Effect of endogenous CO2 overpressure on the wine yeasts proteome during the second fermentation in cava elaboration

Durante los últimos años, el consumo y exportación de vinos espumosos está en auge. La elaboración de vino espumoso siguiendo el método tradicional o “Champenoise” es un proceso altamente tecnificado que está formado por dos etapas fermentativas. En la primera, el mosto procedente del prensado de la uva es transformado en un vino base, al que se adiciona azúcar y se inocula con levaduras seleccionadas en botella cerrada para llevar a cabo la segunda fermentación. Una vez finaliza esta segunda etapa fermentativa, las levaduras se someten a un largo período de envejecimiento sobre lías. La etapa global que comprende la segunda fermentación y el período de crianza recibe el nombre de “prise de mousse” o toma de espuma. Además de los estreses típicos de una fermentación, como alto contenido en etanol y falta de nutrientes, durante la segunda fermentación también afectan otros factores a las levaduras, como baja temperatura y sobrepresión de CO2 endógeno. Las cepas de segunda fermentación deben ser capaces de adaptarse y tolerar estos estreses para mantener la viabilidad y asegurar el éxito de esta etapa. Para ello las células disponen de mecanismos como la autofagia, proceso inducido bajo condiciones de falta de nutrientes que les permite sobrevivir mediante el reciclaje de sus propios componentes intracelulares. Una vez los compuestos tóxicos como el etanol incrementan en el vino, el papel que juegan orgánulos como la mitocondria resultaría de gran importancia para la adaptación y resistencia a estas condiciones. Sin embargo, la mayoría de las células mueren en los primeros meses de envejecimiento y su contenido intracelular es liberado al vino a través de la acción de enzimas hidrolíticas, en el proceso conocido como autolisis. Dicho proceso contribuye enormemente a la mejora de la calidad y propiedades organolépticas de los vinos espumosos. Durante la autolisis, la rotura de la pared celular de las levaduras y liberación de sus componentes es un hecho importante. En este contexto, las manoproteínas son esenciales desde el punto de vista enológico, mejorando la estabilidad de los vinos y potenciando sus propiedades organolépticas. Además de la tolerancia a los distintos estreses, la capacidad de floculación de las cepas es un criterio de selección que permite una buena separación de las levaduras durante la etapa de removido. La respuesta de las levaduras a la sobrepresión de CO2 se ha estudiado principalmente desde el punto de vista proteómico, ya que este enfoque permite analizar el comportamiento de las levaduras ante condiciones de estrés de una manera global. Aunque es un enfoque interesante, la mayoría de las investigaciones sobre la segunda fermentación de los vinos espumosos se basan en enfoques transcriptómicos y metabolómicos. Por lo tanto, los resultados obtenidos en este trabajo aportarían nueva información y complementarían a los trabajos ya existentes. En esta Tesis Doctoral se estudió y comparó el comportamiento del proteoma de dos cepas de levadura industriales, Saccharomyces cerevisiae P29 y G1, bajo condiciones de sobrepresión por CO2, típicas de la elaboración de vino espumoso. Mientras la cepa P29 es comúnmente usada en la elaboración de estos vinos y por ello está perfectamente adaptada a estas condiciones tan especiales, la cepa G1 es una levadura de velo de flor responsable de la crianza biológica de los vinos finos, no familiarizada por tanto con estas condiciones fermentativas. Aunque mostraron pautas similares en su cinética de fermentación, el comportamiento a nivel proteómico fue diferente bajo condiciones de presión. Estas diferencias vienen dadas por la plasticidad del proteoma bajo condiciones de estrés, el cual permite a las levaduras adaptarse de distinta manera, pero igual de eficiente para lograr su supervivencia. Proteínas relacionadas con mecanismos adaptativos como la autofagia, la respuesta a estrés y remodelamiento e integridad de la pared celular, se detectaron en ambas cepas bajo condiciones de presión. La respuesta proteómica de S. cerevisiae P29 a la sobrepresión por CO2 estuvo caracterizada por un aumento de proteínas de estrés involucradas principalmente en la síntesis de glicerol, la resistencia a metabolitos tóxicos, la eliminación de ROS y la acumulación de energía. También se encontraron proteínas mitocondriales requeridas para la cadena respiratoria y la síntesis de aminoácidos ramificados asociados a la formación de compuestos aromáticos, bajo estas condiciones. Estos resultados podrían explicarse debido a la respuesta causada por la acumulación de etanol a lo largo de la segunda fermentación, más que a la sobrepresión por CO2 endógeno. Por otro lado, la respuesta proteómica de la levadura de velo de flor S. cerevisiae G1 estuvo marcada por una alta abundancia de manoproteínas, un mayor requerimiento de proteínas responsables de la