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• W2617551877 abstract "The metabolic engineering of Saccharomyces cerevisiae for the production of succinic acid has progressed dramatically, and a series of high-producing hosts are available. At low cultivation pH and high titers, the product transport can become bidirectional, i.e. the acid is reentering the cell and is again exported or even catabolized. Here, a quantitative approach for the identification of product recycling fluxes is developed.The metabolic flux distributions at two time-points of the fermentation process were analyzed. 13C labeled succinic acid was added to the extracellular space and intracellular enrichments were measured and subsequently used for the estimation of metabolic fluxes. The labeling was introduced by a labeling switch experiment, leading to an immediate labeling of about 85% of the acid while keeping the total acid concentration constant. Within 100 s significant labeling enrichment of the TCA cycle intermediates fumarate, iso-citrate and α-ketoglutarate was observed, while no labeling was detected for malate and citrate. These findings suggest that succinic acid is rapidly exchanged over the cellular membrane and enters the oxidative TCA cycle. Remarkably, in the oxidative direction malate 13C enrichment was not detected, indicating that there is no flux going through this metabolite pool. Using flux modeling and thermodynamic assumptions on compartmentation it was concluded that malate must be predominantly cytosolic while fumarate and iso-citrate were more dominant in the mitochondria.Adding labeled product without changing the extracellular environment allowed to quantify intracellular metabolic fluxes under high producing conditions and identify product degradation cycles. In the specific case of succinic acid production, compartmentation was found to play a major role, i.e. the presence of metabolic activity in two different cellular compartments lead to intracellular product degradation reducing the yield. We also observed that the flux from glucose to succinic acid branches at two points in metabolism: (1) At the level of pyruvate, and (2) at cytosolic malate which was not expected." @default.
• W2617551877 created "2017-06-05" @default.
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• W2617551877 date "2017-05-23" @default.
• W2617551877 modified "2023-10-13" @default.
• W2617551877 title "Intracellular product recycling in high succinic acid producing yeast at low pH" @default.
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• W2617551877 doi "https://doi.org/10.1186/s12934-017-0702-0" @default.
• W2617551877 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5442661" @default.
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