Project description:Exploring molecular details of carbon utilization trade-offs in galactose-evolved yeast Adaptively evolved yeast mutants on galactose for around 400 generations showed diminished growth and carbon uptake rates on glucose. Genome-scale approaches were applied to characterize the molecular genetic basis of these trade-offs in carbon source utilization. Engineered mutants showing trade-offs in a specific carbon uptake rate between both carbons were used as controls. The transcriptional responses of the evolved mutants were almost identical during growth on both carbon sources. These carbon-independent conserved patterns were clearly observed in specific pathways and genes. Up-regulation of PGM2, a confirmed beneficial genetic change for improving galactose utilization was preserved on both carbons. In addition, HXK1, GLK1 and genes involved in reserve carbohydrate metabolism were up-regulated, while HXK2 was down-regulated. Genes that have a transcription factor binding site for Gis1p, Rph1p, Msn2/4p and Nrg1p were up-regulated. These results indicated changes in the metabolic pathways involved in metabolism of both carbons and in nutrient signaling pathway. The concentration profile of trehalose and glycogen supported these findings. Mutations in RAS2 and ERG5 genes were selected because of their beneficial and neutral effect on galactose utilization, respectively in our previous study. Site-directed mutants containing galactose-beneficial mutations in RAS2 only resulted in a significant decrease in glucose utilization. Integration of all these analyses clearly suggest an antagonistic pleiotropic trade-off in carbon source utilization caused by changes in regulatory region, and we hereby demonstrate how systems biology can be used to gain insight into evolutionary processes at the molecular level. Yeast galactose evolved mutants having improved galactose availability were grown on aerobic batch with glucose as carbon source
Project description:Exploring molecular details of carbon utilization trade-offs in galactose-evolved yeast Adaptively evolved yeast mutants on galactose for around 400 generations showed diminished growth and carbon uptake rates on glucose. Genome-scale approaches were applied to characterize the molecular genetic basis of these trade-offs in carbon source utilization. Engineered mutants showing trade-offs in a specific carbon uptake rate between both carbons were used as controls. The transcriptional responses of the evolved mutants were almost identical during growth on both carbon sources. These carbon-independent conserved patterns were clearly observed in specific pathways and genes. Up-regulation of PGM2, a confirmed beneficial genetic change for improving galactose utilization was preserved on both carbons. In addition, HXK1, GLK1 and genes involved in reserve carbohydrate metabolism were up-regulated, while HXK2 was down-regulated. Genes that have a transcription factor binding site for Gis1p, Rph1p, Msn2/4p and Nrg1p were up-regulated. These results indicated changes in the metabolic pathways involved in metabolism of both carbons and in nutrient signaling pathway. The concentration profile of trehalose and glycogen supported these findings. Mutations in RAS2 and ERG5 genes were selected because of their beneficial and neutral effect on galactose utilization, respectively in our previous study. Site-directed mutants containing galactose-beneficial mutations in RAS2 only resulted in a significant decrease in glucose utilization. Integration of all these analyses clearly suggest an antagonistic pleiotropic trade-off in carbon source utilization caused by changes in regulatory region, and we hereby demonstrate how systems biology can be used to gain insight into evolutionary processes at the molecular level.
Project description:Aims: We performed an analysis of maltotriose utilization by 52 Saccharomyces yeast strains able to ferment maltose efficiently and correlated the observed phenotypes with differences in the copy number of genes possibly involved in maltotriose utilization by yeast cells. Methods and Results: The analysis of maltose and maltotriose utilization by laboratory and industrial strains of the species Saccharomyces cerevisiae and Saccharomyces pastorianus (a natural S. cerevisiae/Saccharomyces bayanus hybrid) was carried out using microscale liquid cultivation, as well as in aerobic batch cultures. All strains utilize maltose efficiently as a carbon source, but three different phenotypes were observed for maltotriose utilization: efficient growth, slow/delayed growth and no growth. Through microarray karyotyping and pulsed-field gel electrophoresis blots, we analysed the copy number and localization of several maltose-related genes in selected S. cerevisiae strains. While most strains lacked the MPH2 and MPH3 transporter genes, almost all strains analysed had the AGT1 gene and increased copy number of MALx1 permeases. Conclusions: Our results showed that S. pastorianus yeast strains utilized maltotriose more efficiently than S. cerevisiae strains and highlighted the importance of the AGT1 gene for efficient maltotriose utilization by S. cerevisiae yeasts. Significance and Impact of the Study: Our results revealed new maltotriose utilization phenotypes, contributing to a better understanding of the metabolism of this carbon source for improved fermentation by Saccharomyces yeasts.