síntesis y remodelación de la pared celular, y un elevado contenido de enzimas hidrolíticas asociadas con el proceso de autolisis. Estos resultados, junto con su capacidad para flocular y tolerar altas concentraciones de etanol, convierten a la cepa G1 en una alternativa interesante para la mejora y producción de vino espumoso.During the last years, the consumption and export of sparkling wines has been booming. Sparkling wine elaboration following the traditional method or “Champenoise” is a highly technical process which consists of two fermentative stages. In the first phase, the must coming from the pressing of grapes is transformed into a base wine, which is added with sugar and inoculated with selected yeasts to carry out the second fermentation in sealed bottle. Once this fermentative stage is over, yeast cells are subjected to a large aging period in contact with yeast lees. The whole process involving the second fermentation and aging is called “prise de mousse” or foam production. Apart from the typical fermentation stresses as high ethanol content and nutrient starvation, during the second fermentation, other factors as low temperature and endogenous CO2 overpressure affect yeast cells. Second fermentation strains must be capable of adapting and tolerating these stresses in order to preserve cell viability and ensure the success of this stage. For this purpose, yeast cells have mechanisms such as autophagy, process induced under starvation conditions which allow them to survive via recycling of their own intracellular components. Once toxic compounds as ethanol increase in the wine, the role which play certain organelles as mitochondria would be of great importance for the adaptation and resistance to these conditions. However, most of cells die at the first months of aging and their intracellular content is released into the wine through the action of hydrolytic enzymes, in a process known as autolysis. This process contributes hugely to quality improvement and organoleptic properties of sparkling wines. During this process, the yeast cell wall breakdown and the release of its components is an important fact. In this context, cell wall mannoproteins are essential from the enological point of view, improving the wine stability and enhancing their organoleptic properties. In addition to the stress tolerance, the flocculation capacity of the yeast strains is selection criteria which allow a proper separation of yeast cells during wine clarification. The yeast response to CO2 overpressure has been studied mainly from the proteomic point of view, since this approach allow to analyze the yeast behavior under stress conditions in a global way. Even though it is an interesting approach, most of research about second fermentation of sparkling wine is based on transcriptomic and metabolomic approaches. Therefore, the results obtained in this work would generate new information and complement the existing works. In this Thesis the proteome behavior was studied and compared in two industrial yeast strains, Saccharomyces cerevisiae P29 and G1, under CO2 overpressure conditions, typical of sparkling wine elaboration. Whereas P29 strain is commonly used in the elaboration of these wines and for these reason, it is perfectly adapted to these special conditions, the G1 strain is a flor yeast responsible for biological aging of sherry wines, and therefore, not familiar with these fermentative conditions. Although both strains show similar patterns in their fermentation kinetics, the behavior at proteomic level was different under pressure conditions. These differences come from the proteome plasticity under stress conditions, which allow yeast cells to adapt in a different way, but just as efficient to achieve their survival. Proteins related to adaptive mechanisms such as autophagy, stress response and cell wall remodeling and integrity, were detected in both yeast strains under pressure conditions. The proteomic response of S. cerevisiae P29 to CO2 overpressure was characterized by an increase in stress proteins mainly involved in glycerol synthesis, resistance to toxic metabolites, ROS removing and energy accumulation. Mitochondrial proteins required for respiratory chain and branched-amino acid metabolism associated with aromatic compounds formation, were also found under these conditions. These results could be explained due to the cell response caused by ethanol accumulation along the second fermentation, more than endogenous CO2 overpressure. On the other hand, the proteomic response of the flor yeast S. cerevisiae G1 was marked by a high abundance of mannoproteins, a high requirement of proteins responsible for cell wall synthesis and remodeling, and a high content of hydrolytic enzymes associated with autolysis process. These results, along with its capacity to flocculate and tolerate high ethanol concentrations, make G1 strain an interesting alternative for sparkling wine improvement and production

