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Proteomic response to physiological fermentation stresses in a wild-type wine strain of Saccharomyces cerevisiae.

ABSTRACT: We report a study on the adaptive response of a wild-type wine Saccharomyces cerevisiae strain, isolated from natural spontaneous grape must, to mild and progressive physiological stresses due to fermentation. We observed by two-dimensional electrophoresis how the yeast proteome changes during glucose exhaustion, before the cell enters its complete stationary phase. On the basis of their identification, the proteins representing the S. cerevisiae proteomic response to fermentation stresses were divided into three classes: repressed proteins, induced proteins and autoproteolysed proteins. In an overall view, the proteome adaptation of S. cerevisiae at the time of glucose exhaustion seems to be directed mainly against the effects of ethanol, causing both hyperosmolarity and oxidative responses. Stress-induced autoproteolysis is directed mainly towards specific isoforms of glycolytic enzymes. Through the use of a wild-type S. cerevisiae strain and PMSF, a specific inhibitor of vacuolar proteinase B, we could also distinguish the specific contributions of the vacuole and the proteasome to the autoproteolytic process.

SUBMITTER: Trabalzini L

PROVIDER: S-EPMC1223135 | biostudies-other | 2003 Feb

REPOSITORIES: biostudies-other

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Project description:Saccharomyces cerevisiae is the main microorganism responsible for the fermentation of wine. Nevertheless, in the last years wineries are facing new challenges due to current market demands and climate change effects on the wine quality. New yeast starters formed by non-conventional Saccharomyces species (such as S. uvarum or S. kudriavzevii) or their hybrids (S. cerevisiae x S. uvarum and S. cerevisiae x S. kudriavzevii) can contribute to solve some of these challenges. They exhibit good fermentative capabilities at low temperatures, producing wines with lower alcohol and higher glycerol amounts. However, S. cerevisiae can competitively displace other yeast species from wine fermentations, therefore the use of these new starters requires an analysis of their behavior during competition with S. cerevisiae during wine fermentation. In the present study we analyzed the survival capacity of non-cerevisiae strains in competition with S. cerevisiae during fermentation of synthetic wine must at different temperatures. First, we developed a new method, based on QPCR, to quantify the proportion of different Saccharomyces yeasts in mixed cultures. This method was used to assess the effect of competition on the growth fitness. In addition, fermentation kinetics parameters and final wine compositions were also analyzed. We observed that some cryotolerant Saccharomyces yeasts, particularly S. uvarum, seriously compromised S. cerevisiae fitness during competences at lower temperatures, which explains why S. uvarum can replace S. cerevisiae during wine fermentations in European regions with oceanic and continental climates. From an enological point of view, mixed co-cultures between S. cerevisiae and S. paradoxus or S. eubayanus, deteriorated fermentation parameters and the final product composition compared to single S. cerevisiae inoculation. However, in co-inoculated synthetic must in which S. kudriavzevii or S. uvarum coexisted with S. cerevisiae, there were fermentation performance improvements and the final wines contained less ethanol and higher amounts of glycerol. Finally, it is interesting to note that in co-inoculated fermentations, wine strains of S. cerevisiae and S. uvarum performed better than non-wine strains of the same species.

Project description:During fermentative growth in natural and industrial environments, Saccharomyces cerevisiae must redistribute the available nitrogen from multiple exogenous sources to amino acids in order to suitably fulfill anabolic requirements. To exhaustively explore the management of this complex resource, we developed an advanced strategy based on the reconciliation of data from a set of stable isotope tracer experiments with labeled nitrogen sources. Thus, quantifying the partitioning of the N compounds through the metabolism network during fermentation, we demonstrated that, contrary to the generally accepted view, only a limited fraction of most of the consumed amino acids is directly incorporated into proteins. Moreover, substantial catabolism of these molecules allows for efficient redistribution of nitrogen, supporting the operative de novo synthesis of proteinogenic amino acids. In contrast, catabolism of consumed amino acids plays a minor role in the formation of volatile compounds. Another important feature is that the ?-keto acid precursors required for the de novo syntheses originate mainly from the catabolism of sugars, with a limited contribution from the anabolism of consumed amino acids. This work provides a comprehensive view of the intracellular fate of consumed nitrogen sources and the metabolic origin of proteinogenic amino acids, highlighting a strategy of distribution of metabolic fluxes implemented by yeast as a means of adapting to environments with changing and scarce nitrogen resources.IMPORTANCE A current challenge for the wine industry, in view of the extensive competition in the worldwide market, is to meet consumer expectations regarding the sensory profile of the product while ensuring an efficient fermentation process. Understanding the intracellular fate of the nitrogen sources available in grape juice is essential to the achievement of these objectives, since nitrogen utilization affects both the fermentative activity of yeasts and the formation of flavor compounds. However, little is known about how the metabolism operates when nitrogen is provided as a composite mixture, as in grape must. Here we quantitatively describe the distribution through the yeast metabolic network of the N moieties and C backbones of these nitrogen sources. Knowledge about the management of a complex resource, which is devoted to improvement of the use of the scarce N nutrient for growth, will be useful for better control of the fermentation process and the sensory quality of wines.

Project description:Wineries face unprecedented challenges due to new market demands and climate change effects on wine quality. New yeast starters including non-conventional Saccharomyces species, such as S. kudriavzevii, may contribute to deal with some of these challenges. The design of new fermentations using non-conventional yeasts requires an improved understanding of the physiology and metabolism of these cells. Dynamic modeling brings the potential of exploring the most relevant mechanisms and designing optimal processes more systematically. In this work we explore mechanisms by means of a model selection, reduction and cross-validation pipeline which enables to dissect the most relevant fermentation features for the species under consideration, Saccharomyces cerevisiae T73 and Saccharomyces kudriavzevii CR85. The pipeline involved the comparison of a collection of models which incorporate several alternative mechanisms with emphasis on the inhibitory effects due to temperature and ethanol. We focused on defining a minimal model with the minimum number of parameters, to maximize the identifiability and the quality of cross-validation. The selected model was then used to highlight differences in behavior between species. The analysis of model parameters would indicate that the specific growth rate and the transport of hexoses at initial times are higher for S. cervisiae T73 while S. kudriavzevii CR85 diverts more flux for glycerol production and cellular maintenance. As a result, the fermentations with S. kudriavzevii CR85 are typically slower; produce less ethanol but higher glycerol. Finally, we also explored optimal initial inoculation and process temperature to find the best compromise between final product characteristics and fermentation duration. Results reveal that the production of glycerol is distinctive in S. kudriavzevii CR85, it was not possible to achieve the same production of glycerol with S. cervisiae T73 in any of the conditions tested. This result brings the idea that the optimal design of mixed cultures may have an enormous potential for the improvement of final wine quality.

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Proteomic response to physiological fermentation stresses in a wild-type wine strain of Saccharomyces cerevisiae.

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