Project description:Laboratory strains of Saccharmoyces cerevisiae have been widely used as a model for studying eukaryotic cells and mapping the molecular mechanisms of many different human diseases. Industrial wine yeasts, on the other hand, have been selected over hundreds of years on the basis of their adaptation to stringent environmental conditions and the organoleptic properties they confer to wine. Here, we applied a two-factor design to study the response of a standard laboratory strain, CEN.PK.113-7D, and an industrial wine yeast-strain, EC1118, to growth temperature at 15°C and 30°C under 12 nitrogen-limited, anaerobic steady-state chemostat cultures. Physiological characterization revealed that growth temperature strongly impacted biomass yields in both strains. Moreover, we observed that the wine yeast is better adapted to mobilizing resources for biomass and that the laboratory yeast exhibited higher fermentation rates. To elucidate mechanistic differences controlling the growth temperature response and underlying adaptive mechanisms between strains, DNA microarrays and targeted metabolome analysis were used. We identified 1007 temperature dependent genes and 473 strain dependent genes. The transcriptional response was used to identify highly correlated subnetworks of significantly changing genes in metabolism. We show that temperature differences most strongly affect nitrogen metabolism and the heat shock response. Lack of STRE mediated gene induction, coupled with reduced trehalose levels, indicates a decreased general stress response at 15°C relative to 30°C. Between strains, differential responses are centred around sugar uptake, nitrogen metabolism and expression of genes related to organoleptic properties. Our study provides global insight into how growth temperature exerts a differential physiological and transcriptional response in laboratory and wine strains of S. cerevisiae.
Project description:The aim of this study is to phenotype a collection of 27 S. cerevisiae commercial wine strains growing within temperatures (4-45ºC) in both minimal media (SD) and synthetic must (SM) and, taking into account µmax value, to select two strains with divergent phenotype in their capacity to grow at low temperature. To confirm this differential phenotype, we design a competition between both strains during wine fermentations. As expected, at low temperature fermentation, the strain showing a good performance out-competes to the strain growing badly in cold. Finally we aimed to decipher the molecular basis underlying this divergent phenotype by analyzing the genomic, proteomic and transcriptomic differences between both strains at low temperature (15ºC) and optimum temperature (28ºC).
Project description:The aim of this study is to phenotype a collection of 27 S. cerevisiae commercial wine strains growing within temperatures (4-45ºC) in both minimal media (SD) and synthetic must (SM) and, taking into account µmax value, to select two strains with divergent phenotype in their capacity to grow at low temperature. To confirm this differential phenotype, we design a competition between both strains during wine fermentations. As expected, at low temperature fermentation, the strain showing a good performance out-competes to the strain growing badly in cold. Finally we aimed to decipher the molecular basis underlying this divergent phenotype by analyzing the genomic, proteomic and transcriptomic differences between both strains at low temperature (15ºC) and optimum temperature (28ºC). Two Saccharomyces cerevisiae strains with divergent phenotype in their capacity to grow and ferment at low temperature were analyzed (P5 strain as a candidate with a good performance in fermentations at low temperature (15ºC) and P24 as a candidate with a worse behavior at low temperature). All experiments were performed using triplicates arrays, and Cy5-dCTP and Cy3-dCTP dye-swap assays were performed to reduce dye-specific bias.
Project description:To characterize the ecological interactions among S. cerevisiae strains coming from the same geographical area, we examined the fitness of two natural isolates from San Giovese grapes, alone or in competition, in synthetic wine must (SWM). We performed genome-wide analyses in order to identify the genes involved in yeast competition and cooperation.
Project description:To characterize the ecological interactions among S. cerevisiae strains coming from the same geographical area, we examined the fitness of two natural isolates from San Giovese grapes, alone or in competition, in synthetic wine must (SWM). We performed genome-wide analyses in order to identify the genes involved in yeast competition and cooperation.
