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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

S. cerevisiae engineered chassis for dicarboxylic acid production

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Project description:<p>Chassis strain suitable for producing multiple compounds is a central concept in synthetic biology. Design of a chassis using computational, first-principle, models is particularly attractive due to the predictability and control it offers, including against phenotype reversal due to adaptive mutations. Yet, the theory of model-based chassis design has rarely been put to rigorous experimental test and assessed at multiomics level. Here, we report two <em>Saccharomyces cerevisiae</em> chassis strains for dicarboxylic acid production based on genome-scale metabolic modelling. The chassis strain, harbouring gene knockouts in serine biosynthesis and in pentose-phosphate pathway, is geared for higher flux towards three target products - succinate, fumarate and malate - but does not appreciably secrete any. Introducing modular product-specific mutations resulted in improved secretion of the corresponding acid as predicted by the model. Adaptive laboratory evolution of the chassis-derived producer cells further improved production for succinate and fumarate attesting to the evolutionary robustness of the underlying growth-product coupling. In the case of malate, which exhibited decreased production during evolution, the multiomics analysis revealed flux bypass at peroxisomal malate dehydrogenase that was not accounted in the model. Integration of transcriptomics, proteomics and metabolomics data showed overall concordance with the flux re-routing predicted by the model. Together, our results provide experimental evidence for model-based design of microbial chassis and have implications for computer-aided design of microbial cell factories.</p>

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