Enhancing High-Level Food-Grade Expression of Glutamate Decarboxylase and Its Application in the Production of γ-Aminobutyric Acid

Gamma-aminobutyric acid (GABA), a non-proteinogenic amino acid, exhibits diverse physiological functions and finds extensive applications in food, medicine, and various industries. Glutamate decarboxylase (GAD) can effectively convert L-glutamic acid (L-Glu) or monosodium glutamate (MSG) into GABA. However, the low food-grade expression of GAD has hindered large-scale GABA production. In this study, we aimed to elevate GAD expression in <i>Bacillus subtilis</i> through cofactor synthesis enhancement, CRISPRi-based host strain modification, and fermentation optimization. In a 3-L fermenter, the optimized strain achieved a remarkable GAD activity of 319.62 U/ml without antibiotic selection pressure, representing the highest reported food-grade expression to date. Subsequently, enzymatic property analysis facilitated the optimization of GABA production using MSG and L-Glu as substrates, achieving 100% molar conversion yields of 274.40 g/l and 481.62 g/l, respectively, with the latter yielding an unprecedented productivity of 48.16 g/l/h. Finally, <i>in vitro</i> fermentation demonstrated that GABA supplementation promoted gut microbial growth and increased the relative abundance of <i>Actinobacteriota</i> and <i>Bacteroidota</i>.