Optimization of Aerobic Synthesis of Fumaric Acid from Glucose by a Recombinant Escherichia coli Strain Functioning in a Whole-Cell Biocatalyst Mode

Abstract

Biocatalytic synthesis of fumaric acid from glucose by the previously engineered E. coli strain FUM1.0 (pMW119-kgd) (E. coli MG1655 ∆ackA-pta, ∆ldhA, ∆adhE, ∆ptsG, P_L-glk, P_tac-galP, ∆fumB, ∆fumAC, poxB::P_L-pycA^Bs, pMW119-kgd) was optimized. The maximal yield of the target substance was achieved upon its synthesis through a variant of the tricarboxylic acid cycle mediated by the action of heterologous 2-ketoglutarate decarboxylase. The enhanced expression of the genes encoding components of the succinate dehydrogenase enzymatic complex did not markedly affect the biosynthetic characteristics of the producing strain. A positive effect of decreasing the intracellular ATP supply on the conversion of the carbohydrate substrate into the target product was demonstrated. The activation of the futile cycle of pyruvate–phosphoenolpyruvate–pyruvate due to an increase in the expression of the ppsA gene led to a slight increase in the yield of fumaric acid. Upon uncoupling the H⁺-ATP synthase complex subunits resulting in ATP formation cessation via oxidative phosphorylation, due to deletion of the atpFH genes, the molar yield of fumaric acid from glucose demonstrated by the strain functioning in the whole-cell biocatalyst mode reached 92%.