Saccharomyces cerevisiae
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PROVIDER: PRJNA311716 | ENA |
REPOSITORIES: ENA
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Project description:Extremely low specific growth rates (below 0.01 h-1) represent a largely unexplored area of microbial physiology. Retentostats enable controlled, energy-limited cultivation at near-zero specific growth rates while avoiding starvation. In this study, anaerobic, glucose-limited retentostats were used to analyze physiological and genome-wide transcriptional responses of Saccharomyces cerevisiae to cultivation at near-zero specific growth rates. Cultures at near-zero specific growth rates exhibited several characteristics previously associated with quiescence, including accumulation of storage polymers and an increased expression of genes involved in storage metabolism, autophagy and exit from the replicative cell cycle into G0. Analysis of transcriptome data from glucose-limited retentostat and chemostat cultures showed, as specific growth rate was decreased, quiescence-related transcriptional responses already set in at specific growth rates above 0.025 h-1. Many genes involved in mitochondrial processes were specifically upregulated at near-zero specific growth rates, possibly reflecting an increased turn-over of organelles under these conditions. Prolonged (> 2 weeks) cultivation in retentostat cultures led to induction of several genes that were previously implicated in chronological ageing. These observations stress the need for systematic dissection of physiological responses to slow growth, quiescence, ageing and starvation and indicate that controlled cultivation systems such as retentostats can contribute to this goal.
2011-08-23 | GSE22574 | GEO
Project description:Extremely low specific growth rates (below 0.01 h-1) represent a largely unexplored area of microbial physiology. Retentostats enable controlled, energy-limited cultivation at near-zero specific growth rates while avoiding starvation. In this study, anaerobic, glucose-limited retentostats were used to analyze physiological and genome-wide transcriptional responses of Saccharomyces cerevisiae to cultivation at near-zero specific growth rates. Cultures at near-zero specific growth rates exhibited several characteristics previously associated with quiescence, including accumulation of storage polymers and an increased expression of genes involved in storage metabolism, autophagy and exit from the replicative cell cycle into G0. Analysis of transcriptome data from glucose-limited retentostat and chemostat cultures showed, as specific growth rate was decreased, quiescence-related transcriptional responses already set in at specific growth rates above 0.025 h-1. Many genes involved in mitochondrial processes were specifically upregulated at near-zero specific growth rates, possibly reflecting an increased turn-over of organelles under these conditions. Prolonged (> 2 weeks) cultivation in retentostat cultures led to induction of several genes that were previously implicated in chronological ageing. These observations stress the need for systematic dissection of physiological responses to slow growth, quiescence, ageing and starvation and indicate that controlled cultivation systems such as retentostats can contribute to this goal. Independent duplicate retentostat cultures were subjected to microarray analysis at four time points after switching the effluent line to the filter unit (2, 9, 16 and 22 d). Microarray analysis of independent, triplicate anaerobic glucose-limited chemostat cultures grown at a specific growth rate of 0.025 h-1 (t = 0) were also performed as part of this study, resulting in a dataset of 11 arrays.
2011-08-22 | E-GEOD-22574 | biostudies-arrayexpress
Project description:Saccharomyces cerevisiae is an established microbial host for the production of non-native compounds. The synthesis of these compounds typically demands energy and competes with growth for carbon and energy substrate. Uncoupling product formation form growth would benefit product yields and decrease formation of by-product biomass. Studying non-growing metabolically-active yeast cultures provides a first step towards developing S. cerevisiae as a non-growing, robust cell factory. Non-growing metabolically-active cultures can be obtained in retentostat, a glucose-limited, continuous bioreactor system in which biomass accumulates while spent medium is constantly removed. Hitherto retentostat cultures of S. cerevisiae have only been reported under anaerobiosis, condition inappropriate for the production of energy-demanding products. The present study, using retentostat cultures, explores the physiology of non-dividing, fully respiring S. cerevisiae, focusing on industrially-relevant features. Following model-aided experimental design, retentostat cultivations were optimized for accelerated but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates. During 20 days in retentostat the biomass concentration increased, leading very slow growth rates (specific growth rates below 0.001 h-1) but high culture viability (over 80% of viable cells). The maintenance requirement (mATP) was estimated at 0.64 mmolATP.gX-1.h-1, which is remarkably ca. 35% lower than the mATP measured in anaerobic retentostat cultures. Transcriptional down-regulation of genes involved in biosynthesis and up-regulation of stress-responsive genes towards near-zero growth rates corresponded well with data from anaerobic retentostats. More striking was the extreme heat-shock tolerance of S. cerevisiae, which exceeded by far previously reported heat shock tolerance of notoriously robust yeast cultures such as stationary phase cultures. Furthermore, while the metabolic fluxes in the retentostats were relatively low as a result of extreme caloric restriction, off-line measurements revealed that S. cerevisiae retained a high catabolic capacity. The high viability and extreme heat-shock tolerance revealed the robustness of S. cerevisiae at near-zero growth in retentostat. In addition, the relatively low maintenance requirements and high metabolic capacity under severe calorie restriction underline the potential of S. cerevisiae as a non-dividing microbial cell factory for the production of energy-intensive compounds. The retentostat is a promising tool to identify the molecular basis of this extreme robustness. The goal of the present study is to investigate the physiology of aerobic fully respiring S. cerevsiae at near-zero growth rates. Fundamental but industrially-relevant questions were addressed thanks to the design, implementation and study of aerobic retentostat cultivations enabling a rapid but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates.
