxygen availability strongly influenced population dynamics, but clear species-dependent differences were observed. Our data show that L. thermotolerans required the least oxygen, followed by T. delbrueckii and M. pulcherrima. Distinct species-specific chemical volatile profiles correlated in all cases with increased persistence of non-Saccharomyces yeasts, in particular increases in some higher alcohols and medium chain fatty acids. The results highlight the role of oxygen in regulating the succession of yeasts during wine fermentations and suggests that more stringent aeration strategies would be necessary to support the persistence of non-Saccharomyces yeasts in real must fermentations.

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.

Physical cell-cell contact elicits specific transcriptomic responses in wine yeast species.

Fermenting grape juice provides a habitat for a well-mapped and evolutionarily relevant microbial ecosystem consisting of many natural or inoculated strains of yeasts and bacteria. The molecular nature of many of the ecological interactions within this ecosystem remains poorly understood, with the partial exception of interactions of a metabolic nature such as competition for nutrients and production of toxic metabolites/peptides. Data suggest that physical contact between species plays a significant role in the phenotypic outcome of interspecies interactions. However, the molecular nature of the mechanisms regulating these phenotypes remains unknown. Here, we present a transcriptomic analysis of physical versus metabolic contact between two wine relevant yeast species, Saccharomyces cerevisiae and Lachancea thermotolerans. The data show that these species respond to the physical presence of the other species. In S. cerevisiae, physical contact results in the upregulation of genes involved in maintaining cell wall integrity, cell wall structural components, and genes involved in the production of H2S. In L. thermotolerans, HSP stress response genes were the most significantly upregulated gene family. Both yeasts downregulated genes belonging to the FLO family, some of which play prominent roles in cellular adhesion. qPCR analysis indicates that the expression of some of these genes is regulated in a species-specific manner, suggesting that yeasts adjust gene expression to specific biotic challenges or interspecies interactions. These findings provide fundamental insights into yeast interactions and evolutionary adaptations of these species to the wine ecosystem.IMPORTANCEWithin the wine ecosystem, yeasts are the most relevant contributors to alcoholic fermentation and wine organoleptic characteristics. While some studies have described yeast-yeast interactions during alcoholic fermentation, such interactions remain ill-defined, and little is understood regarding the molecular mechanisms behind many of the phenotypes observed when two or more species are co-cultured. In particular, no study has investigated transcriptional regulation in response to physical interspecies cell-cell contact, as opposed to the generally better understood/characterized metabolic interactions. These data are of direct relevance to our understanding of microbial ecological interactions in general while also creating opportunities to improve ecosystem-based biotechnological applications such as wine fermentation. Furthermore, the presence of competitor species has rarely been considered an evolutionary biotic selection pressure. In this context, the data reveal novel gene functions. This, and further such analysis, is likely to significantly enlarge the genome annotation space.

Redox cofactor metabolism in Saccharomyces cerevisiae and its impact on the production of alcoholic fermentation end-products.

The alcoholic fermentation of organic carbon sources by Saccharomyces cerevisiae produces many by-products, with the most abundant originating from central carbon metabolism. The production of these metabolites involves redox reactions and largely depends on the maintenance of redox homeostasis. Despite the metabolic pathways being mostly conserved across strains of S. cerevisiae, their production of various amounts of metabolic products suggests that their intracellular concentration of redox cofactors and/or redox balance differ. This study explored the redox status dynamics and NAD(H) and NADP(H) cofactor ratios throughout alcoholic fermentation in four S. cerevisiae strains that exhibit different carbon metabolic fluxes. This study focussed on the molecular end-products of fermentation, redox cofactor ratios and the impact thereof on redox homeostasis. Strain-dependent differences were identified in the redox cofactor levels, with NADP(H) ratios and levels remaining stable while NAD(H) levels decreased drastically as the fermentation progressed. Changes in the NAD+/NADH ratio were also observed. Total levels of NAD(H) decreased drastically as the fermentation progressed despite the cells remaining viable until the end of fermentation. NAD+ was found to be favoured initially while NADH was favoured towards the end of the fermentation. The change in the NAD+/NADH redox cofactor ratio during fermentation was linked with the production of end-products. The findings in this study could steer further research in the selection of S. cerevisiae wine strains for desirable aroma contributions based on their intracellular redox balance management.

Differentiation of Geographic Origin of South African Wines from Austrian Wines by IRMS and SNIF-NMR.

