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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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PSY3642_to_PSY3639_SC_aerobic

ABSTRACT: We designed and experimentally validated an in silico gene deletion strategy for engineering endogenous one-carbon (C1) metabolism in yeast. Specifically, a Flux Balance Analysis (FBA) model predicted that five genes ALT2, FDH1, FDH2, FUM1, and ZWF1, when deleted in combination, would cause Saccharomyces cerevisiae to overproduce and secrete formic acid under aerobic growth conditions. Once constructed, the quintuple mutant strain showed the predicted increase in formic acid secretion relative to a formate dehydrogenase mutant (fdh1 fdh2 ), while formic acid secretion in wild-type yeast was undetectable. Gene expression and physiological data generated post hoc identified a mitochondrial deficiency phenotype in the engineered strain and regulatory events that suggest a role for multisite modulation—the coordinated expression of multiple pathway enzymes—in controlling yeast C1 metabolism. Together, these results demonstrate that FBA-based modeling strategies can be useful tools for non-fermentative pathway design and discovery in eukaryotic microbes. In addition, the results indicate that seemingly unrelated mutations can be combined to modulate flux through biochemical reactions that interact at a systems level across subcellular compartments. Samples were processed in biological triplicate with one sample processed with dye orientations reversed to minimize the effects of incorporation bias.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Caleb Kennedy

PROVIDER: E-GEOD-12240 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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PSY3642_to_PSY3639_SC_aerobic

Project description:We designed and experimentally validated an in silico gene deletion strategy for engineering endogenous one-carbon (C1) metabolism in yeast. Specifically, a Flux Balance Analysis (FBA) model predicted that five genes ALT2, FDH1, FDH2, FUM1, and ZWF1, when deleted in combination, would cause Saccharomyces cerevisiae to overproduce and secrete formic acid under aerobic growth conditions. Once constructed, the quintuple mutant strain showed the predicted increase in formic acid secretion relative to a formate dehydrogenase mutant (fdh1 fdh2 ), while formic acid secretion in wild-type yeast was undetectable. Gene expression and physiological data generated post hoc identified a mitochondrial deficiency phenotype in the engineered strain and regulatory events that suggest a role for multisite modulation—the coordinated expression of multiple pathway enzymes—in controlling yeast C1 metabolism. Together, these results demonstrate that FBA-based modeling strategies can be useful tools for non-fermentative pathway design and discovery in eukaryotic microbes. In addition, the results indicate that seemingly unrelated mutations can be combined to modulate flux through biochemical reactions that interact at a systems level across subcellular compartments.

2009-07-24 | GSE12240 | GEO

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