High level production of γ-aminobutyric acid in engineered Escherichia coli by refactoring the glutamate decarboxylase.

Gamma-Aminobutyric acid (GABA), an important bioactive compound, is synthesized through the decarboxylation of L -glutamate (L -Glu) by glutamate decarboxylase (GAD). Particularly, most bacterial GADs are robust only under acidic conditions and sharply lose their activity when shifted to near-neutral pH. In this work, to broaden the pH dependence of GadB from Levilactobacillus brevis , five C-terminal truncated mutants were constructed based on the investigation of its C-terminal loop region, and their catalytic activities were compared at near-neutral pH. Subsequently, site-specific mutagenesis was performed to the selected GadB ΔC11 , and several mutants with substantially improved activity were thus obtained. Among these mutants, GadB ΔC11 K17T/D294G/E312S/Q346H exhibited the highest catalytic efficiency (k cat / K m , 3.91 s−1mM−1), which was 4.95-fold and 17-fold higher than that of WT enzyme (0.79 s−1mM−1) and GadB ΔC11 (0.23 s−1mM−1) at pH 4.8 and 37 °C. Furthermore, the GABA synthesis system using dormant E. coli cells expressing the best mutant GadB ΔC11 K17T/D294G/E312S/Q346H gene as the biocatalyst and pure water as a sole medium was constructed, and its GABA productivity was investigated. After 7.5 h of reaction, GABA production reached a concentration of 270.42 g/L with a 99.9% conversion ratio under the investigated conditions. Overall, refactoring the GAD system of E. coli through broadening the pH dependence of intracellular GadB is an effective approach for improving its GABA production. [Display omitted] • The active pH range of L. brevis GadB was broadened by removing the C-plug. • Improving the catalytic activity of GadB via site-specific mutagenesis. • Enhancing the production of GABA in E. coli by refactoring its GadB. [ABSTRACT FROM AUTHOR]