Metabolic flux analysis of succinic acid production by recombinant Escherichia coli

Abstract

In this study, the possibility of constructing a nonnatural metabolic pathway was evaluated by expression of the malic enzyme in a host which lost fermentation ability. The sfcA gene (1.7 kb) encoding E. coli malic enzyme was cloned by PCR from E coli XL1-Blue. Flask cultures were carried out in LB medium and induced with 0.01 mM IPTG. Higher IPTG concentration resulted in no production of succinic acid. E. coli strain NZN111 harboring pTrcML produced 8 g/L of succinic acid in flask culture. It is 6 times higher than that produced by wild type E. coli. Fermentation studies were carried out at various temperatures and pH. When NZN111 cultured at 30℃ and pH 6.7, 9.5 g/L of succinic acid was produced. Metabolic flux analysis showed that there exist a cyclic pathway among phosphoenolpyruvate carboxylase, pyruvate kinase, and malic enzyme. Overexpression of malic enzyme caused bottle neck, and reduced expression of malic enzyme somewhat resolved the bottleneck problem.