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• The GAD system in Lactococcus lactis subsp. lactis CV56 has been identified. • Recombinant GadB has been biochemically characterized. • Genetic engineering has been employed to increase the expression of GAD system genes in L. lactis NZ9000. • Enhanced production of GABA by using a two-stage pH control strategy. γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). Biosynthesis of GABA by lactic acid bacteria (LAB) has particularly attracted attention, as LAB are generally recognized as safe organisms. In this work, based on identification of biosynthetic gene cluster for GABA in Lactococcus lactis subsp. lactis CV56, the GadB has been heterologously expressed in Escherichia coli , purified and biochemically characterized. The recombinant enzyme existed as a homodimer under native conditions with a molecular mass of 115.03 kDa, and exhibited maximal activity at 50 °C and pH 4.7. The K m value of GadB was 3.9 mM for L-glutamate, with a limiting rate (V max) of 15.1 U/mg. Subsequently, the genetic engineering technique has been employed to increase the levels of expression of key GAD system genes from strain CV56 in the model LAB- L. lactis subsp. cremoris NZ9000. The fermentation results revealed that the coexpression of gadC and gadB made much more contribution to the overall yield of GABA than overexpression of any individual gene. Moreover, a novel two-stage pH control strategy for the selected strain L. lactis NZ9000/pNZ8148- gadBC was developed, and 25.61 g/L of GABA was finally achieved in the batch fermentation. [ABSTRACT FROM AUTHOR]