Dynamic control over feedback regulation identifies pyruvate-ferredoxin oxidoreductase as a central metabolic enzyme in stationary phase E. coli
We demonstrate the use of two-stage dynamic metabolic control to manipulate feedback regulation in central metabolism and improve stationary phase biosynthesis in engineered E. coli . Specifically, we report the impact of dynamic control over two enzymes: citrate synthase, and glucose-6-phosphate dehydrogenase, on stationary phase fluxes. Firstly, reduced citrate synthase levels lead to a reduction in α -ketoglutarate, which is an inhibitor of sugar transport, resulting in increased stationary phase glucose uptake and glycolytic fluxes. Reduced glucose-6-phosphate dehydrogenase activity activates the SoxRS regulon and expression of pyruvate-ferredoxin oxidoreductase, which is in turn responsible for large increases in acetyl-CoA production. The combined reduction in citrate synthase and glucose-6-phosphate dehydrogenase, leads to greatly enhanced stationary phase metabolism and the improved production of citramalic acid enabling titers of 126±7g/L. These results identify pyruvate oxidation via the pyruvate-ferredoxin oxidoreductase as a “central” metabolic pathway in stationary phase E. coli , which coupled with ferredoxin reductase comprise a pathway whose physiologic role is maintaining NADPH levels. Dynamic reduction in α -keto-glutarate pools alleviate inhibition of PTS dependent transport improving stationary phase sugar uptake. Dynamic reduction in glucose-6-phosphate dehydrogenase activates pyruvate flavodoxin/ferredoxin oxidoreductase and improves stationary acetyl-CoA flux. Pyruvate flavodoxin/ferredoxin oxidoreductase is responsible for large stationary phase acetyl-CoA fluxes under aerobic conditions. Production of citramalate to titers 126 ± 7g/L at > 90 % of theoretical yield.
