Metabolic Engineering of Escherichia coli for Biosynthesis of Gamma-Aminobutyric Acid from Glucose Resulting from a Partial Reversal of GABA-Shunt Mediated by the Action of 2-Ketoglutarate Decarboxylase
摘要
Using directly engineered derivatives of previously constructed succinate-producing Escherichia coli strain SUC1.0 (pMW119-kgd) (MG1655 ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, PLglk, PtacgalP, ∆aceBAK, ∆glcB, ∆sdhAB, pMW119-kgd) the feasibility of gamma-aminobutyric acid biosynthesis from glucose by this bacterium resulting from a partial reversal of GABA-shunt was demonstrated. The formation of succinate semialdehyde from 2-ketoglutarate was ensured in the strain resulting from the expression of Mycobacterium tuberculosis 2-ketoglutarate decarboxylase gene. Conversion of succinate semialdehyde to succinic acid was prevented by the inactivation of cellular NAD+- and NADPH+-dependent succinate semialdehyde dehydrogenases. Formation of the target substance by the action of native 4-aminobutyrate aminotransferase was achieved upon the inactivation of glutamate decarboxylases A and B. Enhancement of 4-aminobutyrate aminotransferase gene expression led to an increase in the molar yield of gamma-aminobutyric acid from glucose demonstrated by the strain synthesizing the target product through the partially reversed GABA-shunt from ~11 to ~25%.