Construction of Mini-Chemostats for High-throughput Strain Characterization
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE120188 | GEO | 2019/02/01
REPOSITORIES: GEO
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Project description:Diurnal temperature cycling is an intrinsic characteristic of many exposed microbial ecosystems. However, its influence on yeast physiology and transcriptome has not been studied in detail. In this study, 24-h sinoidal temperature cycles, oscillating between 12 and 30M-BM-0C, were imposed on anaerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. After three diurnal temperature cycles (DTC), concentrations of glucose, and extracellular metabolites, as well as CO2-production rates showed regular, reproducible circadian rhytms. DTC also led to waves of transcriptional activation and repression, which involved one sixth of the yeast genome. A substantial fraction of these DTC-responsive genes appeared to primarily respond to changes in glucose concentration. Elimination of known glucose-responsive genes revealed overrepresentation of previously identified temperature-responsive genes as well as genes involved in cell cycle and de novo purine biosynthesis. Analyses of budding index and flow cytomery demonstrated that DTC led to a partial synchronization of the cell cycle of the yeast populations in the chemostat cultures, which was lost upon release from DTC. Comparison of DTC results with data from steady-state cultures showed that DTC was sufficiently slow to allow S. cerevisiae chemostat cultures to almost completely acclimatize their transcriptome and physiology at the DTC temperature maximum, and to approach acclimation at the DTC temperature minimum. The aim of this study was to investigate the impact of diurnal temperature cycles on the physiology and transcriptome of S. cerevisiae and to assess the extent to which these responses can be predicted from steady-state analyses. To this end, we used a continuous cultivation set-up, in which the yeast was grown under controlled conditions and subjected to 24-h sinoidal temperature cycles. Since the sampling volume was kept within 5% of the reactor volume during 24 h, sampling from two independent duplicate cultures for microarray analysis was spread over the fifth and sixth temperature cycle. Sample points from the fifth and sixth temperature cycle were combined, resulting in six sample points covering one temperature cycle (at temperatures of 30; 21; 14.6; 12; 21 and 27.4M-BM-0C). For the last time point, one additional analytical duplicate sample was taken. Furthermore, steady-state chemostat cultures at constant temperatures of 12M-BM-0C and 30M-BM-0C (independent duplicate cultures at both temperatures) were sampled for microarray analysis. All this resulted in a total array set of 17 arrays.
2014-05-12 | E-GEOD-55372 | 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:The physiology of D. hafniense strain Y51 under close to natural conditions were examined: D. hafniense Y51 was subjected to a variety of limiting conditions (carbon or electron acceptor) or ammonium deprivation, in chemostat. Its physiology was assessed by transcriptomic and proteomic analyses and compared to that in batch culture. Electron donor/acceptor-limited growth conditions cause a switch to alternative metabolic pathways allowing the organism with a capacity to survive in polluted environments and with a readiness to use alternative electron acceptors such as PCE; (ii) nitrogen limitation induces nitrogen fixation implying a robustness vis-a-vis ammonia shortage; (iii) limiting conditions trigger a general stress response that should enable the organism to cope with even more environmental constraints.
2017-11-21 | GSE107146 | GEO
Project description:RNA-seq of chemostat grown yeast (Y11335 BY4742 )
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Project description:We used Affymetrix GeneChips to determine the physiological differences between biofilm and planktonic cells of Bacteroides thetaiotaomicron strain VPI-5482 (ATCC 29148) by comparing gene expression. For this purpose, B. thetaiotaomicron cells were grown in sterile, continuous flow bioreactors fed with tryptone, yeast extract, glucose (TYG) medium. The bioreactors were controlled at a temperature of 37C using a water jacket and a recirculating water heater. After 8 hours post-inoculation, planktonic cells were harvested from the bulk solution in the bioreactor, and after 8 days post-inoculation, the biofilm was scraped from the carbon paper. RNA was harvested from both biofilm and planktonic populations. RNA was extracted by a phenol:chloroform method and purified with a Qiagen RNA Easy mini-kit.
2014-01-01 | GSE38534 | GEO
Project description:Escherichia coli strain MG1655 was grown in parallel chemostat cultures in GGM. In one chemostat the culture was fed adequate Zn; in the other, measures were taken to eliminate Zn from the chemostat vessel and culture medium. Culture volume (120 ml), temperature (37 oC) and stirring speed (437 rpm) were maintained. Steady state values for pH and OD600 were 6.9 and 0.6, respectively. After 50 hours, samples from the adequate Zn and Zn-limited chemostats were harvested into RNAprotect and total RNA was purified using Qiagen’s RNeasy Mini kit (using the supplier’s protocol) prior to use in microarray analysis. Biological experiments (i.e. a comparison of adequate versus low Zn in chemostat culture) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats.
2009-05-29 | GSE11894 | GEO
Project description:Diurnal temperature cycling is an intrinsic characteristic of many exposed microbial ecosystems. However, its influence on yeast physiology and transcriptome has not been studied in detail. In this study, 24-h sinoidal temperature cycles, oscillating between 12 and 30°C, were imposed on anaerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. After three diurnal temperature cycles (DTC), concentrations of glucose, and extracellular metabolites, as well as CO2-production rates showed regular, reproducible circadian rhytms. DTC also led to waves of transcriptional activation and repression, which involved one sixth of the yeast genome. A substantial fraction of these DTC-responsive genes appeared to primarily respond to changes in glucose concentration. Elimination of known glucose-responsive genes revealed overrepresentation of previously identified temperature-responsive genes as well as genes involved in cell cycle and de novo purine biosynthesis. Analyses of budding index and flow cytomery demonstrated that DTC led to a partial synchronization of the cell cycle of the yeast populations in the chemostat cultures, which was lost upon release from DTC. Comparison of DTC results with data from steady-state cultures showed that DTC was sufficiently slow to allow S. cerevisiae chemostat cultures to almost completely acclimatize their transcriptome and physiology at the DTC temperature maximum, and to approach acclimation at the DTC temperature minimum.
2014-05-12 | GSE55372 | GEO
Project description:Low concentrations of pharmaceutical compounds were shown to induce transcriptional responses in isolated microorganisms, which could have consequences on ecosystem dynamics. In order to test if these transcriptional responses could also be observed in complex river microbial communities, biofilm reactors were inoculated with water from two distinct rivers and supplemented with environmentally relevant doses of four pharmaceutical products (erythromycin-ER, gemfibrozil-GM, sulfamethazine-SN and sulfamethoxazole-SL). To follow the expression of functional genes, we constructed a 9,600 features anonymous DNA microarray platform onto which cDNA from the various biofilms was hybridized. The reactor design for biofilm development has been previously described (Lawrence et al., 2004; Lawrence et al., 2000). Two duplicate experiments were carried out, with reactors being inoculated with either water from the WC (nutrient rich) or the SSR (nutrient poor). Treatments consisted in the addition of various pharmaceutical compounds: 1 µg l-1 erythromycin (ER), 1 µg l-1 gemfibrozil (GM), 0.5 µg l-1 sulfamethazine (SN), 0.5 µg l-1 sulfamethoxazole (SL). Nothing was added to control reactors (CO). All treatments were replicated independently three times. A reference sample (composite sample from Wascana Creek reactors used to construct the microarray) was hybridized (Cy5) on each slide.
2010-07-06 | E-GEOD-20501 | biostudies-arrayexpress