Project description:To investigate the glucose regulatory system in Saccharomyces cerevisiae, we conducted a time-course in which glucose-depleted wildtype (WT) cells were inoculated into fresh media (SC, 2% glucose). Their subsequent transcriptional output was monitored over a period of five hours by DNA microarrays: samples for gene expression profiling were taken immediately after, as well as 3, 7.5, 15, 30, 60, 110, 150, and 300 minutes after inoculation into fresh medium. Transcripts upregulated are involved in translational processes such as the GO biological processes “ribosome biogenesis” and “ribosome localization”. Transcripts downregulated are enriched for the GO biological processes “cellular respiration” and various metabolism related processes. The time-course was used to verify the physiological relevance of gene expression profiles determined for individual deletions of glucose regulatory system components. Importantly, transcripts up- or downregulated in WT cells upon the addition of glucose are similarly up- or downregulated in deletion mutants that each lack a component of the glucose regulatory system.
Project description:To investigate the glucose regulatory system in Saccharomyces cerevisiae, we conducted a time-course in which glucose-depleted wildtype (WT) cells were inoculated into fresh media (SC, 2% glucose). Their subsequent transcriptional output was monitored over a period of five hours by DNA microarrays: samples for gene expression profiling were taken immediately after, as well as 3, 7.5, 15, 30, 60, 110, 150, and 300 minutes after inoculation into fresh medium. Transcripts upregulated are involved in translational processes such as the GO biological processes “ribosome biogenesis” and “ribosome localization”. Transcripts downregulated are enriched for the GO biological processes “cellular respiration” and various metabolism related processes. The time-course was used to verify the physiological relevance of gene expression profiles determined for individual deletions of glucose regulatory system components. Importantly, transcripts up- or downregulated in WT cells upon the addition of glucose are similarly up- or downregulated in deletion mutants that each lack a component of the glucose regulatory system. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used for each separate hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Two overnight WT cultures were used to inoculate 50 ml cultures at an OD600 of 0.15. These were depleted of glucose by growing for 24 hours and were used the next day to inoculate 500 ml cultures in fresh medium to an OD600 of 0.15. Samples for expression profiling were taken immediately after, as well as 3, 7.5, 15, 30, 60, 110, 150, and 300 minutes after inoculation into fresh medium.
Project description:Microarrays were conducted to asses the effect of Stb3 deletion in immediate transcriptional induction in response to glucose Experiment Overall Design: Response of Stb3 delete cells was compared to that of wild type (BY4742) in cells grown for 3 days in YPD and cells that received 2% glucose for 10 minutes after the 3 day growth period. Two repetitive samples were done for each timepoint.
Project description:Dynamic transcriptional response of S. cerevisiae cells to copper impulse was investigated in both HO deletion strain used as the reference strain and the mutant strain lacking CCC2 gene which were grown in continuous cultures using a copper-deficient defined medium. Copper was introduced into the medium as an impulse so as to reach a copper sulfate concentration of 0.5 mM. Samples were collected within the first two hours following the copper addition (1st, 5th, 10th, 15th, 20th, 25th, 30th, 60th, 120th minutes), in addition to the steady-state sampling.
Project description:Aim: Analyse inhibitory effects of galacturonic acid, an important constituent of plant biomass hydrolysates, on growing and starving cultures of Saccharomyces cerevisiae CEN.PK113-7D. Method & Results: Biomass yields in aerobic and anaerobic glucose-limited chemostat cultures (pH 3.5) were reduced by 25 and 10%, respectively, upon addition of 10 g∙l-1 galacturonic acid. Genes previously reported to show a transcriptional response to other organic acids were overrepresented in a set of galacturonic-acid responsive genes identified by microarray analysis. These results suggested that galacturonic acid causes weak-acid uncoupling of the yeast plasma membrane pH gradient. Consistent with this hypothesis, galacturonate-accelerated loss of viability in starving cell suspensions was strongly pH dependent. Loss of viability was much slower in a strain in which all HXT (hexose transporter) genes were deleted. Moreover, deletion of HXT genes alleviated growth inhibition on ethanol observed at galacturonic acid concentrations of 10 g∙l-1 and above. Conclusions: At low pH, galacturonic acid negatively affects the physiology of S. cerevisiae. Reduced sensitivity of hexose-transporter mutants indicated that one or more HXT transporters are involved in transport of galacturonic acid. Significance and Impact: This study shows that galacturonic acid toxicity should be taken into account in process development for yeast-based fermentative conversion of pectin-rich feedstocks such as sugar beet pulp and citrus peel. Involvement of hexose transporters in galacturonic acid toxicity provides leads for improving tolerance. To investigate the impact of galacturonic acid on S. cerevisiae, a DNA microarray-based transcriptome analysis was performed on aerobic, glucose-limited chemostat cultures grown in the presence and absence of 10 g∙l-1 galacturonic acid at pH3.5.
Project description:Aim: Analyse inhibitory effects of galacturonic acid, an important constituent of plant biomass hydrolysates, on growing and starving cultures of Saccharomyces cerevisiae CEN.PK113-7D. Method & Results: Biomass yields in aerobic and anaerobic glucose-limited chemostat cultures (pH 3.5) were reduced by 25 and 10%, respectively, upon addition of 10 g∙l-1 galacturonic acid. Genes previously reported to show a transcriptional response to other organic acids were overrepresented in a set of galacturonic-acid responsive genes identified by microarray analysis. These results suggested that galacturonic acid causes weak-acid uncoupling of the yeast plasma membrane pH gradient. Consistent with this hypothesis, galacturonate-accelerated loss of viability in starving cell suspensions was strongly pH dependent. Loss of viability was much slower in a strain in which all HXT (hexose transporter) genes were deleted. Moreover, deletion of HXT genes alleviated growth inhibition on ethanol observed at galacturonic acid concentrations of 10 g∙l-1 and above. Conclusions: At low pH, galacturonic acid negatively affects the physiology of S. cerevisiae. Reduced sensitivity of hexose-transporter mutants indicated that one or more HXT transporters are involved in transport of galacturonic acid. Significance and Impact: This study shows that galacturonic acid toxicity should be taken into account in process development for yeast-based fermentative conversion of pectin-rich feedstocks such as sugar beet pulp and citrus peel. Involvement of hexose transporters in galacturonic acid toxicity provides leads for improving tolerance.