Project description:In the present study transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at genome-wide level how signalling and carbon catabolite repression differed in cells grown on either glucose or xylose. The more detailed knowledge about is xylose sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is it rather recognised as a non-fermentable carbon source is important in achieving understanding for further engineering this yeast for more efficient anaerobic fermentation of xylose.
Project description:In this study we focus on two Saccharomyces cerevisiae strains with varying production of heterologous α-amylase and we compare the metabolic fluxes and transcriptional regulation at aerobic and anaerobic conditions, in particular with the objective to identify the final electron acceptor for protein folding. We found that anaerobic conditions showed high amount of amylase productions when comparing to aerobic conditions and the genome-scale transcriptional analysis suggested that genes related to the endoplasmic reticulum (ER), lipid synthesis and stress responses were generally up-regulated at anaerobic conditions. Moreover, we proposed a model for the electron transfer from ER to the final electron acceptor, fumarate under anaerobic conditions.
Project description:Physiological effects of carbon dioxide and impact on genome-wide transcript profiles were analysed in chemostat cultures of Saccharomyces cerevisiae. In anaerobic, glucose-limited chemostat cultures grown at atmospheric pressure, cultivation under CO2-saturated conditions had only a marginal (<10%) impact on the biomass yield. Conversely, a 25% decrease of the biomass yield was found in aerobic, glucose-limited chemostat cultures aerated with a mixture of 79% CO2 and 21% O2. This observation indicated that respiratory metabolism is more sensitive to CO2 than fermentative metabolism. Consistent with the more pronounced physiological effects of CO2 in respiratory cultures, the number of CO2-responsive transcripts was higher in aerobic cultures than in anaerobic cultures. Many genes involved in mitochondrial functions showed a transcriptional response to elevated CO2 concentrations. This is consistent with an uncoupling effect of CO2 and/or intracellular bicarbonate on the mitochondrial inner membrane. Other transcripts that showed a significant transcriptional response to elevated CO2 included NCE103 (probably encoding carbonic anhydrase), PCK1 (encoding PEP carboxykinase) and members of the IMD gene family (encoding isozymes of inosine monophosphate dehydrogenase Keywords: Dose reponse
Project description:In the present study transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at genome-wide level how signalling and carbon catabolite repression differed in cells grown on either glucose or xylose. The more detailed knowledge about is xylose sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is it rather recognised as a non-fermentable carbon source is important in achieving understanding for further engineering this yeast for more efficient anaerobic fermentation of xylose. Experiment Overall Design: Three aerobic batch fermentations were carried out both on 50 g l-1 glucose and on 50 g l-1 xylose to compare the yeast transcriptome and proteome of cells growing on xylose with that of glucose repressed and glucose derepressed cells. Samples of the xylose-grown cells were harvested at 72 h from the start of the xylose cultures with 32 g l-1 of residual xylose present. Samples of the glucose repressed cells were harvested at 5 h from the start of the glucose cultures with 37 g l-1 of residual glucose present. Samples of the glucose derepressed cells were harvested at 24 h from the start of the glucose cultures containing no glucose but 13 g l-1 of accumulated ethanol.
Project description:In this study we focus on two Saccharomyces cerevisiae strains with varying production of heterologous M-NM-1-amylase and we compare the metabolic fluxes and transcriptional regulation at aerobic and anaerobic conditions, in particular with the objective to identify the final electron acceptor for protein folding. We found that anaerobic conditions showed high amount of amylase productions when comparing to aerobic conditions and the genome-scale transcriptional analysis suggested that genes related to the endoplasmic reticulum (ER), lipid synthesis and stress responses were generally up-regulated at anaerobic conditions. Moreover, we proposed a model for the electron transfer from ER to the final electron acceptor, fumarate under anaerobic conditions. Three Saccharomyces cerevisiae strains with varied amylase productions were selected at early glucose phase in batch fermentations for RNA extraction and hybridization on Affymetrix microarrays. Biological triplicates were applied, and strains with empty plasmid (no amylase productions) were used as control strain.
Project description:The inhibitors hydroxymethylfurfural (HMF) and furfural were added to the feed-medium of carbon-limited anaerobic chemostat cultures. Samples were taken for transcriptome analysis at steady-state from cultures with inhibitors and without inhibitors.
Project description:In this study we focus on two Saccharomyces cerevisiae (CEN. PK series) strains producing either insulin precursor or amylase and we compare the transcriptional regulation at different dilution rates, in particular with the objective to identify the relationship between cell metabolism and recombinant protein production. We found that anaerobic conditions showed high amount of amylase productions when comparing to aerobic conditions and the genome-scale transcriptional analysis suggested that genes related to the endoplasmic reticulum (ER), lipid synthesis and stress responses were generally up-regulated at anaerobic conditions. Moreover, we proposed a model for the electron transfer from ER to the final electron acceptor, fumarate under anaerobic conditions.
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.
Project description:In some of the earliest uses of genome-wide gene-expression microarrays and array-based Comparative Genomic Hybridization (aCGH), a set of diploid yeasts that had undergone experimental evolution under aerobic glucose limitation was used to explore how gene expression and genome structure had responded to this selection pressure. To more deeply understand how adaptation to one environment might constrain or enhance performance in another we have now identified the adaptive mutations in this set of clones using whole-genome sequencing, and have assessed whether the evolved clones had become generalists or specialists by assaying their fitness under three contrasting growth environments: aerobic and anaerobic glucose limitation and aerobic acetate limitation. Additionally, evolved clones and their common ancestor were assayed for gene expression, biomass estimates and residual substrate levels under the alternative growth conditions. Relative fitnesses were evaluated by competing each clone against a common reference strain in each environment. Unexpectedly, we found that the evolved clones also outperformed their ancestor under strictly fermentative and strictly oxidative growth conditions. We conclude that yeasts evolving under aerobic glucose limitation become generalists for carbon limitation, as the mutations selected for in one environment are advantageous in others. High-throughput sequencing of the evolved clones uncovered mutations in genes involved in glucose sensing, signaling, and transport that in part explain these physiological phenotypes, with different sets of mutations found in independently-evolved clones. Earlier gene expression data from aerobic glucose-limited cultures had revealed a shift from fermentation towards respiration in all evolved clones explaining increased fitness in that condition. However, because the evolved clones also show higher fitness under strictly anaerobic conditions and under conditions requiring strictly respirative growth, this switch cannot be the sole source of adaptive benefit. Furthermore, because independently evolved clones are genetically distinct we conclude that there are multiple mutational paths leading to the generalist phenotype. Strain Name: Parental strain (CP1AB) or evolved clones (E1 - E5) Media: aerobic / anaerobic