Dynamic control over feedback regulation improves stationary phase fluxes in engineered 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 -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. HighlightsO_LIDynamic reduction in -keto-glutarate pools alleviate inhibition of PTS dependent transport improving stationary phase sugar uptake. C_LIO_LIDynamic reduction in glucose-6-phosphate dehydrogenase activates pyruvate flavodoxin/ferredoxin oxidoreductase and improves stationary acetyl-CoA flux. C_LIO_LIPyruvate flavodoxin/ferredoxin oxidoreductase is responsible for large stationary phase acetyl-CoA fluxes under aerobic conditions. C_LIO_LIProduction of citramalate to titers 126 {+/-} 7g/L at > 90 % of theoretical yield. C_LI