RESUMO
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.
Assuntos
Fermentação , NAD , Niacina , Oxirredução , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Niacina/metabolismo , NAD/metabolismo , Etanol/metabolismo , Coenzimas/metabolismoRESUMO
The use of non-Saccharomyces yeasts in the winemaking process may have several positive outcomes. Kluyveromyces marxianus has recently been revealed as a promising species for this industry. While the majority of studies mention the use of K. marxianus in various industries including food production (e.g. dairy and cocoa), recent studies have also shown that its aroma and pectinase production make it a suitable yeast for the wine industry. Nevertheless, only particular strain, IWBT Y885, was investigated. In this study, five different K. marxianus strains as well as one protoplast fusant (BF2020) were compared to strain Y885. These comparisons focused on various oenological traits such as fermentation performance, fermentation metabolites, hydrogen sulfide, and pectinase production. Throughout the study, variations were found between the K. marxianus strains investigated. Indeed, although common traits such as high pectinase activity appeared conserved among K. marxianus strains, a fairly large phenotypic diversity was also evident. Using cluster analysis, strain groupings emerged with strains L01, L05, Y885, and BF2020 grouping together. Similarly, strains L02 and L04 grouped together while strain L03 appearing to show the most variation between the strains investigated. Variation between strains was observed regardless of the original source of isolation.
Assuntos
Kluyveromyces , Poligalacturonase , Fermentação , Kluyveromyces/genética , Kluyveromyces/metabolismo , Poligalacturonase/metabolismo , Leveduras/metabolismoRESUMO
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.
Assuntos
Kluyveromyces/metabolismo , Nitrogênio/metabolismo , Odorantes/análise , Saccharomycetales/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Vinho/microbiologia , Álcoois/metabolismo , Fermentação , Filogenia , Saccharomyces cerevisiae/metabolismo , Vitis/metabolismo , Vitis/microbiologia , Compostos Orgânicos Voláteis/análise , Vinho/análiseRESUMO
Microbial multispecies ecosystems are responsible for many biotechnological processes and are particularly important in food production. In wine fermentations, in addition to the natural microbiota, several commercially relevant yeast species may be co-inoculated to achieve specific outcomes. However, such multispecies fermentations remain largely unpredictable because of multilevel interactions between naturally present and/or co-inoculated species. Understanding the nature of such interactions has therefore become essential for successful implementation of such strategies. Here we investigate interactions between strains of Saccharomyces cerevisiae and Lachancea thermotolerans. Co-fermentations with both species sharing the same bioreactor (physical contact) were compared to co-fermentations with physical separation between the species in a membrane bioreactor ensuring free exchange of metabolites. Yeast culturability, viability and the production of core metabolites were monitored. The previously reported negative interaction between these two yeast species was confirmed. Physical contact greatly reduced the culturability and viability of L. thermotolerans and led to earlier cell death, compared to when these yeasts were co-fermenting without cell-cell contact. In turn, in the absence of cell-cell contact, L. thermotolerans metabolic activity led to an earlier decline in culturability in S. cerevisiae. Cell-cell contact did not result in significant differences in the major fermentation metabolites ethanol, acetic acid and lactic acid, but impacted on the production of some volatile compounds.
Assuntos
Comunicação Celular/fisiologia , Fermentação , Filogenia , Saccharomyces cerevisiae/metabolismo , Ácido Acético/metabolismo , Reatores Biológicos , Técnicas de Cocultura , Ecossistema , Etanol/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Vitis , VinhoRESUMO
Sulfur dioxide is generally used as an antimicrobial in wine to counteract the activity of spoilage yeasts, including Brettanomyces bruxellensis. However, this chemical does not exert the same effectiveness on different B. bruxellensis yeasts since some strains can proliferate in the final product leading to a negative sensory profile due to 4-ethylguaiacol and 4-ethylphenol. Thus, the capability of deciphering the general molecular mechanisms characterizing this yeast species' response in presence of SO2 stress could be considered strategic for a better management of SO2 in winemaking. A RNA-Seq approach was used to investigate the gene expression of two strains of B. bruxellensis, AWRI 1499 and CBS 2499 having different genetic backgrounds, when exposed to a SO2 pulse. Results revealed that sulphites affected yeast culturability and metabolism, but not volatile phenol production suggesting that a phenotypical heterogeneity could be involved for the SO2 cell adaptation. The transcriptomics variation in response to SO2 stress confirmed the strain-related response in B. bruxellensis and the GO analysis of common differentially expressed genes showed that the detoxification process carried out by SSU1 gene can be considered as the principal specific adaptive response to counteract the SO2 presence. However, nonspecific mechanisms can be exploited by cells to assist the SO2 tolerance; namely, the metabolisms related to sugar alcohol (polyols) and oxidative stress, and structural compounds.
