Phosphoglycerate mutase knock-out mutant Saccharomyces cerevisiae: physiological investigation and transcriptome analysis
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ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe Saccharomyces cerevisiae
PROVIDER: GSE22122 | GEO | 2011/01/24
SECONDARY ACCESSION(S): PRJNA127587
REPOSITORIES: GEO
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Project description:Characterize the gpm1 mutant growth on dual substrate of ethanol and glycerol We used microarrays to detail the responses of the gpm1 mutant compare with reference
2011-01-24 | E-GEOD-22122 | biostudies-arrayexpress
Project description:Glycerol offers several advantages as a substrate for biotechnological applications. An important step towards using the popular production host Saccharomyces cerevisiae for glycerol-based bioprocesses have been recent studies in which commonly used S. cerevisiae strains were engineered to grow in synthetic medium containing glycerol as the sole carbon source. In order to boost extensive S. cerevisiae metabolic engineering incentives aiming at the use of glycerol, we realized that promoters with predictable expression levels in synthetic glycerol medium were required. In the current study, we used transcriptome analysis and a yECitrine-based fluorescence reporter assay to select and characterize useful 25 promoters for driving expression of target genes in S. cerevisiae under the given conditions. The promoters of the genes ALD4 and ADH2 showed 4.2- and 3-fold higher activities compared to the well-known strong TEF1 promoter. Moreover, the collection contains promoters with graded activities in synthetic glycerol medium and different degrees of glucose repression. To demonstrate the general applicability of the promoter collection, we successfully used a subset of the characterized promoters with graded activities in order to optimize growth on glycerol in an engineered derivative of CEN.PK, in which glycerol catabolism exclusively occurs via a non-native DHA pathway.
2018-03-31 | GSE109036 | GEO
Project description:Rsf1p is a putative transcription factor required for efficient growth using glycerol as sole carbon source but not for growth on the alternative respiratory carbon source ethanol. We use microarrays to determine the differences in the transcriptional program between the delta-rsf1 mutant and the wild type during respiratory growth on glycerol as well as the transition to growth on glycerol as sole carbon source. Experiment Overall Design: delta-rsf1or the isogenic parent strain were grown to early log (A600=0.6) in YPD and then washed twice in prewarmed YPG (30 C) and returned to the air shaker in YPG for 15, 30 or 60 minutes. "Limit" conditions were provided by harvesting cells grown in YPD to early log phase without shifting to YPG and by harvesting cells grown in YPG to early log phase. Since the delta-rsf1 mutant and its isogenic parent strain grow equally well on the respiratory carbon source ethanol, cells were also harvested after being grown in ethanol to early log phase.
2009-06-11 | E-GEOD-10031 | biostudies-arrayexpress
Project description:Saccharomyces spp. are widely used for ethanol production however fermentation productivity is negatively affected by the impact of ethanol accumulation on yeast metabolic rate and viability. This study used microarray and statistical two-way ANOVA analysis to compare and evaluate gene expression profiles of two previously generated ethanol-tolerant mutants, CM1 and SM1, with their parent, S. cerevisiae W303-1A, in the presence and absence of ethanol stress. Although sharing the same parentage, the mutants were created differently; SM1 by adaptive evolution involving long-term exposure to ethanol stress, and CM1 using chemical mutagenesis followed by adaptive evolution-based screening. Compared to the parent, differences in the expression levels of genes associated with a number of GO categories in the mutants suggest that their improved ethanol stress response is a consequence of increased mitochondrial and NADH oxidation activities, stimulating glycolysis and energy production. This leads to increased activity of energy-demanding processes associated with the production of proteins and plasma membrane components, which are necessary for acclimation to ethanol stress. It is suggested that a key function of the ethanol stress response is restoration of the NAD+/NADH redox balance, which increases glyceraldehyde-3-phosphate dehydrogenase activity, and higher glycolytic flux in the ethanol-stressed cell. Both mutants achieved this by a constitutive increase in carbon flux in the glycerol pathway as a means of increasing NADH oxidation.
