Flux Optimization of TCA Cycle for Efficient Production of Fumaric Acid

Abstract

Fumaric acid is an important C4-dicarboxylic acid widely used inchemical, food, and pharmaceutical industries. Rational metabolicengineering together with flux optimization were performed for thedevelopment of an Escherichia coli strain capable of efficiently producingfumaric acid. The initial engineered strain, CWF4N overexpressingphosphoenolpyruvate carboxylase (PPC), produced 5.30 g/L of fumaricacid. Optimization of PPC flux by examining 24 types of synthetic PPCexpression vectors further increased the titer up to 5.72 g/L with a yieldof 0.432 g/g·glucose. Overexpression of the succinate dehydrogenasecomplex (sdhCDAB) led to an increase in carbon yield up to 0.493g/g·glucose. Based on this mutant strain, citrate synthase(CS) wascombinatorially overexpressed and balanced with PPC using 48 types ofsynthetic expression vectors. As a result, 6.24 g/L of fumaric acid wasproduced with a yield of 0.500 g/g·glucose. Fed-batch culture of this finalstrain allowed production of 25.5 g/L of fumaric acid with a yield of0.366 g/g·glucose. Deletion of the aspA gene encoding aspartase andsupplementation of aspartic acid further increased the fumaric acid titerto 35.1 g/L with a yield of 0.490 g/g·glucose. (Technology DevelopmentProgram to Solve Climate Changes on Systems Metabolic Engineeringfor Biorefineries from the Ministry of Science, ICT and Future Planning(MSIP) through the National Research Foundation (NRF) of Korea