Assuntos
Brettanomyces/genética , Brettanomyces/metabolismo , Fermentação , Estresse Fisiológico , Dióxido de Enxofre/metabolismo , Vinho/microbiologia , Microbiologia de Alimentos , Perfilação da Expressão Gênica , RNA-Seq , TranscriptomaRESUMO
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.
Assuntos
Compostos de Amônio/metabolismo , Kluyveromyces/metabolismo , Nitrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminoácidos/metabolismo , Fermentação , Perfilação da Expressão Gênica , Kluyveromyces/genética , Redes e Vias Metabólicas/fisiologia , Saccharomyces cerevisiae/genética , Vitis/microbiologia , Vinho/microbiologiaRESUMO
Commercial wine fermentation is commonly conducted by inoculated strains of Saccharomyces cerevisiae. However, other non-Saccharomyces yeast species have recently become popular co-inoculants. Co-inoculated yeast species compete with each other for nutrients, and such competition may impact fermentation kinetics and aroma production. Understanding the specific nutrient requirements of non-Saccharomyces strains therefore is essential to better characterize the competitive potential of each strain, and to support rational decision making for nutrient supplementation during wine making. This study investigated the nitrogen source preference of commercial non-Saccharomyces yeasts by conducting pure culture and sequential culture fermentations in synthetic grape musts with adjusted nitrogen contents. Amino acid and ammonium uptake varied between yeast species. Lachancea thermotolerans and Torulaspora delbrueckii assimilated more nitrogen at a faster rate than Pichia kluyveri and Metschnikowia pulcherrima. Significant variation in amino acid preference between species was observed. Sequential fermentations confirmed the more competitive behaviour of L. thermotolerans and T. delbrueckii, with consequences for fermentation kinetics and aroma production. Furthermore, the data suggest that declining populations of non-Saccharomyces yeasts release nitrogen and supports the activity of S. cerevisiae. The data provide the most detailed assessment of nitrogen utilisation by the investigated yeast strains in a wine environment.
Assuntos
Compostos de Nitrogênio/metabolismo , Vinho/análise , Leveduras/metabolismo , Aminoácidos/metabolismo , Compostos de Amônio/metabolismo , Técnicas de Cocultura , Fermentação , Sucos de Frutas e Vegetais/análise , Cinética , Interações Microbianas , Nitrogênio/metabolismo , Especificidade da Espécie , Vitis , Compostos Orgânicos Voláteis/análise , Vinho/microbiologia , Leveduras/classificação , Leveduras/fisiologiaRESUMO
Protein haze formation in bottled wines is a significant concern for the global wine industry, and wine clarification before bottling is therefore a common but expensive practice. Previous studies have shown that wine yeast strains can reduce haze formation through the secretion of certain mannoproteins, but it has been suggested that other yeast-dependent haze protective mechanisms exist. On the other hand, the addition of chitin has been shown to reduce haze formation, likely because grape chitinases have been shown to be the major contributors to haze. In this study, Chardonnay grape must fermented by various yeast strains resulted in wines with different protein haze levels, indicating differences in haze-protective capacities of the strains. The cell wall chitin levels of these strains were determined, and a strong correlation between cell wall chitin levels and haze protection capability was observed. To further evaluate the mechanism of haze protection, Escherichia coli-produced green fluorescent protein (GFP)-tagged grape chitinase was shown to bind efficiently to yeast cell walls in a cell wall chitin concentration-dependent manner, while commercial chitinase was removed from synthetic wine in quantities that also correlated with the cell wall chitin levels of the strains. Our findings suggest a new mechanism of reducing wine haze, and we propose a strategy for optimizing wine yeast strains to improve wine clarification.IMPORTANCE In this study, we establish a new mechanism by which wine yeast strains can impact the protein haze formation of wines, and we demonstrate that yeast cell wall chitin binds grape chitinase in a chitin concentration-dependent manner. We also show that yeast can remove this haze-forming protein from wine. Chitin has in the past been shown to efficiently reduce wine haze formation when added to the wine in high concentration as a clarifying agent. Our data suggest that the selection of yeast strains with high levels of cell wall chitin can reduce protein haze. We also investigate how yeast cell wall chitin levels are affected by environmental conditions.
