Abstract
Escherichia coli was engineered for efficient aerobic conversion of glucose to fumaric acid. A novel design for biosynthesis of the target product through the modified TCA cycle rather than via glyoxylate shunt, implying oxaloacetate formation from pyruvate and artificial channelling of 2-ketoglutarate towards succinic acid via succinate semialdehyde formation, was implemented. The main fumarases were inactivated in the core strain MSG1.0 (∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, P(L)-glk, P(tac)-galP) by the deletion of the fumA, fumB, and fumC genes. The Bacillus subtilis pycA gene was expressed in the strain to ensure pyruvate to oxaloacetate conversion. The Mycobacterium tuberculosis kgd gene was expressed to enable succinate semialdehyde formation. The resulting strain was able to convert glucose to fumaric acid with a yield of 0.86 mol/mol, amounting to 86% of the theoretical maximum. The results demonstrated the high potential of the implemented strategy for development of efficient strains for bio-based fumaric acid production.