Project description:Bacteria have evolved different mechanisms to catabolize carbon sources from a mixture of nutrients. They first consume their preferred carbon source, before others are used. Regulatory mechanisms adapt the metabolism accordingly to maximize growth and to outcompete other organisms. The human pathogen Campylobacter jejuni is an asaccharolytic Gram-negative bacterium that catabolizes amino acids and organic acids for growth. It prefers serine and aspartate as carbon sources, however it lacks all regulators known to be involved in regulating carbon source utilization in other organisms. In which manner C. jejuni adapts its metabolism towards the presence or absence of preferred carbon sources is unknown. In this study, we show with transcriptomic analysis and enzyme assays how C. jejuni adapts its metabolism in response to its preferred carbon source serine. In the presence of serine as well as lactate and pyruvate C. jejuni represses the utilization of other carbon sources, by repressing the expression of a number of central metabolic enzymes. The regulatory proteins RacR, Cj1000 and CsrA play a role in the regulation of these metabolic enzymes. This metabolism dependent transcriptional repression correlates with an accumulation of intracellular succinate. Hence, we propose a demand-based catabolite repression mechanism in C. jejuni, which depends on the intracellular succinate level.
Project description:In order to understand LBG derived galacto-manno-oligosaccharides utilization by a probiotic bacterium, Lactobacillus plantarum WCFS1, we have grown Lactobacillus plantarum WCFS1 (in duplicates) till mid log phase (OD600nm ~0.5, 10 h) in carbon free MRS (de Man, Rogosa Sharpe ) media containing either galacto-manno-oligosaccharides, mannose, glucose or galactose (1% w/v) as the sole carbon source.
Project description:We have a cDNA microarray to investigate changes in gene expression following transfer of fungal cultures from growth on glucose to growth on pectin or no carbon source. Our goal was to asses the roles of release from carbon catabolite repression and specific induction on proteins needed for metabolism (or utilization) of a single class of complex polysaccharide. Keywords = Aspergillus Keywords = Pectin Keywords = central metabolism Keywords = pectin Keywords = carbon catabolite repression Keywords = polysaccharide Keywords = exopolygalacturonase Keywords: time-course
Project description:Investigation of whole genome gene expression level changes in Streptomyces sp. SirexAA-E (ActE) when grown on different carbon sources. The results of this study demonstrate that ActE upregulates a small number of genes specific for the utilization of the avaliable carbon source. Cellulolytic Streptomyces sp. SirexAA-E (ActE), isolated from the pinewood-boring wasp Sirex noctilio, has a genome enriched for biomass utilization. The secreted proteomes obtained from growth on pure polysaccharides catalyzed hydrolysis of cellulose, mannan, and xylan with specific activities comparable to Spezyme CP, a commercial cellulase preparation. During reaction of an ActE secretome with cellulose, reducing sugar release was markedly stimulated in the presence of O2. ActE also expresses and secretes an expanded repertoire of enzymes during growth on natural and pre-treated biomass. These results indicate a new microbial contribution to biomass utilization that is widely distributed in natural environments by insects Streptomyces sp. ActE was grown in minimal medium supplimented with 0.5% carbon source (glucose, sigmacell-20, xylan, chitin, cellobiose, or AFEX). Cells were grown for 7 days and total RNA was extracted from the cell pellet. At least 3 biological replicates were performed for each carbon source (glucose, 3; sigmacell, 3; xylan, 5; chitin, 3; cellobiose 3; AFEX 3). Each biological replicate contained 3 technical replicates. The complete dataset were RMA Background Corrected, quantile normalized, the RMA algorithm was utilized by DNAStar ArrayStar.
Project description:Histidine is a good source of nutrient for many bacteria, but its utilization poses a significant challenge as it delivers excess nitrogen over carbon. The rate of histidine catabolism (hut) must thus be tightly regulated. Here, in Pseudomonas fluorescens SBW25, we show that the CbrAB two-component system directly activates the transcription of hut genes, while mediating succinate-induced carbon catabolite repression of hut at the translational level via the CbrAB-CrcYZ-Hfq/Crc regulatory cascade. When growing in minimal salt medium supplemented with histidine and succinate (the most preferred carbon source), the CbrAB-mediated promoter activity is weak; and the global nitrogen regulator NtrBC plays the dominant role in directly activating hut transcription.