Metabolic Engineering of Escherichia coli for High Yield Production of Succinic Acid Driven by Methanol

Methanol is increasingly becoming an attractive carbon feedstock for the production of various biochemicals due to its high abundance and low price. In this study, when methanol assimilation module was introduced into succinic acid producing Escherichia coli by employing the NAD-dependent methanol dehydrogenase from Bacillus methanolicus and ribulose monophosphate pathway from different donor organisms, succinic acid yield was increased from 0.91 ± 0.08 g/g to 0.98 ± 0.11 g/g with methanol as an auxiliary substrate under the anaerobic fermentation. Further ¹³ C-labeling experiments showed that the recombinant strain successfully converted methanol into succinic acid, as the carbon atom of carboxyl group in succinic acid was labeled by ¹³ C. It was found that the NADH generated by methanol oxidation would benefit succinate production, as the NADH/NAD ⁺ ratio in vivo was decreased from 0.67 to 0.45 in the engineered strain, indicating that the efficiency of succinic acid synthesis was significantly improved when driven by methanol. This study represents a successful case for the development of reducing chemical production using methanol as an auxiliary substrate.