ABSTRACT: Expression Differences in Ethanol Stress Response Between Saccharomyces cerevisiae Parent W303-1A Strain and its Two Ethanol Tolerant Mutants
Project description:Saccharomyces spp. are widely used for ethanol production however fermentation productivity is negatively affected by the impact of ethanol accumulation on yeast metabolic rate and viability. This study used microarray and statistical two-way ANOVA analysis to compare and evaluate gene expression profiles of two previously generated ethanol-tolerant mutants, CM1 and SM1, with their parent, S. cerevisiae W303-1A, in the presence and absence of ethanol stress. Although sharing the same parentage, the mutants were created differently; SM1 by adaptive evolution involving long-term exposure to ethanol stress, and CM1 using chemical mutagenesis followed by adaptive evolution-based screening. Compared to the parent, differences in the expression levels of genes associated with a number of GO categories in the mutants suggest that their improved ethanol stress response is a consequence of increased mitochondrial and NADH oxidation activities, stimulating glycolysis and energy production. This leads to increased activity of energy-demanding processes associated with the production of proteins and plasma membrane components, which are necessary for acclimation to ethanol stress. It is suggested that a key function of the ethanol stress response is restoration of the NAD+/NADH redox balance, which increases glyceraldehyde-3-phosphate dehydrogenase activity, and higher glycolytic flux in the ethanol-stressed cell. Both mutants achieved this by a constitutive increase in carbon flux in the glycerol pathway as a means of increasing NADH oxidation.
Project description:Saccharomyces spp. are widely used for ethanol production however fermentation productivity is negatively affected by the impact of ethanol accumulation on yeast metabolic rate and viability. This study used microarray and statistical two-way ANOVA analysis to compare and evaluate gene expression profiles of two previously generated ethanol-tolerant mutants, CM1 and SM1, with their parent, S. cerevisiae W303-1A, in the presence and absence of ethanol stress. Although sharing the same parentage, the mutants were created differently; SM1 by adaptive evolution involving long-term exposure to ethanol stress, and CM1 using chemical mutagenesis followed by adaptive evolution-based screening. Compared to the parent, differences in the expression levels of genes associated with a number of GO categories in the mutants suggest that their improved ethanol stress response is a consequence of increased mitochondrial and NADH oxidation activities, stimulating glycolysis and energy production. This leads to increased activity of energy-demanding processes associated with the production of proteins and plasma membrane components, which are necessary for acclimation to ethanol stress. It is suggested that a key function of the ethanol stress response is restoration of the NAD+/NADH redox balance, which increases glyceraldehyde-3-phosphate dehydrogenase activity, and higher glycolytic flux in the ethanol-stressed cell. Both mutants achieved this by a constitutive increase in carbon flux in the glycerol pathway as a means of increasing NADH oxidation. Two conditions: 0% and 6.5 % (v/v) ethanol added to the culture growth medium for each strain. One set of triplicates with one dye swap for each condition. Each triplicate was prepared from different biological replicate (i.e. different culture).
Project description:Transcriptional profiling of Saccharomyces cerevisiae cells comparing the W303-1A wildtype with the W303-1A double mutant for MSN2 and MSN4 during zinc deficient conditions Keywords: Genetic modification with zinc limitation
Project description:Transcriptional profiling of Saccharomyces cerevisiae cells comparing the W303-1A wildtype with the W303-1A double mutant for MSN2 and MSN4 during zinc deficient conditions Keywords: Genetic modification with zinc limitation Two condition experiment, W303-1A vs W303-1A delta MSN2, MSN4. Biological replicates: 2 wildtype, 2 knock-out, independently grown and harvested.
Project description:Transcriptional profiling of ethanol tolerant strains Ets2 and Ets3 comparing control Saccharomyces cerevisiae L3262 with ethanol tolerant strains Ets2 and Ets3, through screening a mutant library of SPT15 of Saccharomyces cerevisiae L3262.
Project description:Proteomic analysis of the extracellular matrix of Saccharomyces cerevisiae W303-1A Wt and the isogenic mutant strain gup1Δ during the development of multicellular overlays.
Project description:To identify genes involved in acclimatization of yeast to non-lethal ethanol stress, S. cerevisiae strain W303-1A was grown separately at 0% and 6.5% (v/v) ethanol, and gene expression profiles were compared using microarray analysis. A t-test with a p-value of 0.05 was applied for differences in gene expression across unstressed and stressed replicates of W303-1A. There were 1049 genes with significant differences in expression. Among the genes that passed the t-test, 46 genes were up-regulated 5-fold and more in ethanol stress and 149 by more than 3-fold. There were 40 genes down-regulated by more than a factor of 5 and 161 by more than a factor of 3; the remaining genes that passed the t-test had expression level differences lower than 3-fold. GenowizTM bioanalysis results that identify GO categories comprising stress induced (more than 3-fold) genes show the two highest ethanol stress induced categories to be Retrotransposition Nucleocapsid (z-score of 15.69) and Transposition RNA Mediated (z-score of 15.54). These categories were not reported in previous gene expression studies on ethanol stress (Alexandre et al. 2001, Chandler et al. 2004, Fujita et al. 2004, Hirasawa et al. 2007). Our further investigaition identifies lack of annotation and omission of the genes from the retrotransposition-associated categories in slide printing as possible causes of this inconsistency.
Project description:Genomic DNA from Saccharomyces cerevisiae W303-1A was isolated along with nucleosomal DNA from W303-1A and the yeast strain GAL:orc2-1. Nucleosomal DNA was isolated following a 2h release from a nocodazole block in which the medium was changed from galactose (YPAG) to glucose (YPAD).
Project description:Evolutionary engineering strategy was used for selection of ethanol-tolerant Saccharomyces cerevisiae clones under gradually increasing ethanol stress levels. Clones B2 and B8 were selected based on their higher ethanol-tolerance and higher ethanol production levels. Whole genome microarray analysis was used for identifying the gene expression levels of these two evolved clones compared to the reference strain.
