Dataset Information

Role of Transcriptional Regulation in Controlling Fluxes in Central Carbon Metabolism of Saccharomyces cerevisiae

ABSTRACT: In contrast to batch cultivation, chemostat cultivation allows the identification of carbon source responses without interference by carbon-catabolite repression, accumulation of toxic products, and differences in specific growth rate. This study focuses on the yeast Saccharomyces cerevisiae, grown in aerobic, carbon-limited chemostat cultures. Genome-wide transcript levels and in vivo fluxes were compared for growth on two sugars, glucose and maltose, and for two C2-compounds, ethanol and acetate. In contrast to previous reports on batch cultures, few genes (180 genes) responded to changes of the carbon source by a changed transcript level. Very few transcript levels were changed when glucose as the growth-limiting nutrient was compared with maltose (33 transcripts), or when acetate was compared with ethanol (16 transcripts). Although metabolic flux analysis using a stoichiometric model revealed major changes in the central carbon metabolism, only 117 genes exhibited a significantly different transcript level when sugars and C2-compounds were provided as the growthlimiting nutrient. Despite the extensive knowledge on carbon source regulation in yeast, many of the carbon source-responsive genes encoded proteins with unknown or incompletely characterized biological functions. In silico promoter analysis of carbon source-responsive genes confirmed the involvement of several known transcriptional regulators and suggested the involvement of additional regulators. Transcripts involved in the glyoxylate cycle and gluconeogenesis showed a good correlation with in vivo fluxes. This correlation was, however, not observed for other important pathways, including the pentose-phosphate pathway, tricarboxylic acid cycle, and, in particular, glycolysis. These results indicate that in vivo fluxes in the central carbon metabolism of S. cerevisiae grown in steadystate, carbon-limited chemostat cultures are controlled to a large extent via post-transcriptional mechanisms. Keywords: medium composition

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE8895 | GEO | 2007/08/31

SECONDARY ACCESSION(S): PRJNA102283

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:In contrast to batch cultivation, chemostat cultivation allows the identification of carbon source responses without interference by carbon-catabolite repression, accumulation of toxic products, and differences in specific growth rate. This study focuses on the yeast Saccharomyces cerevisiae, grown in aerobic, carbon-limited chemostat cultures. Genome-wide transcript levels and in vivo fluxes were compared for growth on two sugars, glucose and maltose, and for two C2-compounds, ethanol and acetate. In contrast to previous reports on batch cultures, few genes (180 genes) responded to changes of the carbon source by a changed transcript level. Very few transcript levels were changed when glucose as the growth-limiting nutrient was compared with maltose (33 transcripts), or when acetate was compared with ethanol (16 transcripts). Although metabolic flux analysis using a stoichiometric model revealed major changes in the central carbon metabolism, only 117 genes exhibited a significantly different transcript level when sugars and C2-compounds were provided as the growthlimiting nutrient. Despite the extensive knowledge on carbon source regulation in yeast, many of the carbon source-responsive genes encoded proteins with unknown or incompletely characterized biological functions. In silico promoter analysis of carbon source-responsive genes confirmed the involvement of several known transcriptional regulators and suggested the involvement of additional regulators. Transcripts involved in the glyoxylate cycle and gluconeogenesis showed a good correlation with in vivo fluxes. This correlation was, however, not observed for other important pathways, including the pentose-phosphate pathway, tricarboxylic acid cycle, and, in particular, glycolysis. These results indicate that in vivo fluxes in the central carbon metabolism of S. cerevisiae grown in steadystate, carbon-limited chemostat cultures are controlled to a large extent via post-transcriptional mechanisms. Experiment Overall Design: Cultivation of microorganisms in chemostats offers numerous advantages for studying the structure and regulation of metabolic networks (11). In chemostat cultures, individual culture parameters can be changed, while keeping other relevant physical and chemical culture parameters (composition of synthetic medium, pH, temperature, aeration, etc.) constant. An especially important parameter in this respect is the specific growth rate, which, in a chemostat, is equal to the dilution rate, which can be accurately controlled. This allows the experimenter to investigate the effects of environmental changes or genetic interventions at a fixed specific growth rate, even if these changes result in different specific growth rates in batch cultures. In a chemostat, growth can be limited by a single, selected nutrient. The very low residual concentrations of this growth-limiting nutrient in chemostat cultures alleviate effects of catabolite repression and inactivation. Furthermore, these low residual substrate concentrations prevent substrate toxicity, which, for example, occurs when S. cerevisiae is grown on ethanol or acetate as the carbon source in batch cultures . Experiment Overall Design: The central goal of the present study is to assess to what extent carbon source-dependent regulation of fluxes through central carbon metabolism in S. cerevisiae is regulated at the level of transcription. To this end, we compare the transcriptome of carbon-limited, aerobic chemostat cultures grown on four different carbon sources: glucose, maltose, ethanol, and acetate. Data from the transcriptome analysis are compared with flux distribution profiles calculated with a stoichiometric metabolic network model. Questions that will be addressed are as follows: (i) does glucose-limited aerobic cultivation lead to a complete alleviation of glucose-catabolite repression; (ii) how (in)complete is our understanding of the genes involved in the transcriptional response of S. cerevisiae to four of the most common carbon sources for this yeast; and (iii) to what extent do transcriptome analyses with microarrays provide a reliable indication of flux distribution in metabolic networks?

