CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase

摘要

In wild-type Escherichia coli, 1 mol of CO2 was fixated in 1 mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD~+ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD~+ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, IdhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72h, 14.5gL~(-1) of glucose was consumed to generate 12.08gL~(-1) of succinic acid. Furthermore, under optimized condition of CO2 supply, the succinic acid productivity and the CO2 fixation rate reached 223.88 mg L~(-1) h~(-1) and 83.48 mgL~(-1) h~(-1), respectively.