Production of Fumaric Acid from Glucose By a Metabolically Engineered Escherichia coli

In this study, Escherichia coli was metabolically engineered to produce a fumaric acid, a naturally synthesized organic acid as an intermediate of the TCA cycle, under aerobic condition. To redirect carbon flux through the glyoxylate shunt, the iclR gene was deleted, and the fumA, fumB, and fumC genes were also deleted to enhance fumaric acid formation. The resulting strain was able to produce 1.45 g/L of fumaric acid from 15 g/L of glucose in flask culture. Additionally, in silico flux response anaylsis was performed and the native ppc gene (encoding phosphoenolpyruvate carboxylase (PPC)) was overexpressed on plasmid level from the strong tac promoter. This resulting strain produced 4.09 g/L of fumaric acid. Moreover, the arcA and ptsG genes were deleted to reinforce the oxidative TCA cycle flux, and the aspA gene was deleted to prevent the fumaric acid from converting into L-aspartic acid. For enhanced glucose uptake rate and fumaric acid productivity, the native promoter of the galP gene was replaced with the strong trc promoter and the lacI gene was also deleted to avoid the use of inducer. Fed-batch culture of the final strain CWF812 produced 28.2 g/L fumaric acid in 63 h with the overall yield and productivity of 0.389 g fumaric acid/g glucose and 0.448 g/L/h, respectively. (This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557)).