Assuntos
Quitina/química , Glicoproteínas de Membrana/metabolismo , Vinho/microbiologia , Parede Celular/química , Quitinases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Biossíntese de Proteínas , Estabilidade Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vitis/química , Vinho/análise , Leveduras/genética , Leveduras/metabolismoRESUMO
Non-Saccharomyces yeasts impact wine fermentations and can diversify the flavor profiles of wines. However, little information is available on the metabolic networks of most of these species. Here we show that unlike the main wine yeast Saccharomyces cerevisiae, Torulaspora delbrueckii and to a lesser extent Lachancea thermotolerans produce significant concentrations of C5 and C6 polyols under wine fermentation conditions. In particular, D-arabitol, D-sorbitol and D-mannitol were produced at significant levels. Their release into the extracellular matrix started when that of glycerol ceased. The data also show that polyol production is influenced by initial sugar concentration, repressed by acetic acid and induced in ethanol supplemented media. Moreover, unlike glycerol and sorbitol, mannitol was partially re-assimilated when populations started to decline. The findings suggest that polyol synthesis is a physiological adaptation to stressful conditions characteristic of alcoholic fermentation and that these polyols may serve a similar purpose as glycerol production in S. cerevisiae, including osmoadaptation and redox balancing.
Assuntos
Polímeros/metabolismo , Torulaspora/metabolismo , Vinho/análise , Vinho/microbiologia , Ácido Acético/química , Meios de Cultura/química , Etanol/química , Fermentação , Cinética , Polímeros/química , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Álcoois Açúcares/química , Álcoois Açúcares/metabolismo , Açúcares/química , Torulaspora/crescimento & desenvolvimento , Vitis/metabolismoRESUMO
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.
Assuntos
Fermentação , Nitrogênio/metabolismo , Odorantes/análise , Vinho/microbiologia , Leveduras/metabolismo , Acetatos/análise , Álcoois/análise , Ésteres/análise , Saccharomyces cerevisiaeRESUMO
The perception of haze in wine is brought about when pathogenesis-related proteins become unstable and aggregate, subsequently resulting in crosslinking until it develops into light-dispersing particles. Elimination of these proteins is usually achieved via bentonite fining, which, although effective, suffers from several drawbacks. The utilization of proteases has been proposed as an ideal alternative. In a previous study, an aspartic protease (MpAPr1) from the yeast Metschnikowia pulcherrima was purified and shown to be partially active against grape proteins in synthetic medium. In this study, the effects of pure MpAPr1 supplemented to Sauvignon Blanc juice on subsequent fermentation were investigated. The juice was incubated for 48 h and thereafter inoculated with Saccharomyces cerevisiae. Results revealed that the enzyme had no observable effects on fermentation performance and retained activity throughout. Protein degradation could be detected and resulted in a significant modification of the wine composition and an increase in the presence of certain volatile compounds, especially those linked to amino acid metabolism.
Assuntos
Ácido Aspártico Proteases/metabolismo , Metschnikowia/enzimologia , Proteínas de Plantas/metabolismo , Vitis/metabolismo , Vinho/normas , Fermentação , Microbiologia de Alimentos , Saccharomyces cerevisiae/metabolismo , Vitis/química , Vinho/análiseRESUMO
The wine matrix contains limited carbon compounds to sustain microbial life. Brettanomyces bruxellensis is one of very few yeast species that has adapted to this environment. Indeed, the presence of growth-inhibiting compounds and conditions do not prevent its proliferation. Literature regarding the nutritional requirements of this yeast is surprisingly poor, given the observation that B. bruxellensis produces biomass with apparently less nutrients than other yeasts. In this study, various carbon sources were screened in a synthetic wine medium, under anaerobic and semi-aerobic growth conditions, in order to determine which compounds B. bruxellensis assimilates. Slight differences were observed between strains but overall, B. bruxellensis produced biomass from limited nutrients consumed in a specific order regardless of the oxygen conditions. Upon initial consumption of the simple sugars, B. bruxellensis was able to remain viable, by concurrently utilising ethanol (only in the presence of oxygen) and malic acid. Although initially beneficial, oxygen was found detrimental in the long term. Formation of volatile phenols occurred during the consumption of the sugars but not as a mechanism to help correct the redox imbalance. The study confirms that B. bruxellensis is able to survive using limited amount of nutrients, making this yeast a challenge for winemakers.