Project description:Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport. Experiment Overall Design: In a recent study (25) we analyzed glucose efflux upon exposure of S. cerevisiae to excess maltose, with yeast cells originating from âyoungâ chemostat cultures (<20 generations). In these experiments no cell lysis was observed upon exposure to excess maltose. However, in further work on this subject, we observed an apparent effect of chemostat culture age on transport capacity. The aim of the present study was to further investigate the effect of prolonged maltose-limited chemostat cultivation on the physiology of S. cerevisiae. To this end we monitored the affinity for maltose, genome-wide transcript levels, activities of key enzymes, and physiological responses to maltose excess during long-term cultivation in maltose-limited chemostat cultures. Experiment Overall Design: Jansen, M. L. A., J. H. de Winde, and J. T. Pronk. 2002. Hxt-carrier-mediated glucose efflux upon exposure of Saccharomyces cerevisiaeSaccharomyces cerevisiae to excess maltose. Appl. Environ. Microbiol. 68:4259-4265.

Project description:The global transcriptional response of Saccharomyces cerevisiae was investigated in low temperature chemostat cultures grown in carbon or nitrogen limitation. During steady state chemostats, the growth rates and in vivo fluxes were kept constant however the growth-limiting nutrient was significantly higher at 12oC than at 30oC and had significant effects on transcriptional responses. Growth at 12oC resulted in a rearrangement of transporters for the limiting nutrient, where hexose transporters (HXTs) and ammonium permeases (MEPs) were differentially expressed in cultures grown at 30oC in carbon and nitrogen limitations, respectively. In addition, we found repression of genes encoding proteins in reserve carbohydrates metabolism and metabolism of alternative carbon or nitrogen sources other than glucose or ammonia. However, there were also similar responses when the transcriptional response was evaluated regardless of the growth-limiting nutrient. In particular, induction of ribosome biogenesis genes emphasizes the significance of transcription and translational adaptation at low temperature. In contrast, genes encoding proteins during stress response were downregulated. This down-regulation of stress elements better known as environmental stress response (ESR) is in contradiction with previous low temperature transcriptome analyses. During continuous steady state low temperature cultivation, ESR no longer plays an integral role in S. cerevisiaeM-bM-^@M-^Ys response to temperature change. Similarly, trehalose accumulation, consistent with its gene expression, was not indispensable for growth at 12oC. This response, however, does not exclude that ESR may be required for transition phase in low temperature growth when cells are transferred from one temperature to another. Keywords: chemostat temperature 12 degree celsuis 30 degree celsius The global transcriptional response of Saccharomyces cerevisiae was investigated in low temperature chemostat cultures grown in carbon or nitrogen limitation at a dilution rate of 0.03h-1

Dataset Information

Role of Transcriptional Regulation in Controlling Fluxes in Central Carbon Metabolism of Saccharomyces cerevisiae

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure