Cofactor Metabolic Engineering of Escherichia coli for Aerobic L-Malate Production with Lower CO2 Emissions

Abstract

Escherichia coli has been engineered for L-malate production via aerobic cultivation. However, the maximum yield obtained through this mode is inferior to that of anaerobic fermentation due to massive amounts of CO2 emissions. Here, we aim to address this issue by reducing CO2 emissions of recombinant E. coli during aerobic L-malate production. Our findings indicated that NADH oxidation and ATP-synthesis-related genes were down-regulated with 2 g/L of YE during aerobic cultivations of E. coli E23, as compared to 5 g/L of YE. Then, E23 was engineered via the knockout of nuoA and the introduction of the nonoxidative glycolysis (NOG) pathway, resulting in a reduction of NAD+ and ATP supplies. The results demonstrate that E23 (ΔnuoA, NOG) exhibited decreased CO2 emissions, and it produced 21.3 g/L of L-malate from glucose aerobically with the improved yield of 0.43 g/g. This study suggests that a restricted NAD+ and ATP supply can prompt E. coli to engage in incomplete oxidization of glucose, leading to the accumulation of metabolites instead of utilizing them in cellular respiration.