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γ-Aminobutyrate (GABA) is an important chemical in pharmaceutical field. The use of lactic acid bacteria as biocatalysts for the conversion of l-monosodium glutamate (MSG) into GABA opens interesting perspectives for the production of this functional compound. In this work, an engineered GABA high-producing strain Lactobacillus brevis GadA ΔC14 was constructed by overexpressing a C-terminally truncated GadA mutant, which is active in expanded pH range. After comparison with agar and κ-carrageenan, gellan gum was selected as the optimal immobilization support, and the properties of L. brevis GadA ΔC14  cells encapsulated in this hydrogel were examined. The optimum pH and temperature of immobilized cells were found to be 40°C and pH 4.4, respectively. It was also observed that operational and thermal stabilities of the cells were increased with immobilization. After ten consecutive reaction cycles, the total amounts of GABA produced by the immobilized cells summed up to 87.56 g/L under the optimum experimental conditions. Taken together, the improved stability and good usability make the immobilized L. brevis GadA ΔC14  cells more valuable for industrial applications., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Gamma-aminobutyrate (GABA) is an important chemical in the pharmaceutical field. GABA-producing lactic acid bacteria (LAB) offer the opportunity of developing this health-oriented product. In this study, the gadA, gadB, gadC, gadCB, and gadCA gene segments of Lactobacillus brevis were cloned into pMG36e, and strain Lb. brevis/pMG36e-gadA was selected for thorough characterization in terms of GABA production after analysis of GAD activities. Subsequently, a physiology-oriented engineering strategy was adopted to construct an F o F 1 -ATPase deficient strain NRA6 with higher GAD activity. As expected, strain NRA6 could produce GABA at a concentration of 43.65 g/L with a 98.42% GABA conversion rate in GYP fermentation medium, which is 1.22-fold higher than that obtained by the wild-type strain in the same condition. This work demonstrates how the acid stress response mechanisms of LAB can be employed to develop cell factories with improved production efficiency and contributes to research into the development of the physiology-oriented engineering.

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]