Assuntos
Brettanomyces/crescimento & desenvolvimento , Carbono/metabolismo , Meios de Cultura/química , Vinho/microbiologia , Brettanomyces/metabolismo , Carbono/análise , Meios de Cultura/metabolismo , Etanol/metabolismo , Fermentação , Malatos/metabolismo , Oxigênio/metabolismoRESUMO
BACKGROUND: MpAPr1, encoding an acid protease from the wine yeast Metschnikowia pulcherrima IWBT Y1123, was previously isolated and shown to display potential activity against casein and grape proteins. However, its characterisation remained partial. RESULTS: MpAPr1 was cloned into the pGAPZαA vector and transformed into Komagataella pastoris X33 for heterologous expression. After verification of activity, the enzyme properties were characterised. Protease activity within the concentrated supernatant was retained over a pH range of 3.0 to 5.0 and between 10 °C and 50 °C. Optimal conditions for protease activity were found at 40 °C and pH 4.5. Activity was mostly unaffected by the presence of metal ions with the exception of Cu2+ and Ni2+ . Furthermore, proteolytic activity was retained in the presence of sugar and ethanol. pH and temperature conditions for MpAPr1 expression in K. pastoris were optimised. Purification was achieved by means of cation exchange chromatography and kinetic parameters (Km and Vmax ) were determined. MpAPr1 activity against grape proteins was confirmed, but the extent of the degradation was dependent on the nature of these proteins and the environmental conditions. CONCLUSION: Overall, the results suggest that MpAPr1 could be applied in food biotechnology processes such as winemaking. © 2017 Society of Chemical Industry.
Assuntos
Ácido Aspártico Proteases/química , Proteínas Fúngicas/química , Metschnikowia/enzimologia , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/isolamento & purificação , Ácido Aspártico Proteases/metabolismo , Estabilidade Enzimática , Etanol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Metschnikowia/química , Metschnikowia/genética , Metschnikowia/metabolismo , Transporte Proteico , Vitis/metabolismo , Vitis/microbiologia , Vinho/análise , Vinho/microbiologiaRESUMO
Brettanomyces bruxellensis is a common red wine spoilage yeast. Yet, in addition to wine, it has been isolated from other ecological niches that are just as nutritionally deficient as wine. B. bruxellensis can therefore be regarded as a survivor, well adapted to colonise harsh environments not often inhabited by other yeasts. This review is focused on the nutritional requirements of B. bruxellensis and the relevance thereof for its adaptation to the different matrices within which it occurs. Furthermore, the environmental conditions necessary (e.g. aerobic or anaerobic conditions) for the assimilation of the carbon or nitrogenous sources are discussed in this review. From literature, several confusing inconsistencies, regarding nutritional sources necessary for B. bruxellensis survival, in these specialist ecological niches are evidenced. The main focus of this review is wine but other products and niches that B. bruxellensis inhabits namely beer, cider, fruit juices and bioethanol production plants are also considered. This review highlights the lack of knowledge regarding B. bruxellensis when considering its nutritional requirements in comparison to Saccharomyces cerevisiae. However, there is a large enough body of evidence showing that the nutritional needs of B. bruxellensis are meagre, explaining its ability to colonise harsh environments.