Geographic origin and terroir are very important parameters for wine and significantly impact price. Incorrect declarations are known to occur intentionally to increase profit, thus, measures for control are required. Accompanying paperwork has been shown to be unreliable, thus, control of the product itself is required. Here we investigate and compare the stable isotope pattern of South African (Western Cape Province) wine, and evaluate its potential for discrimination from Central European/Austrian wine. The results show that the isotope values of the investigated South African wine samples differ significantly from the values of average Austrian (Central European) wines. Thus, a differentiation of the products from these two regions by stable isotope analysis is generally straightforward. However, the data suggest that vintages from years with exceptionally hot and dry summer weather in Europe may reduce the differentiation between these regions. Therefore, this method is a potent tool for the discrimination of Austrian (Central European) and South African wines under current climatic conditions, although drier and hotter summer weather in Europe, which is likely to occur more frequently due to global climate change, may require further method adjustments in the future.

The Grapevine and Wine Microbiome: Insights from High-Throughput Amplicon Sequencing.

From the time when microbial activity in wine fermentation was first demonstrated, the microbial ecology of the vineyard, grape, and wine has been extensively investigated using culture-based methods. However, the last 2 decades have been characterized by an important change in the approaches used for microbial examination, due to the introduction of DNA-based community fingerprinting methods such as DGGE, SSCP, T-RFLP, and ARISA. These approaches allowed for the exploration of microbial community structures without the need to cultivate, and have been extensively applied to decipher the microbial populations associated with the grapevine as well as the microbial dynamics throughout grape berry ripening and wine fermentation. These techniques are well-established for the rapid more sensitive profiling of microbial communities; however, they often do not provide direct taxonomic information and possess limited ability to detect the presence of rare taxa and taxa with low abundance. Consequently, the past 5 years have seen an upsurge in the application of high-throughput sequencing methods for the in-depth assessment of the grapevine and wine microbiome. Although a relatively new approach in wine sciences, these methods reveal a considerably greater diversity than previously reported, and identified several species that had not yet been reported. The aim of the current review is to highlight the contribution of high-throughput next generation sequencing and metagenomics approaches to vineyard microbial ecology especially unraveling the influence of vineyard management practices on microbial diversity.

The Impact of Saccharomyces cerevisiae on a Wine Yeast Consortium in Natural and Inoculated Fermentations.

Natural, also referred to as spontaneous wine fermentations, are carried out by the native microbiota of the grape juice, without inoculation of selected, industrially produced yeast or bacterial strains. Such fermentations are commonly initiated by non-Saccharomyces yeast species that numerically dominate the must. Community composition and numerical dominance of species vary significantly between individual musts, but Saccharomyces cerevisiae will in most cases dominate the late stages of the fermentation and complete the process. Nevertheless, non-Saccharomyces species contribute significantly, positively or negatively, to the character and quality of the final product. The contribution is species and strain dependent and will depend on each species or strain's absolute and relative contribution to total metabolically active biomass, and will therefore, be a function of its relative fitness within the microbial ecosystem. However, the population dynamics of multispecies fermentations are not well understood. Consequently, the oenological potential of the microbiome in any given grape must, can currently not be evaluated or predicted. To better characterize the rules that govern the complex wine microbial ecosystem, a model yeast consortium comprising eight species commonly encountered in South African grape musts and an ARISA based method to monitor their dynamics were developed and validated. The dynamics of these species were evaluated in synthetic must in the presence or absence of S. cerevisiae using direct viable counts and ARISA. The data show that S. cerevisiae specifically suppresses certain species while appearing to favor the persistence of other species. Growth dynamics in Chenin blanc grape must fermentation was monitored only through viable counts. The interactions observed in the synthetic must, were upheld in the natural must fermentations, suggesting the broad applicability of the observed ecosystem dynamics. Importantly, the presence of indigenous yeast populations did not appear to affect the broad interaction patterns between the consortium species. The data show that the wine ecosystem is characterized by both mutually supportive and inhibitory species. The current study presents a first step in the development of a model to predict the oenological potential of any given wine mycobiome.

Sequence-based Analysis of the Vitis vinifera L. cv Cabernet Sauvignon Grape Must Mycobiome in Three South African Vineyards Employing Distinct Agronomic Systems.

