Engineering the level of metabolic cofactors to manipulate metabolic flux is emerging to be an attractive strategy for bioprocess applications. We present the metabolic consequences of increasing NADH in the cytosol of the mitochondria of Saccharomyces cerevisiae. In a strain that was disabled to metabolize formate, we overexpressed the native NAD(+)-dependent formate dehydrogenase either in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence from the CYB2 gene. Upon exposure to formate, the mutant strains readily consumed formate and induced fermentative metabolism even under glucose de-repressed conditions. Cytosolic overexpression of formate dehydrogenase resulted in the production of glycerol while when this enzyme was directed into the mitochondria, we observed glycerol and ethanol production. Clearly, these results point towards different compartmental regulation of redox homeostasis. When pulsed with formate, S. cerevisiae growing at a steady-state on glucose immediately consumed formate. However, formate consumption ceased after 20 minutes. Our analysis revealed that metabolites at key branch points of metabolic pathways were affect the most by the genetic perturbations, while the intracellular concentration of sugar phosphates were specifically affected by time. In conclusion, the results have implications in the design of metabolic networks in yeast for industrial applications.
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