2010-07-01 | GSE21120 | GEO
Project description:When the yeast Saccharomyces cerevisiae is subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from being purely respiratory to mixed respiratory and fermentative. This shift is characterized by ethanol production, a phenomenon known as the Crabtree effect due to its analogy with lactate overflow in cancer cells. It is well known that at high glycolytic fluxes there is glucose repression of respiratory pathways resulting in a decrease in the respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect (or overflow metabolism) is due to a limited respiratory capacity or caused by glucose-mediated repression of respiration. We addressed this issue by increasing respiration in S. cerevisiae by introducing a heterologous alternative oxidase, and observed reduced aerobic ethanol formation. In contrast, increasing non-respiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, while NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, while alternative oxidase is directed to the mitochondria. The onset of aerobic ethanol formation is demonstrated to be a consequence of an imbalance in mitochondrial redox balancing. In addition to answering fundamental physiological questions, our findings are relevant for all biomass derived applications of S. cerevisiae. Keywords: Genetic Modification
2007-02-01 | GSE6277 | GEO
Project description:A putative yeast mitochondrial upstream activating sequence (UAS) was used in a one-hybrid screening procedure that identified the YJR127C ORF on chromosome X. This gene was previously designated ZMS1 and is listed as a transcription factor on the SGD website. Real time RT-PCR assays showed that expression of YJR127C/ZMS1 was glucose-repressible, and a deletion mutant for the gene showed a growth defect on glycerol-based but not on glucose- or ethanol-based medium. Real time RT-PCR analyses identified severely attenuated transcript levels from GUT1 and GUT2 to be the source of that growth defect, the products of GUT1 and GUT2 are required for glycerol utilization. mRNA levels from a large group of mitochondria- and respiration-related nuclear genes also were shown to be attenuated in the deletion mutant. Importantly, transcript levels from the mitochondrial OLI1 gene, which has an associated organellar UAS, were attenuated in the DeltaYJR127C mutant during glycerol-based growth, but those from COX3 (OXI2), which lacks an associated mitochondrial UAS, were not. Transcriptome analysis of the glycerol-grown deletion mutant showed that genes in several metabolic and other categories are affected by loss of this gene product, including protein transport, signal transduction, and others. Thus, the product of YJR127C/ZMS1 is involved in transcriptional control for genes in both cellular genetic compartments, many of which specify products required for glycerol-based growth, respiration, and other functions.
2011-05-26 | GSE29530 | GEO
Project description:The implementation of a cost-effective lignocellulosic ethanol production requires developing efficient high gravity processes (i.e. working at high substrate concentrations). During the fermentation processes, the presence of high concentration of insoluble solids leads to lower glucose consumption rates, reduced ethanol volumetric productivities, and the accumulation of intracellular reactive oxygen species (ROS). Major repressed biological processes include cell cycle progression, trehalose and glycogen biosynthesis, DNA repair mechanisms, and certain genes involved in the general cell stress response. On the other hand, genes related to the glutathione, thioredoxin and methionine scavenging systems are induced.
2019-08-23 | GSE115460 | GEO
Project description:Transcriptomic reprogramming of Ethanol adapted vs Ethanol non adapted culture
| PRJNA115857 | ENA
Project description:Background: Anaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol-production processes are not constant, performance of engineered strains was studied in slow-growing cultures. Results: In slow-growing anaerobic chemostat cultures (D = 0.05 h-1), an engineered PRK-RuBisCO strain produced 80-fold more acetaldehyde and 30-fold more acetate than a reference strain. This observation suggested an imbalance between in vivo activities of PRK-RuBisCO and formation of NADH in biosynthesis. Lowering the copy number of the RuBisCO-encoding cbbm expression cassette from 15 to 2 reduced acetaldehyde and acetate yields by 67% and 29%, respectively. Additional C-terminal fusion of a 19 amino-acid tag to PRK reduced its protein level by 13-fold while acetaldehyde and acetate production decreased by 94% and 61%, respectively, relative to the 15x cbbm strain. These modifications did not affect glycerol production at 0.05 h-1 but caused a 4.6 fold higher glycerol production per amount of biomass in fast-growing (0.29 h-1) anaerobic batch cultures than observed for the 15x cbbm strain. In another strategy, the promoter of ANB1, whose transcript level positively correlated with growth rate, was used to control PRK synthesis in a 2x cbbm strain. At 0.05 h-1, this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15x cbbm strain, without affecting glycerol yield. The maximum growth rate of the resulting strain equalled that of the reference strain, while its glycerol yield was 72% lower. Conclusions: Acetaldehyde and acetate formation by slow-growing cultures of engineered S. cerevisiae strains carrying a PRK-RuBisCO bypass of yeast glycolysis was attributed to an in vivo overcapacity of PRK and RuBisCO. Reducing the capacity of PRK and/or RuBisCO was shown to mitigate this undesirable byproduct formation. Use of a growth-rate-dependent promoter for PRK expression highlighted the potential of modulating gene expression in engineered strains to respond to growth-rate dynamics in industrial batch processes.
2023-05-17 | GSE221392 | GEO