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Genomic analysis of Saccharomyces cerevisiae isolates that grow optimally with glucose as the sole carbon source.

ABSTRACT: A population of Saccharomyces cerevisiae was cultured for approximately 450 generations in the presence of high glucose to select for genetic variants that grew optimally under these conditions. Using the parental strain BY4741 as the starting population, an evolved culture was obtained after aerobic growth in a high glucose medium for approximately 450 generations. After the evolution period, three single colony isolates were selected for analysis. Next-generation Ion Torrent sequencing was used to evaluate genetic changes. Greater than 100 deletion/insertion changes were found with approximately half of these effecting genes. Additionally, over 180 SNPs were identified with more than one-quarter of these resulting in a nonsynonymous mutation. Affymetrix DNA microarrays and RNseq analysis were used to determine differences in gene expression in the evolved strains compared to the parental strain. It was established that approximately 900 genes demonstrated significantly altered expression in the evolved strains relative to the parental strain. Many of these genes showed similar alterations in their expression in all three evolved strains. Interestingly, genes with altered expression in the three evolved strains included genes with a role in oxidative metabolism. Overall these results are consistent with the physiological observations of optimal growth with glucose as the carbon source. Namely, the decreased ethanol production suggest that the underlying metabolism switched from fermentation to respiration during the selection for optimal growth on glucose.

SUBMITTER: Aragon AD

PROVIDER: S-EPMC3822012 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Genomic analysis of Saccharomyces cerevisiae isolates that grow optimally with glucose as the sole carbon source.

Aragon Anthony D AD Torrez-Martinez Norah N Edwards Jeremy S JS

Electrophoresis 20121108 23

A population of Saccharomyces cerevisiae was cultured for approximately 450 generations in the presence of high glucose to select for genetic variants that grew optimally under these conditions. Using the parental strain BY4741 as the starting population, an evolved culture was obtained after aerobic growth in a high glucose medium for approximately 450 generations. After the evolution period, three single colony isolates were selected for analysis. Next-generation Ion Torrent sequencing was use ...[more]

PMID: 23135695

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Project description:BackgroundSaccharomyces cerevisiae, engineered for L-lactic acid production from glucose and xylose, is a promising production host for lignocellulose-to-lactic acid processes. However, the two principal engineering strategies-pyruvate-to-lactic acid conversion with and without disruption of the competing pyruvate-to-ethanol pathway-have not yet resulted in strains that combine high lactic acid yields (YLA) and productivities (QLA) on both sugar substrates. Limitations seemingly arise from a dependency on the carbon source and the aeration conditions, but the underlying effects are poorly understood. We have recently presented two xylose-to-lactic acid converting strains, IBB14LA1 and IBB14LA1_5, which have the L-lactic acid dehydrogenase from Plasmodium falciparum (pfLDH) integrated at the pdc1 (pyruvate decarboxylase) locus. IBB14LA1_5 additionally has its pdc5 gene knocked out. In this study, the influence of carbon source and oxygen on YLA and QLA in IBB14LA1 and IBB14LA1_5 was investigated.ResultsIn anaerobic fermentation IBB14LA1 showed a higher YLA on xylose (0.27 g g Xyl-1 ) than on glucose (0.18 g g Glc-1 ). The ethanol yields (YEtOH, 0.15 g g Xyl-1 and 0.32 g g Glc-1 ) followed an opposite trend. In IBB14LA1_5, the effect of the carbon source on YLA was less pronounced (~?0.80 g g Xyl-1 , and 0.67 g g Glc-1 ). Supply of oxygen accelerated glucose conversions significantly in IBB14LA1 (QLA from 0.38 to 0.81 g L-1 h-1) and IBB14LA1_5 (QLA from 0.05 to 1.77 g L-1 h-1) at constant YLA (IBB14LA1 ~?0.18 g g Glc-1 ; IBB14LA1_5 ~?0.68 g g Glc-1 ). In aerobic xylose conversions, however, lactic acid production ceased completely in IBB14LA1 and decreased drastically in IBB14LA1_5 (YLA aerobic???0.25 g g Xyl-1 and anaerobic?~?0.80 g g Xyl-1 ) at similar QLA (~?0.04 g L-1 h-1). Switching from aerobic to microaerophilic conditions (pO2?~?2%) prevented lactic acid metabolization, observed for fully aerobic conditions, and increased QLA and YLA up to 0.11 g L-1 h-1 and 0.38 g g Xyl-1 , respectively. The pfLDH and PDC activities in IBB14LA1 were measured and shown to change drastically dependent on carbon source and oxygen.ConclusionEvidence from conversion time courses together with results of activity measurements for pfLDH and PDC show that in IBB14LA1 the distribution of fluxes at the pyruvate branching point is carbon source and oxygen dependent. Comparison of the performance of strain IBB14LA1 and IBB14LA1_5 in conversions under different aeration conditions (aerobic, anaerobic, and microaerophilic) further suggest that xylose, unlike glucose, does not repress the respiratory response in both strains. This study proposes new genetic engineering targets for rendering genetically engineering S. cerevisiae better suited for lactic acid biorefineries.

Project description:BackgroundControl effective flux (CEF) of a reaction is the weighted sum of all fluxes through that reaction, derived from elementary flux modes (EFM) of a metabolic network. Change in CEFs under different environmental conditions has earlier been proven to be correlated with the corresponding changes in the transcriptome. Here we use this to investigate the degree of transcriptional regulation of fluxes in the metabolism of Saccharomyces cerevisiae. We do this by quantifying correlations between changes in CEFs and changes in transcript levels for shifts in carbon source, i.e. between the fermentative carbon source glucose and nonfermentative carbon sources like ethanol, acetate, and lactate. The CEF analysis is based on a simple stoichiometric model that includes reactions of the central carbon metabolism and the amino acid metabolism.ResultsThe effect of the carbon shift on the metabolic fluxes was investigated for both batch and chemostat cultures. For growth on glucose in batch (respiro-fermentative) cultures, EFMs with no by-product formation were removed from the analysis of the CEFs, whereas those including any by-products (ethanol, glycerol, acetate, succinate) were omitted in the analysis of growth on glucose in chemostat (respiratory) cultures. This resulted in improved correlations between CEF changes and transcript levels. A regression correlation coefficient of 0.60 was obtained between CEF changes and gene expression changes in the central carbon metabolism for the analysis of 5 different perturbations. Out of 45 data points there were no more than 6 data points deviating from the correlation. Additionally, up- or down-regulation of at least 75% of the genes were in qualitative agreement with the CEF changes for all perturbations studied.ConclusionThe analysis indicates that changes in carbon source are associated with a high degree of hierarchical regulation of metabolic fluxes in the central carbon metabolism as the change in fluxes are correlating directly with the change in transcript levels of genes encoding their corresponding enzymes. For amino acid biosynthesis there was, however, not found to exist a similar correlation, and this may point to either post-transcriptional and/or metabolic regulation, or be due to the absence of a direct perturbation on the amino acid pathways in these experiments.

Dataset Information

Genomic analysis of Saccharomyces cerevisiae isolates that grow optimally with glucose as the sole carbon source.

Publications

Genomic analysis of Saccharomyces cerevisiae isolates that grow optimally with glucose as the sole carbon source.

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