Assuntos
Brettanomyces/metabolismo , Vinho/microbiologia , Bebidas Alcoólicas/microbiologia , Cerveja/microbiologia , Brettanomyces/imunologia , Carbono/metabolismo , Fermentação , Microbiologia de Alimentos , Nitrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Vinho/análiseRESUMO
Aspartic proteases are a relatively small group of proteolytic enzymes that are active in acidic environments and are found across all forms of life. Certain microorganisms secrete such proteases as virulence agents and/or in order to break down proteins thereby liberating assimilable sources of nitrogen. Some of the earlier applications of these proteolytic enzymes are found in the manufacturing of cheese where they are used as milk-clotting agents. Over the last decade, they have received tremendous research interest because of their involvement in human diseases. Furthermore, there has also been a growing interest on these enzymes for their applications in several other industries. Recent research suggests in particular that they could be used in the wine industry to prevent the formation of protein haze while preserving the wines' organoleptic properties. In this mini-review, the properties and mechanisms of action of aspartic proteases are summarized. Thereafter, a brief overview of the industrial applications of this specific class of proteases is provided. The use of aspartic proteases as alternatives to clarifying agents in various beverage industries is mentioned, and the potential applications in the wine industry are thoroughly discussed.
Assuntos
Ácido Aspártico Proteases/metabolismo , Biotecnologia/métodos , Microbiologia de Alimentos/métodosRESUMO
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.
Assuntos
Saccharomyces cerevisiae , Vinho , Saccharomyces cerevisiae/metabolismo , Vinho/análise , NAD/análise , NAD/metabolismo , Glicerol/metabolismo , Fermentação , NADP/análise , NADP/metabolismo , Filogenia , Oxirredução , Açúcares/metabolismoRESUMO
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.
Assuntos
Fermentação , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae , Transcriptoma , Vinho , Vinho/microbiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Perfilação da Expressão Gênica , Vitis/microbiologia , Vitis/genética , Parede Celular/metabolismo , Parede Celular/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Interações MicrobianasRESUMO
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.
Assuntos
NAD , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , NAD/metabolismo , Fermentação , NADP/metabolismo , OxirreduçãoRESUMO
The structure of yeast cell wall (CW) mannoproteins (MPs) influences their impact on wine properties. Yeast species produce a diverse range of MPs, but the link between properties and specific structural features has been ill-characterized. This study compared the protein and polysaccharide moieties of MP-rich preparations from four strains of four different enologically relevant yeast species, named Saccharomyces boulardii (SB62), Saccharomyces cerevisiae (SC01), Metschnikowia fructicola (MF77), and Torulaspora delbrueckii (TD70), and a commercial MP preparation. Monosaccharide determination revealed that SB62 MPs contained the highest mannose/glucose ratio followed by SC01, while polysaccharide size distribution analyses showed maximum molecular weights ranging from 1349 kDa for MF77 to 483 kDa for TD70. Protein identification analysis led to the identification of unique CW proteins in SB62, SC01, and TD70, as well as some proteins shared between different strains. This study reveals MP composition diversity within wine yeasts and paves the way toward their industrial exploitation.
Assuntos
Saccharomyces cerevisiae , Vinho , Saccharomyces cerevisiae/metabolismo , Vinho/análise , Filogenia , Fermentação , Polissacarídeos/metabolismoRESUMO
The extracellular acid proteases of non-Saccharomyces wine yeasts may fulfill a number of roles in winemaking, which include increasing the available nitrogen sources for the growth of fermentative microbes, affecting the aroma profile of the wine, and potentially reducing protein haze formation. These proteases, however, remain poorly characterized, especially at genetic level. In this study, two extracellular aspartic protease-encoding genes were identified and sequenced, from two yeast species of enological origin: one gene from Metschnikowia pulcherrima IWBT Y1123, named MpAPr1, and the other gene from Candida apicola IWBT Y1384, named CaAPr1. In silico analysis of these two genes revealed a number of features peculiar to aspartic protease genes, and both the MpAPr1 and CaAPr1 putative proteins showed homology to proteases of yeast genera. Heterologous expression of MpAPr1 in Saccharomyces cerevisiae YHUM272 confirmed that it encodes an aspartic protease. MpAPr1 production, which was shown to be constitutive, and secretion were confirmed in the presence of bovine serum albumin (BSA), casein, and grape juice proteins. The MpAPr1 gene was found to be present in 12 other M. pulcherrima strains; however, plate assays revealed that the intensity of protease activity was strain dependent and unrelated to the gene sequence.