Project description:Prolonged cultivation of Saccharomyces cerevisiae in aerobic, glucose-limited chemostat cultures (dilution rate, 0Â·10 hâ1) resulted in a progressive decrease of the residual glucose concentration (from 20 to 8 mg lâ1 after 200 generations). This increase in the affinity for glucose was accompanied by a fivefold decrease of fermentative capacity, and changes in cellular morphology. These phenotypic changes were retained when single-cell isolates from prolonged cultures were used to inoculate fresh chemostat cultures, indicating that genetic changes were involved. Kinetic analysis of glucose transport in an âevolvedâ strain revealed a decreased Km, while Vmax was slightly increased relative to the parental strain. Apparently, fermentative capacity in the evolved strain was not controlled by glucose uptake. Instead, enzyme assays in cell extracts of the evolved strain revealed strongly decreased capacities of enzymes in the lower part of glycolysis. This decrease was corroborated by genome-wide transcriptome analysis using DNA microarrays. In aerobic batch cultures on 20 g glucose lâ1, the specific growth rate of the evolved strain was lower than that of the parental strain (0Â·28 and 0Â·37 hâ1, respectively). Instead of the characteristic instantaneous production of ethanol that is observed when aerobic, glucose-limited cultures of wild-type S. cerevisiae are exposed to excess glucose, the evolved strain exhibited a delay of 90 min before aerobic ethanol formation set in. This study demonstrates that the effects of selection in glucose-limited chemostat cultures extend beyond glucose-transport kinetics. Although extensive physiological analysis offered insight into the underlying cellular processes, the evolutionary âdriving forceâ for several of the observed changes remains to be elucidated Experiment Overall Design: A crucial feature of bakers' yeast is its capacity to produce CO2, referred to as fermentative capacity (van Hoek et al., 1998). After prolonged glucose-limited cultivation of S. cerevisiae, in addition to an increased affinity for glucose, we observed a dramatic decrease in fermentative capacity. Consequently, the aim of the present study was to perform an integral analysis of the long-term adaptation of S. cerevisiae during prolonged glucose-limited, aerobic cultivation in chemostat cultures, with special emphasis on the regulation of glucose transport and glycolytic capacity. To this end, we applied an integrated approach that combined transcriptome analysis, measurement of fermentative capacity and activities of glucose transport and glycolytic enzymes, and characterization of cellular morphology.

Project description:This SuperSeries is composed of the following subset Series: GSE3205: Homeostatic Adjustment and Metabolic Remodeling in Glucose-limited Yeast Cultures Time Course 1 GSE3206: Homeostatic Adjustment and Metabolic Remodeling in Glucose-limited Yeast Cultures Time Course 2 Abstract: We studied the physiological response to glucose limitation in batch and steady-state (chemostat) cultures of Saccharomyces cerevisiae by following global patterns of gene expression. Glucose-limited batch cultures of yeast go through two sequential exponential growth phases, beginning with a largely fermentative phase, followed by an essentially completely aerobic use of residual glucose and evolved ethanol. Judging from the patterns of gene expression, the state of the cells growing at steady state in glucose-limited chemostats corresponds most closely with the state of cells in batch cultures just before they undergo this "diauxic shift." Essentially the same pattern was found between chemostats having a fivefold difference in steady-state growth rate (the lower rate approximating that of the second phase respiratory growth rate in batch cultures). Although in both cases the cells in the chemostat consumed most of the glucose, in neither case did they seem to be metabolizing it primarily through respiration. Although there was some indication of a modest oxidative stress response, the chemostat cultures did not exhibit the massive environmental stress response associated with starvation that also is observed, at least in part, during the diauxic shift in batch cultures. We conclude that despite the theoretical possibility of a switch to fully aerobic metabolism of glucose in the chemostat under conditions of glucose scarcity, homeostatic mechanisms are able to carry out metabolic adjustment as if fermentation of the glucose is the preferred option until the glucose is entirely depleted. These results suggest that some aspect of actual starvation, possibly a component of the stress response, may be required for triggering the metabolic remodeling associated with the diauxic shift. Refer to individual Series

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 36 hybridizations

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

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 Experiment Overall Design: Knowledge on the genome-wide transcriptional response of S. cerevisiae to high CO2 concentrations may provide a deeper insight into the molecular mechanisms of CO2 stress. Such insight is essential to develop metabolic-engineering strategies for improving CO2 tolerance. Furthermore, identification of âsignature transcriptsâ that uniquely respond to CO2 stress may be applicable for diagnosing the CO2 status of industrial fermentations. It has recently been demonstrated that the combination of chemostat cultivation with DNA-microarray-based transcriptome analysis offers a powerful and reproducible approach to identify the transcriptional responses of yeasts to environmental parameters For this reason, in the present study we used chemostat cultures of S. cerevisiae to quantify the effect of CO2 on respiring and fermenting cells, and to determine the genome-wide transcriptional responses of this yeast to high CO2 concentrations.

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Prolonged selection in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae

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