First Proteomic Approach to Identify Cell Death Biomarkers in Wine Yeasts during Sparkling Wine Production

Apoptosis and later autolysis are biological processes which take place in Saccharomyces cerevisiae during industrial fermentation processes, which involve costly and time-consuming aging periods. Therefore, the identification of potential cell death biomarkers can contribute to the creation of a long-term strategy in order to improve and accelerate the winemaking process. Here, we performed a proteomic analysis based on the detection of possible apoptosis and autolysis protein biomarkers in two industrial yeast strains commonly used in post-fermentative processes (sparkling wine secondary fermentation and biological aging) under typical sparkling wine elaboration conditions. Pressure had a negatively effect on viability for flor yeast, whereas the sparkling wine strain seems to be more adapted to these conditions. Flor yeast strain experienced an increase in content of apoptosis-related proteins, glucanases and vacuolar proteases at the first month of aging. Significant correlations between viability and apoptosis proteins were established in both yeast strains. Multivariate analysis based on the proteome of each process allowed to distinguish among samples and strains. The proteomic profile obtained in this study could provide useful information on the selection of wine strains and yeast behavior during sparkling wine elaboration. Additionally, the use of flor yeasts for sparkling wine improvement and elaboration is proposed

A Differential Proteomic Approach to Characterize the Cell Wall Adaptive Response to CO2 Overpressure during Sparkling Wine-Making Process

In this study, a first proteomic approach was carried out to characterize the adaptive response of cell wall-related proteins to endogenous CO2 overpressure, which is typical of second fermentation conditions, in two wine Saccharomyces cerevisiae strains (P29, a conventional second fermentation strain, and G1, a flor yeast strain implicated in sherry wine making). The results showed a high number of cell wall proteins in flor yeast G1 under pressure, highlighting content at the first month of aging. The cell wall proteomic response to pressure in flor yeast G1 was characterized by an increase in both the number and content of cell wall proteins involved in glucan remodeling and mannoproteins. On the other hand, cell wall proteins responsible for glucan assembly, cell adhesion, and lipid metabolism stood out in P29. Over-represented proteins under pressure were involved in cell wall integrity (Ecm33p and Pst1p), protein folding (Ssa1p and Ssa2p), and glucan remodeling (Exg2p and Scw4p). Flocculation-related proteins were not identified under pressure conditions. The use of flor yeasts for sparkling wine elaboration and improvement is proposed. Further research based on the genetic engineering of wine yeast using those genes from protein biomarkers under pressure alongside the second fermentation in bottle is required to achieve improvements

Autophagic Proteome in Two Saccharomyces cerevisiae Strains During Second Fermentation for Sparkling Wine Elaboration

A correlation between autophagy and autolysis has been proposed in order to accelerate the acquisition of wine organoleptic properties during sparkling wine elaboration. In this context, a proteomic analysis was carried out in two industrial Saccharomyces cerevisiae strains (P29, conventional sparkling wine strain and G1, implicated in sherry wine elaboration) with the aim of studying the autophagy-related proteome and comparing the effect of CO2 overpressure during sparkling wine elaboration. In general, a detrimental effect of pressure and second fermentation development on autophagy-related proteome was observed in both strains, although it was more pronounced in flor yeast strain G1. Proteins mainly involved in autophagy regulation and autophagosome formation in flor yeast G1, and those required for vesicle nucleation and expansion in P29 strain, highlighted in sealed bottle. Proteins Sec2 and Sec18 were detected 3-fold under pressure conditions in P29 and G1 strains, respectively. Moreover, ‘fingerprinting’ obtained from multivariate data analysis established differences in autophagy-related proteome between strains and conditions. Further research is needed to achieve more solid conclusions and design strategies to promote autophagy for an accelerated autolysis, thus reducing cost and time production, as well as acquisition of good organoleptic properties