2016-06-01 | E-GEOD-77842 | biostudies-arrayexpress
Project description:Saccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limited fed-batch reactors which, due to constraints in oxygen transfer and cooling capacities, have to be operated at low specific growth rates. Because intracellular levels of key metabolites and cellular energy status are growth-rate dependent, slow growth can significantly affect biomass-specific productivity. Using an engineered Saccharomyces cerevisiae strain expressing a heterologous pathway for resveratrol production as a model energy-requiring product, the impact of specific growth rate on yeast physiology and productivity was investigated in aerobic, glucose-limited chemostat cultures. Stoichiometric analysis revealed that de novo resveratrol production from glucose requires a net input of 2 moles of ATP per mole of produced resveratrol. The biomass-specific production rate of resveratrol showed a strong positive correlation with the specific growth rate. At low growth rates, a substantial fraction of the carbon source was invested in cellular maintenance-energy requirements (e.g., 27% at 0.03 h-1). This distribution of resources was unaffected by resveratrol production. Formation of the by-products coumaric, phloretic and cinnamic acid had no detectable effect on maintenance energy requirement and yeast physiology in the chemostats. Expression of the heterologous pathway led to marked differences in transcript levels in the resveratrol-producing strain, including increased expression levels of genes involved in pathways for precursor supply (e.g., ARO7 and ARO9 involved in phenylalanine biosynthesis). The observed strong differential expression of many glucose-responsive genes in the resveratrol producer as compared to a congenic reference strain could be explained from higher residual glucose concentrations and higher relative growth rates in cultures of the resveratrol producer. De novo resveratrol production by engineered S. cerevisiae is an energy demanding process. Resveratrol production by an engineered strain exhibited a strong correlation with specific growth rate. Since industrial production in fed-batch reactors typically involves low specific growth rates, this study emphasizes the need for uncoupling growth and product formation via energy-requiring pathways. The goal of the present study is to investigate the impact of specific growth rate on biomass-specific productivity, product yield, by-product formation and host strain physiology of an S. cerevisiae strain that was previously engineered for de novo production of resveratrol from glucose. To this end, (by)product formation, physiology and transcriptome were analysed in steady-state, glucose-limited chemostat cultures grown at different dilution rates.
2015-12-22 | E-GEOD-65942 | biostudies-arrayexpress
Project description:This paper describes the molecular and physiological adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state using carbon-limited retentostat cultivation. Metabolic and transcriptomic analyses revealed that metabolic patterns shifted between homolactic and mixed-acid fermentation during the retentostat cultivation, which appeared to be controlled at the transcription level of the corresponding pyruvate-dissipation enzyme pathway encoding genes. Furthermore, during extended retentostat cultivation, cells continued to consume several amino acids, but also produced specific amino acids subsets, which may derive from the conversion of glycolytic intermediates. Under conditions of extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly up-regulated, which was confirmed by enhanced initial acidification rates on various sugar substrates in cells obtained from near-zero growth cultures. Moreover, the expression of genes involved in multiple stress response mechanisms was gradually induced during extended retentostat cultivation, supporting the strong molecular focus on maintenance of cellular function and viability. The present integrated transcriptome and metabolome study provides molecular understanding of the adaptation of Lactococcus lactis KF147 to near-zero growth rate conditions, and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates.
