Project description:We exploited natural diversity in 165 Saccharomyces cerevisiae strains isolated from diverse geographical and ecological niches, to identify mechanisms of hydrolysate-toxin tolerance. We performed genome-wide association (GWA) analysis to identify genetic variants underlying toxin tolerance, and gene knockouts and allele-swap experiments to validate the involvement of implicated genes. In the process of this work, we uncovered a surprising difference in genetic architecture depending on strain background: in all but one case, knockout of implicated genes had a significant effect on toxin tolerance in one strain, but no effect in another strain.
Project description:Ethanol is a valuable industrial product and a common metabolite used by many cell types. However, this molecule produces high levels of cytotoxicity affecting cellular performance at several levels. In the presence of ethanol, cells must adjust some of their components, such as the membrane lipids to maintain homeostasis. In the case of microorganism as Saccharomyces cerevisiae, ethanol is one of the principal products of their metabolism and is the main stress factor during fermentation. Although many efforts have been made, mechanisms of ethanol tolerance are not fully understood and very little evidence is available to date for specific signaling by ethanol in the cell. This work studied two Saccharomyces cerevisiae strains, CECT10094 and Temohaya-26, isolated from flor wine and traditional fermentations respectively, which differ in ethanol tolerance, in order to understand the molecular mechanisms underlying the ethanol stress response and the reasons for different ethanol tolerance. The transcriptome was analyzed after ethanol stress and, among others, an increased activation of genes related with the unfolded protein response (UPR) and its transcription factor, Hac1p, was observed in the tolerant strain CECT10094. We observed that this strain also resist more UPR agents than Temohaya-26 and the UPR-ethanol stress correlation was corroborated observing growth of 15 more strains and discarding UPR correlation with other stresses as thermal or oxidative stress. Furthermore, higher activation of UPR pathway in the tolerant strain CECT10094 was observed using a UPR mCherry reporter. Finally, we observed UPR activation in response to ethanol stress in other S. cerevisiae ethanol tolerant strains as the wine strains T73 and EC1118. Overall design: Two condition experiment and two strains. CECT10094 and Temohaya-26 strains were harvested at 1h and after exponential phase in presence of 0% and 10% (v/v)ethanol in media. All samples were labeled with Cy5 dye, while a reference pool prepared by pooling the RNA extracted in all samples was labeled with Cy3. Three biological replicates were combined and indirect comparison between slides belonging to stressed and unstressed samples to identify genes whose variation is due to the effect of ethanol was performed. Biological triplicates are included. One replicate per array.
Project description:Metabolic engineering of Saccharomyces cerevisiae for efficient monoterpenes production was mostly restricted by the limited tolerance to these chemicals. Understanding of the molecular mechanisms underlying the tolerance of S. cerevisiae to monoterpenes was essential for the de novo biosynthesis these chemicals in S. cerevisiae. In this study, commercial oligonucleotide microarray assays were performed to investigate the global response of S. cerevisiae to typical monoterpene D-limonene under transcriptional level. Yeast cell treated with sublethal dose of D-liomonene, gene change profiles were investigated at transcription level and the microarry data were also verified with quantitative real time PCR. D-limonene induced gene expression in Saccharomyces cerevisiae at early logarithmic phase was measured at 2 hours after exposure to doses of 0.02% (v/v) D-limonene. Three independent experiments were performed for each experiment (control or 2 hours).
Project description:The environmental stresses and inhibitors encounted by Saccharomyces cerevisiae strains are main limiting factors in bioethanol fermentation. Investigation of the molecular mechanisms underlying the stresses-related phenotypes diversities within and between S. cerevisiae populations could guide the construction of yeast strains with improved stresses tolerance and fermentation performances. Here, we explored the genetic characteristics of the bioethanol S. cerevisiae strain YJS329, and elucidated the genetic variations correlated with its advantaged traits (higher ethanol yield under sever conditions and better tolerance to multiple stresses compared to an S288c derived laboratory strain BYZ1). Firstly, pulse-field gel electrophoresis combined with array-comparative genomic hybridization was used to compare the genome structure of YJS329 and the laboratory strain BYZ1. Yeast cells were cultured in YPD medium. Genome DNA of YJS329 and BYZ1 was isolated and sonicated. The average length of DNA fragments was 200-1000bp. The shearing DNA was labeled with Cy5/Cy3 and hybridized to NimbleGen S.cerevisiae Whole-Genome Tiling arrays, which is single array design containing all chromosomes with 32bp median probe spacing and totally covered by ~385,000 probes. Scanning was performed with the Axon GenePix 4000B microarray scanner.
Project description:Diploid and haploid strains often exhibit different tolerance to variety of stresses. Transcriptome of acclimation to ethanol stress in diploid and haploid strain of Saccharomyces cerevisiae was analyzed. We analyzed transcriptome profiles of diploid and haploid strains in the presence of ethanol. Overall design: Haploid and diploid strains were cultured in YEPD media with 0%, 3% and 7% ethanol(v/v) in fermentors. The samples were collected at the growth stage for each strain under different conditions.
Project description:Contains a collection of wildtype Saccharomyces cerevisiae strains for estimating the biological variation. Wildtypes are obtained from the "yeast wildtypes - platereader, wt pool bkg set" HybSet, by randomly taking 100 wt vs. refpool and 100 refpool vs. wt hybridizations.
Project description:Contains a collection of wildtype Saccharomyces cerevisiae strains for estimating the biological variation. Wildtypes are obtained from the yeast wildtypes - wt pool background set HybSet, by randomly taking 100 wt vs. refpool (pooled wts) and 100 refpool vs. wt hybridizations
Project description:Diploid and haploid strains often exhibit different tolerance to variety of stresses. Transcriptome of acclimation to ethanol stress in diploid and haploid strain of Saccharomyces cerevisiae was analyzed. We analyzed transcriptome profiles of diploid and haploid strains in the presence of ethanol. Haploid and diploid strains were cultured in YEPD media with 0%, 3% and 7% ethanol(v/v) in fermentors. The samples were collected at the growth stage for each strain under different conditions.
Project description:Contains a collection of wildtype matA Saccharomyces cerevisiae strains for estimating the biological variation. Two channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. Two independent cultures were hybridized on two separate microarrays.
Project description:Gene expression is regulated both by cis elements, which are DNA segments closely linked to the genes they regulate, and by trans activating factors, which are usually proteins capable of diffusing to unlinked genes. Understanding the patterns and sources of regulatory variation is crucial for understanding phenotypic and genome evolution. Here, we investigate the global patterns of gene expression evolution in Saccharomyces cerivisiae. We report statistical methods useful in quantifying cis and trans regulation using next generation sequencing data. Using these methods, measured genome-wide allele-specific expression by deep sequencing to investigate the genetic architecture of gene regulatory variation between two strains of Saccharomyces cerevisiae. We find that expression polymorphism in yeast is common for both cis and trans regulation, though trans variation is more common. Our detailed analyses of the effects of functional constraint on expression variation as indicated by measures such as protein connectivity, gene essentiality, and the ratio of nonsynonymous substitutions to synonymous substitutions clearly reveal that both classes of variation are under purifying selection, but trans variation is more sensitive to selective constraint. Comparing interspecific expression divergence between S. cerevisiae and S. paradoxus to our intraspecific variation suggests that natural selection strongly influences the patterns of variation we observe. Further analyses revealed that cis divergence is more frequently mediated by positive Darwinian selection than trans divergence, which is compatible with neutral evolution. Overall design: Study the gene expression patterns in two strains of yeast (BY and RM)