Recent microbiomic research of agricultural habitats has highlighted tremendous microbial biodiversity associated with such ecosystems. Data generated in vineyards have furthermore highlighted significant regional differences in vineyard biodiversity, hinting at the possibility that such differences might be responsible for regional differences in wine style and character, a hypothesis referred to as "microbial terroir." The current study further contributes to this body of work by comparing the mycobiome associated with South African (SA) Cabernet Sauvignon grapes in three neighboring vineyards that employ different agronomic approaches, and comparing the outcome with similar data sets from Californian vineyards. The aim of this study was to fully characterize the mycobiomes associated with the grapes from these vineyards. The data revealed approximately 10 times more fungal diversity than what is typically retrieved from culture-based studies. The Biodynamic vineyard was found to harbor a more diverse fungal community (H = 2.6) than the conventional (H = 2.1) and integrated (H = 1.8) vineyards. The data show that ascomycota are the most abundant phylum in the three vineyards, with Aureobasidium pullulans and its close relative Kabatiella microsticta being the most dominant fungi. This is the first report to reveal a high incidence of K. microsticta in the grape/wine ecosystem. Different common wine yeast species, such as Metschnikowia pulcherrima and Starmerella bacillaris dominated the mycobiome in the three vineyards. The data show that the filamentous fungi are the most abundant community in grape must although they are not regarded as relevant during wine fermentation. Comparison of metagenomic datasets from the three SA vineyards and previously published data from Californian vineyards revealed only 25% of the fungi in the SA dataset was also present in the Californian dataset, with greater variation evident amongst ubiquitous epiphytic fungi.

Hanseniaspora uvarum from Winemaking Environments Show Spatial and Temporal Genetic Clustering.

Hanseniaspora uvarum is one of the most abundant yeast species found on grapes and in grape must, at least before the onset of alcoholic fermentation (AF) which is usually performed by Saccharomyces species. The aim of this study was to characterize the genetic and phenotypic variability within the H. uvarum species. One hundred and fifteen strains isolated from winemaking environments in different geographical origins were analyzed using 11 microsatellite markers and a subset of 47 strains were analyzed by AFLP. H. uvarum isolates clustered mainly on the basis of their geographical localization as revealed by microsatellites. In addition, a strong clustering based on year of isolation was evidenced, indicating that the genetic diversity of H. uvarum isolates was related to both spatial and temporal variations. Conversely, clustering analysis based on AFLP data provided a different picture with groups showing no particular characteristics, but provided higher strain discrimination. This result indicated that AFLP approaches are inadequate to establish the genetic relationship between individuals, but allowed good strain discrimination. At the phenotypic level, several extracellular enzymatic activities of enological relevance (pectinase, chitinase, protease, ß-glucosidase) were measured but showed low diversity. The impact of environmental factors of enological interest (temperature, anaerobia, and copper addition) on growth was also assessed and showed poor variation. Altogether, this work provided both new analytical tool (microsatellites) and new insights into the genetic and phenotypic diversity of H. uvarum, a yeast species that has previously been identified as a potential candidate for co-inoculation in grape must, but whose intraspecific variability had never been fully assessed.

Characterization of novel killer toxins secreted by wine-related non-Saccharomyces yeasts and their action on Brettanomyces spp.

Wine spoilage associated with Brettanomyces bruxellensis is a major concern for winemakers. An effective and reliable method to control the proliferation of this yeast is therefore of utmost importance. To achieve this purpose, sulphur dioxide (SO2) is commonly employed but the efficiency of this chemical compound is subject to wine composition and it can elicit allergic reactions in some consumers. Biological alternatives are therefore actively sought. The current study focused on identifying and characterizing killer toxins which are antimicrobial compounds that show potential in inhibiting B. bruxellensis in wine. Two killer toxins, CpKT1 and CpKT2, from the wine isolated yeast Candida pyralidae were identified and partially characterized. The two proteins had a molecular mass above 50kDa and exhibited killer activity against several B. bruxellensis strains especially in grape juice. They were active and stable at pH3.5-4.5, and temperatures between 15 and 25°C which are compatible with winemaking conditions. Furthermore, the activity of these killer toxins was not affected by the ethanol and sugar concentrations typically found in grape juice and wine. In addition, these killer toxins inhibited neither the Saccharomyces cerevisiae nor the lactic acid bacteria strains tested. These preliminary results indicated that the application of these toxins will have no effect on the main microbial agents that drive alcoholic and malolactic fermentations and further highlight the potential of using these toxins as agents to control the development of B. bruxellensis in grape juice or wine.