Project description:Yeast mannoproteins contribute to several aspects of wine quality by protecting wine against protein haze, reducing astringency, retaining aroma compounds and stimulating growth of lactic-acid bacteria. The selection of a yeast strain simultaneously overproducing mannoproteins and showing good fermentative characteristics is a difficult task. In this work, a Saccharomyces cerevisiae x Saccharomyces cerevisiae hybrid bearing the two oenologically relevant features was constructed and a reduction in the amount of bentonite necessary for wine stabilization was observed for wines fermented with the generated strain. Additionally, different copy numbers of some genes probably related with these physiological features were detected in this hybrid. Hybrid share with parental Sc1 similar copy number of genes SPR1, SWP1, MNN10 and YPS7 related to cell wall integrity and with parental Sc2 similar copy number of some glycolytic genes as GPM1 and HXK1 as well as genes involved in hexose transport as HXT9, HXT11 and HXT12. This work demonstrates that artificial hybridization and stabilization in winemaking conditions constitute an effective approach to obtain yeast strains with desirable physiological features as mannoprotein overproducing capacity and improved fermentation performance, characteristics genetically depending on the coordinated expression of a multitude of different genes. In this work, genetically stable mannoprotein overproducing Saccharomyces cerevisiae strains simultaneously showing excellent fermentation capacities were obtained by hybridization methods giving rise to non-GMO strains. The potential relationship between the copy number of specific genes and the improved features was also evaluated by means of aCGH analysis of parental and hybrid strains.
Project description:By an evolutionary approach based on long-term culture on gluconate as the sole carbon source, a Saccharomyces cerevisiae wine strains with enhanced flux through the pentose phosphate (PP) pathway were obtained. One of these evolved strains, ECA5, exhibited several novel properties with great potential for wine making, including a higher than wild-type fermentation rate and altered production of acetate and aroma compounds. To describe the mechanisms underlying this complex phenotype, we performed a comparative analysis of transcriptomic profiles between ECA5 and its ancestral strain, EC1118, under low nitrogen, wine fermentation conditions.
Project description:Saccharomyces cerevisiae has been used as a secretion host for production of various products, including pharmaceuticals. However, few antibody molecules have been functionally expressed in S. cerevisiae due to the incompatible surface glycosylation. Our laboratory previously isolated a group of yeast mutant strains with different α-amylase secretory capacities, and these evolved strains have showed advantages for production of some heterologous proteins. However, it is not known whether these secretory strains are generally suitable for pharmaceutical protein production. Here, three non-glycosylated antibody fragments with different configurations (Ran-Fab fragment Ranibizumab, Pex-the scFv peptide Pexelizumab, and Nan-a single V-type domain) were successfully expressed and secreted in three background strains with different secretory capacities, including HA (wild type), MA (evolved strain), and LA (evolved strain). However, the secretion of Ran and Nan were positively correlated with the strains’ secretory capacity, while Pex was most efficiently secreted in the parental strain. Therefore, transcriptional analysis was performed to explore the fundamental changes triggered by the expression of the different pharmaceutical proteins in these selected yeast strains.
Project description:Elucidation of the mechanism of temperature tolerance in yeast is essential for enhancing cellular robustness of strains, providing more economically and sustainable sound industrial processes. We investigated the differential responses of three distinct Saccharomyces cerevisiae strains, an industrial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and an industrial bioethanol strain, Ethanol Red, grown at sub- (12 ºC) and-supra (39 ºC) optimal temperatures under chemostat conditions, in which specific growth rate–dependent changes are eliminated. In contrast to previous studies, we employed anaerobic conditions, mimicking the industrial processes. The proteomic profile of these strains was performed by SWATH-MS, allowing the quantification of 997 proteins. Our analysis revealed that temperature response differs between the strains, highlighting the precision with which each strain responds to changes in its environment; however, we also found some common responsive proteins among the strains, revealing that the response to temperature involves general stress and specific mechanisms. Overall, sub-optimal temperature conditions involved a higher remodeling of the proteome than supra-optimal temperature. Further, the proteomic data evidenced that the cold response involves a strong repression of translation related proteins as well as induction of amino acid metabolism, together with components related to protein folding and degradation. On the other hand, the high temperature response mainly recruits amino acid metabolism. Our study provides a global and thorough insight into how growth temperature affects yeast proteome, which can be a step forward for research regarding the comprehension and the improvement of yeast thermotolerance.