Optimization of the Anaerobic Production of Pyruvic Acid from Glucose by Recombinant Escherichia coli strains with Impaired Fermentation Ability via Enforced ATP Hydrolysis

Anaerobic production of pyruvic acid from glucose by recombinant Escherichia coli strains with impaired fermentation ability during respiration with nitrate as an external terminal electron acceptor was studied. During nitrate respiration in a minimal salt medium lacking ammonium ions, the core E. coli strain MG1655 increment ackA-pta, increment poxB, increment ldhA, increment adhE, increment ptsG, P(L)glk, P(tac)galP, increment frdAB, increment pflB, increment sdhAB, increment aceEF converted glucose into pyruvic acid with a yield of 1.72 mol/mol, secreting lactic acid as the only detected byproduct. The deletion of the lldD and dld genes blocked the secretion of this byproduct. The corresponding strain lacking the respiratory L- and D-lactate dehydrogenases LldD and Dld synthesized pyruvic acid from glucose with a yield of 1.76 mol/mol, consuming the available carbohydrate substrate incompletely. Enforced ATP hydrolysis due to the action of the pyruvic acid-oxaloacetic acid-malic acid-pyruvic acid or pyruvic acid-phosphoenolpyruvate-pyruvic acid futile cycles led to a drastic increase in glucose consumption by recombinants while maintaining the levels of substrate to the target product conversion. As a result, during anaerobic nitrate respiration and enforced ATP hydrolysis pyruvic acid was produced from glucose with a yield of 1.77-1.78 mol/mol with almost exhaustive consumption of the substrate by recombinants and no or minimal byproduct formation.