Metabolic engineering has been broadly applied in the biosynthesis of a wide variety of products, including biofuels, pharmaceuticals and food chemicals. Citramalate is an important precursor to the industrial chemicals itaconic acid and methyl methacrylate, as well as an intermediate in branched-chain amino acid biosynthesis. In our work, synergistic dynamic control over glucose transport as well as the pyruvate-ferredoxin oxidoreductase activity leads to significantly improved acetyl-CoA and citramalate production in engineered E. coli. The implementation of this approach relies on the combined use of controlled proteolysis and gene silencing, using degron tags and CRISPR interference, respectively. A dynamic reduction in citrate synthase activity leads to decreased alpha-ketoglutarate levels which in turn activate PTS-dependent glucose transport and glycolytic flux. A dynamic reduction in glucose-6-phosphate dehydrogenase activity leads to decreased NADPH levels which in turn activate the SoxRS regulon and expression of pyruvate-ferredoxin oxidoreductase, generating larger increases in acetyl-CoA production. In this work, dynamic deregulation of metabolism enhances acetyl-CoA fluxes as demonstrated by improved production of citramalic acid enabling titers of 126±7g/L. These results identify a role for alpha-ketoglutarate in regulating sugar uptake and metabolism in phosphate limited stationary phase. We also identify pyruvate oxidation via the pyruvate-ferredoxin oxidoreductase (YdbK) as a potentially “central” metabolic pathway in stationary phase E. coli.

Dynamic control over central metabolic pathways improves stationary phase acetyl-CoA flux and citramalate production in engineered E. coli

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.