Optimization of Aerobic Synthesis of Succinic Acid from Glucose by Recombinant Escherichia coli Strains Through the Variant Tricaboxylic Acid Cycle Mediated by the Action of 2-ketoglutarate-decarboxylase

The biosynthesis of succinic acid from glucose by the previously engineered E. coli strain SUC1.0 (pMW119-kgd) (MG1655 ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, P_Lglk, P_tacgalP, ∆aceBAK, ∆glcB, ∆sdhAB, pMW119-kgd) was optimized. The yield of the target substance was increased, upon the activation in the strain of the variant tricarboxylic acid cycle, mediated by the action of heterologous 2-ketoglutarate decarboxylase, due to the intensification of the anaplerotic formation of oxaloacetic acid. Inactivation of the nonspecific thioesterase YciA in the strain did not considerably change the biosynthetic characteristics of the producer. The enhancement of the expression of native phosphoenolpyruvate carboxylase led to an increase in the yield of the target compound by the recombinant synthesizing succinic acid via the reactions of the native tricarboxylic acid cycle from 25 to 42%, and from 67 to 75% upon the induced expression of Mycobacterium tuberculosis 2-ketoglutarate decarboxylase. Expression in the strain of pyruvate carboxylase gene from Bacillus subtilis resulted in an increase in the yield of succinic acid up to 84%. Functioning in whole-cell biocatalyst mode, the engineered strain SUC1.0 P_L-pycA (pMW119-kgd) demonstrated a substrate to target product conversion ratio reaching 93%, approaching the corresponding theoretical maximum.