High-level production of γ-aminobutyric acid via efficient co-expression of the key genes of glutamate decarboxylase system in Escherichia coli

Biosynthesis of the functional factor γ-aminobutyric acid (GABA) in bacteria involves two key proteins an intracellular glutamate decarboxylase (GadB) and a membrane-bound antiporter (GadC). Efficient co-expression of suitable GadB and GadC candidates is crucial for improving GABA productivity. In this study, gadB_ΔC11 of Lactiplantibacillus plantarum and gadC_ΔC41 of Escherichia coli were inserted into the designed double promoter (P_T7lac and P_BAD) expression system. Then, E. coli Lemo21(DE3) was chosen as the host to minimize the toxic effects of GadC_ΔC41 overexpression. Furthermore, a green and high-efficiency GABA synthesis system using dormant engineered Lemo21(DE3) cells as biocatalysts was developed. The total GABA yield reached 829.08 g/L with a 98.7% conversion ratio within 13 h, when engineered E. coli Lemo21(DE3) cells were concentrated to an OD₆₀₀ of 20 and reused for three cycles in a 3 M L-glutamate solution at 37 °C, which represented the highest GABA productivity ever reported. Overall, expanding the active pH ranges of GadB and GadC toward physiological pH and employing a tunable expression host for membrane-bound GadC production is a promising strategy for high-level GABA biosynthesis in E. coli.