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Physiological and transcriptional responses to high concentrations of lactic acid in anaerobic chemostat cultures of Saccharomyces cerevisiae.

ABSTRACT: Based on the high acid tolerance and the simple nutritional requirements of Saccharomyces cerevisiae, engineered strains of this yeast are considered biocatalysts for industrial production of high-purity undissociated lactic acid. However, high concentrations of lactic acid are toxic to S. cerevisiae, thus limiting its growth and product formation. Physiological and transcriptional responses to high concentrations of lactic acid were studied in anaerobic, glucose-limited chemostat cultures grown at different pH values and lactic acid concentrations, resulting in a 50% decrease in the biomass yield. At pH 5, the yield decrease was caused mostly by osmotically induced glycerol production and not by the classic weak-acid action, as was observed at pH 3. Cultures grown at pH 5 with 900 mM lactic acid revealed an upregulation of many genes involved in iron homeostasis, indicating that iron chelation occurred at high concentrations of dissociated lactic acid. Chemostat cultivation at pH 3 with 500 mM lactate, resulting in lower anion concentrations, showed an alleviation of this iron homeostasis response. Six of the 10 known targets of the transcriptional regulator Haa1p were strongly upregulated in lactate-challenged cultures at pH 3 but showed only moderate induction by high lactate concentrations at pH 5. Moreover, the haa1Delta mutant exhibited a growth defect at high lactic acid concentrations at pH 3. These results indicate that iron homeostasis plays a major role in the response of S. cerevisiae to high lactate concentrations, whereas the Haa1p regulon is involved primarily in the response to high concentrations of undissociated lactic acid.

SUBMITTER: Abbott DA

PROVIDER: S-EPMC2547041 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Physiological and transcriptional responses to high concentrations of lactic acid in anaerobic chemostat cultures of Saccharomyces cerevisiae.

Abbott Derek A DA Suir Erwin E van Maris Antonius J A AJ Pronk Jack T JT

Applied and environmental microbiology 20080801 18

Based on the high acid tolerance and the simple nutritional requirements of Saccharomyces cerevisiae, engineered strains of this yeast are considered biocatalysts for industrial production of high-purity undissociated lactic acid. However, high concentrations of lactic acid are toxic to S. cerevisiae, thus limiting its growth and product formation. Physiological and transcriptional responses to high concentrations of lactic acid were studied in anaerobic, glucose-limited chemostat cultures grown ...[more]

PMID: 18676708

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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.

Project description:Raw expression values (CHP data) for transcriptional profiling of the response of Saccharomyces cerevisiae to challenges with lactic acid at pH 3 and pH 5. Experiment Overall Design: The laboratory reference strain CEN.PK 113-7D (MATa) was grown at 30 Â°C in 1.5-L chemostat fermentors (Applikon, Schiedam, The Netherlands) with a working volume of 1-L using an electronic level sensor to maintain a constant volume. All cultures, including the reference, were fed with minimal medium as described by Verduyn et al. (1992) with 25 g L-1 glucose as the limiting nutrient and 0.15 ml L-1 silicone antifoam (BDH, Poole, England) to prevent excessive foaming. The dilution rate was set to 0.10 h-1 and the pH was controlled at 5.0 with the automatic addition (ADI 1031 bio controller, Applikon) of 2 M KOH. The stirrer speed was set at 800 RPM and anaerobicity was maintained by sparging the fermentor with N2 gas at 500 ml min-1. To prevent diffusion of oxygen, the fermentor was equipped with Norprene tubing and Viton O-rings and the medium vessel was also flushed with N2 gas. A comparable degree of weak acid uncoupling was ensured by decreasing the biomass yield to approximately 50% of the reference condition (no organic acids added) with the addition of the appropriate concentration of lactic acid to the reservoir media. Experiment Overall Design: Sampling of chemostat cultures, probe preparation and hybridization to Affymetrix GeneChip microarrays was performed as described previously (Piper et al., 2002), but with the following modifications. Double-stranded cDNA synthesis was carried out using 15 Î¼g of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix). The double-stranded cDNA was purified (Genechip Sample Cleanup Module, Qiagen) before in vitro transcription and labeling (GeneChip IVT Labeling Kit, Affymetrix). Finally, labeled cRNA was purified (GeneChip Sample Cleanup Module) prior to fragmentation and hybridization of 15 Î¼g of biotinylated cRNA. Experiment Overall Design: Data acquisition was performed using the Affymetrix scanner 3000, quantification of array images and data filtering were performed with the Affymetrix software packages Microarray Suite v5.0, MicroDB v3.0 and Data Mining Tool v3.0.

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Physiological and transcriptional responses to high concentrations of lactic acid in anaerobic chemostat cultures of Saccharomyces cerevisiae.

Publications

Physiological and transcriptional responses to high concentrations of lactic acid in anaerobic chemostat cultures of Saccharomyces cerevisiae.

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