2014-01-01 | GSE51494 | GEO
Project description:Saccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limited fed-batch reactors which, due to constraints in oxygen transfer and cooling capacities, have to be operated at low specific growth rates. Because intracellular levels of key metabolites and cellular energy status are growth-rate dependent, slow growth can significantly affect biomass-specific productivity. Using an engineered Saccharomyces cerevisiae strain expressing a heterologous pathway for resveratrol production as a model energy-requiring product, the impact of specific growth rate on yeast physiology and productivity was investigated in aerobic, glucose-limited chemostat cultures. Stoichiometric analysis revealed that de novo resveratrol production from glucose requires a net input of 2 moles of ATP per mole of produced resveratrol. The biomass-specific production rate of resveratrol showed a strong positive correlation with the specific growth rate. At low growth rates, a substantial fraction of the carbon source was invested in cellular maintenance-energy requirements (e.g., 27% at 0.03 h-1). This distribution of resources was unaffected by resveratrol production. Formation of the by-products coumaric, phloretic and cinnamic acid had no detectable effect on maintenance energy requirement and yeast physiology in the chemostats. Expression of the heterologous pathway led to marked differences in transcript levels in the resveratrol-producing strain, including increased expression levels of genes involved in pathways for precursor supply (e.g., ARO7 and ARO9 involved in phenylalanine biosynthesis). The observed strong differential expression of many glucose-responsive genes in the resveratrol producer as compared to a congenic reference strain could be explained from higher residual glucose concentrations and higher relative growth rates in cultures of the resveratrol producer. De novo resveratrol production by engineered S. cerevisiae is an energy demanding process. Resveratrol production by an engineered strain exhibited a strong correlation with specific growth rate. Since industrial production in fed-batch reactors typically involves low specific growth rates, this study emphasizes the need for uncoupling growth and product formation via energy-requiring pathways.
2015-12-22 | GSE65942 | GEO
Project description:Cellular responses of Saccharomyces cerevisiae at near-zero growth rates: transcriptome analysis of anaerobic retentostat cultures
| PRJNA128419 | ENA
Project description:This paper describes the molecular and physiological adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state using carbon-limited retentostat cultivation. Metabolic and transcriptomic analyses revealed that metabolic patterns shifted between homolactic and mixed-acid fermentation during the retentostat cultivation, which appeared to be controlled at the transcription level of the corresponding pyruvate-dissipation enzyme pathway encoding genes. Furthermore, during extended retentostat cultivation, cells continued to consume several amino acids, but also produced specific amino acids subsets, which may derive from the conversion of glycolytic intermediates. Under conditions of extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly up-regulated, which was confirmed by enhanced initial acidification rates on various sugar substrates in cells obtained from near-zero growth cultures. Moreover, the expression of genes involved in multiple stress response mechanisms was gradually induced during extended retentostat cultivation, supporting the strong molecular focus on maintenance of cellular function and viability. The present integrated transcriptome and metabolome study provides molecular understanding of the adaptation of Lactococcus lactis KF147 to near-zero growth rate conditions, and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates. loop design of the samples including two shortcuts
2014-01-01 | E-GEOD-51494 | biostudies-arrayexpress
Project description:Saccharomyces cerevisiae is an established microbial host for the production of non-native compounds. The synthesis of these compounds typically demands energy and competes with growth for carbon and energy substrate. Uncoupling product formation form growth would benefit product yields and decrease formation of by-product biomass. Studying non-growing metabolically-active yeast cultures provides a first step towards developing S. cerevisiae as a non-growing, robust cell factory. Non-growing metabolically-active cultures can be obtained in retentostat, a glucose-limited, continuous bioreactor system in which biomass accumulates while spent medium is constantly removed. Hitherto retentostat cultures of S. cerevisiae have only been reported under anaerobiosis, condition inappropriate for the production of energy-demanding products. The present study, using retentostat cultures, explores the physiology of non-dividing, fully respiring S. cerevisiae, focusing on industrially-relevant features. Following model-aided experimental design, retentostat cultivations were optimized for accelerated but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates. During 20 days in retentostat the biomass concentration increased, leading very slow growth rates (specific growth rates below 0.001 h-1) but high culture viability (over 80% of viable cells). The maintenance requirement (mATP) was estimated at 0.64 mmolATP.gX-1.h-1, which is remarkably ca. 35% lower than the mATP measured in anaerobic retentostat cultures. Transcriptional down-regulation of genes involved in biosynthesis and up-regulation of stress-responsive genes towards near-zero growth rates corresponded well with data from anaerobic retentostats. More striking was the extreme heat-shock tolerance of S. cerevisiae, which exceeded by far previously reported heat shock tolerance of notoriously robust yeast cultures such as stationary phase cultures. Furthermore, while the metabolic fluxes in the retentostats were relatively low as a result of extreme caloric restriction, off-line measurements revealed that S. cerevisiae retained a high catabolic capacity. The high viability and extreme heat-shock tolerance revealed the robustness of S. cerevisiae at near-zero growth in retentostat. In addition, the relatively low maintenance requirements and high metabolic capacity under severe calorie restriction underline the potential of S. cerevisiae as a non-dividing microbial cell factory for the production of energy-intensive compounds. The retentostat is a promising tool to identify the molecular basis of this extreme robustness.
2016-06-01 | GSE77842 | GEO
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. Keywords: Evolution
2007-08-31 | GSE8897 | GEO