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

Using directly engineered derivatives of previously constructed succinate-producing Escherichia coli strain SUC1.0 (pMW119- kgd ) (MG1655 ∆ ackA-pta , ∆ poxB , ∆ ldhA , ∆ adhE , ∆ ptsG , P L glk , P tac galP , ∆ 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%.