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:Adaptively evolved mutants of yeast on galactose were characterized by feremtation physiology and tools from systems biology. Three evolved mutants of yeast grown on aerobic batch with galactose as carbon source
Project description:Trade-offs often occurs during experimental evolution. For example, the degeneration of growth in glucose was found in a galactose adapted yeast. Here, we isolated one Lactococcus lactis mutant using experimental on maltose. The mutant grows normally on glucose, but faster than the wild-type on maltose and galactose. DNA microarray analysis and whole genome re-sequencing were applied to disclose the crucial points that determine the phenotype.
Project description:Cells must sense and respond to available nutrients to survive. Microbes evolved mechanisms to activate genes necessary to utilize a carbon source specifically when it is present. To efficiently grow in mixed carbohydrate environments, microbes repress genes necessary to utilize carbon sources that require substantial resources to catabolize when a simpler carbon source is present, known as carbon catabolite repression. We investigated nutrient utilization in the basidiomycete, oleaginous yeast Rhodotorula (Rhodosporidium) toruloides. R. toruloides is a saprophytic fungus that utilizes the building blocks of complex polysaccharides found in plant cell walls. A transcription factor homologous to the cellulose degradation regulator CLR-2/ClrB in Ascomycete filamentous fungi, which we named Cbr1, is required for cellobiose utilization in R. toruloides. Although the role of CLR-2/ClrB is limited to regulating expression of genes involved in cellulose and hemicellulose utilization, Cbr1 is also involved in fucose and tricarboxylic acid cycle intermediate utilization. Additionally, Cbr1 inhibits carbon catabolite repression specifically during utilization of glucose-glucose disaccharides, which may have evolved to limit the glucose-mediated repression of genes encoding proteins that cleave disaccharides into glucose. In fungi, it is thought carbon source-specific transcription factors activate genes necessary for carbon source utilization, while carbon catabolite repression is regulated by transcription factors that broadly repress all nonpreferred carbon source utilization genes when a preferred carbon source is present. Our data provide evidence for a noncanonical carbon catabolite repression mechanism and suggest transcriptional mechanisms regulating carbon catabolite repression may be less broadly conserved than previously thought.
Project description:Efficient utilization of lignocellulosic biomass-derived sugars is essential to improve the economics of biorefinery. While Pseudomonas putida is a promising microbial host, its usage is limited because this strain cannot utilize xylose or galactose as a sole carbon source. To address this issue, we heterologously introduced a xylose utilizing gene (xylD) from Caulobacter crescentus and a galactose operon (galETKM) from E. coli MG1655. To improve the utilization further, we evolved the engineered strains in minimal medium conditions. After the evolution, they acquired better fitnesses on the non-native sugars. To understand transcriptional changes after the evolution, the transcriptomes of few evolved isolates were analyzed.
Project description:We attempted to improve the resistance of taxadiene-producing yeast strain to oxidative stress to develop a more robust yeast cell factory for improved Taxol® drug oxyenated taxanes precursors production from taxadiene. To this end, we evolved a yeast strain on H2O2-containing defined growth medium, supplemented with galactose as carbon source to induce the heterologous taxadiene biosynthesis pathway genes in that strain. The oxidative stress re-induction effect on the expression profiles of the superior evolved yeast strain (E_LRS5) was then studied before (steady state I) and during its continous use (steady state II) in galactose-limited chemostats, in parallel with the parent strain (LRS5).
Project description:Carbon source is the basic nutrition and is essential for yeast growth. We grew the yeast cells (BY4741 strain) under different carbon sources including glucose with different concentration, galactose and raffinose. We generated bulk-cell RNA-seq data and investigated the dynamics of gene expression profiles under different growth conditions. We also generated single-cell RNA-seq data for yeast cells under 2% glucose, and explored the heterogeneity of gene expression within a cell population.
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.