Global gene expression of Ruegeria pomeroyi strain DSS3 in response to glucose, ammonium, phosphate, or sulfate limitation
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ABSTRACT:
ORGANISM(S): Ruegeria pomeroyi DSS-3
SUBMITTER: Shalabh Sharma
PROVIDER: E-GEOD-27032 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We used the previously designed oligonucleotide microarrays (BM-CM-<rgmann et al., 2007, Environmental Microbiology, 9: 2742-2755) to detect the mRNA transcripts of R. pomeroyi DSS-3 when the cells were cultured under steady-state conditions limited with ammonium (NH4Cl, 0.26 mM) but with an excess of D-ribose-5-phosphate (C5H9Na2O8P*2H2O, 0.5 mM), methylphosphonic acid (CH5PO3, 0.5 mM), or potassium phosphate (KH2PO4, 0.5 mM), or during ammonium excess (NH4Cl, 2.8 mM) but were limited with potassium phosphate (KH2PO4, 9.2 M-NM-<M). A total of 13 microarray hybridizations were performed: three biological replicates each from ribose phosphate, methylphosphonate, or potassium phosphate excess growth regimes, three biological replicates from potassium phosphate limited growth regime, and one technical replicate for the potassium phosphate excess growth regime. Data for the technical replicates were averaged and combined, resulted in a total of 12 samples.
2014-06-02 | E-GEOD-38740 | biostudies-arrayexpress
Project description:The Saccharomyces cerevisiae SFP1 is required for proper regulation of ribosome biogenesis and cell size in response to nutrients. A mutant deleted for SFP1 shows specific traits among which a slow growth phenotype, which is particularly evident during growth on glucose. To assess the effects of nutrients on the activity of Sfp1 independent by growth rate related feedback we grew an sfp1Πmutant and its isogenic reference strain in chemostat cultures, at the same specific growth rate, under glucose/ethanol-limitation. Our data show that Sfp1 is involved in the modulation of cell size and RiBi gene expression and that these two functions are differently influenced by nutrients. The continuous cultures were then pulsed with a glucose excess generating a situation of batch growth similar to shake flask cultures. The dynamic analysis of the metabolic and transcriptional response following the glucose addition suggested that Sfp1 plays a role at the crossroads of ribosome biogenesis and central carbon metabolism regulation. Finally, we show that the down-regulation of RP genes, which was observed in an sfp1Πstrain during shake flask growth, cannot be directly ascribed to the absence of Sfp1 but is most probably a secondary effect due to the low growth potential of the mutant strain. Experiment Overall Design: After ten volume changes, few seconds after the samples for the steady state analysis were collected, the anaerobic glucose pulse experiments were started by sparging the medium reservoir and the fermenter with pure nitrogen gas (airflow of 0.5 L min-1, Hoek-Loos, Schiedam, <5 ppm O2). NorpreneTM tubing and butyl septa were used to minimize oxygen diffusion into the anaerobic culture. Two minutes after nitrogen sparging and just before the addition of glucose, the medium and the effluent pumps were switched off. At this time point (which we will refer to as time T=0) the 200 mM glucose pulse was injected aseptically through a rubber septum. Experiment Overall Design: Sampling from chemostats, total RNA extraction, probe preparation and hybridization to Affymetrix Genechip® microarrays were performed as previously described (1). Samples were collected at steady state and then at 5, 10, 30, 60 and 120 minutes after the pulse. The results relative to steady state samples were derived from three independent cultures, those relative to the time course analysis were derived from two independent cultures. Experiment Overall Design: 1) Cipollina C., van den Brink J., Daran-Lapujade P., Pronk J.T., Vai M. and de Winde J.H. (2007) Revisiting the role of yeast Sfp1 in ribosome biogenesis and cell size control: A chemostat study. Microbiology. In press.
2008-06-18 | E-GEOD-9644 | biostudies-arrayexpress
Project description:S. cerevisiae strain FY1679 homozygous for HO deletion by KanMX4 was grown in a series of nutrient limited continuous cultures carbon, nitrogen, phosphorus and sulfur limitation to determine genes which are growth rate regulated and those which are nutrient specific regulated.
2007-05-02 | E-MEXP-115 | 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.
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Project description:Many studies in yeast have observed correlations between changes in phosphorylated adenosine and guanosine nucleotide concentrations, and changes in cell physiology or gene expression. Evidence for a causal relationship is however sparse. To investigate this further under controlled conditions, we have developed strains of S. cerevisiae containing inducible pathways which increase use of ATP or GTP. Analysis of the transcriptional response following induction of these strains during continuous culture in chemostats allowed the effects of specifically increasing the demand for each purine nucleotide triphosphate to be determined independently of growth rate and nutritional changes.
2018-08-23 | E-MTAB-5174 | biostudies-arrayexpress
Project description:The protrotophic laboratory strain CEN.PK113-7D (MAT a) was grown in laboratory fermentors with a working volume of 1 litre at dilution rate (D) of 0.20 per hour (in duplicate for each nitrogen (glutamine and ammonium) limited condition). After cultivation for few hundred generations in ammonium, samples from 2 continuous cultures were taken for array analysis. After cultivation for few hundred generations in glutamine, one evolved strain was picked and cultivated in 2 biological replicate chemostats until steady state was reached, and samples for array analysis were collected. All collected cell samples were cooled below 2 degree C within ten seconds by mixing 40% sample and 60% crushed ice.
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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.
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