Engineering Escherichia coli for respiro-fermentative production of pyruvate from glucose under anoxic conditions.

An Escherichia coli K-12 MG1655-derived strain was engineered for respiro-fermentative production of pyruvate from glucose under anoxic conditions, which is preferred for industrial-scale microbial synthesis of valuable chemicals. The pathways of anaerobic pyruvate dissimilation were blocked in the strain by the deletion of the ackA, pta, poxB, ldhA, adhE, and pflB genes. The phosphoenolpyruvate-dependent phosphotransferase system of glucose transport and phosphorylation was substituted by an alternative ATP-dependent system resulting from the overexpression of galP and glk upon deletion of ptsG. The channelling of pyruvate towards the oxidative branch of the TCA cycle under respiratory conditions was prevented in the strain due to the deletion of aceEF genes, encoding components of pyruvate dehydrogenase, while the operation of the entire reductive branch of the TCA cycle was interrupted by knocking out frdAB and sdhAB. Reoxidation of glycolytic NADH was ensured via anaerobic respiration with nitrate serving as an external electron acceptor. To enforce anaerobic ATP hydrolysis, an ATP-consuming futile cycle of pyruvate-oxaloacetate-malate-pyruvate was established in the strain by expressing the Bacillus subtilis pycA gene, encoding pyruvate carboxylase. In the presence of sufficient amounts of an external electron acceptor and CO2 source, the engineered strain was able to efficiently utilise glucose and convert it to pyruvate anaerobically with a yield of 1.73 mol/mol, amounting to 87% of the theoretical maximum. The implemented strategy offers the potential for the development of highly efficient processes of bio-based pyruvate production.