Coordinated Regulation of Glutamate-GABA Flux Enables High-Titre GABA Production in Enterococcus faecium

The efficient microbial conversion of monosodium glutamate (MSG) to γ-aminobutyric acid (GABA) is central to the development of sustainable fermentation platforms for functional foods and biopharmaceutical applications. In this study, the effects of key physicochemical and nutritional parameters on GABA production by Enterococcus faecium were systematically evaluated to define the optimal conditions for intensifying the process. The incubation time, inoculum density, and initial pH were first examined to determine their influence on biomass formation and the redistribution of MSG-derived carbon and nitrogen flux between glutamine accumulation and GABA biosynthesis. Alkaline conditions markedly enhanced glutamate decarboxylase-mediated conversion, whereas slowly metabolized carbon sources and ammonium-based nitrogen supplementation supported sustained decarboxylation. Multivariate analysis further identified the initial pH, carbon source complexity, and nitrogen form as the major determinants of GABA productivity. Response surface methodology based on a central composite design confirmed that pH ≈ 10, starch supplementation at 20-25 g L⁻¹, NH₄Cl at 15-20 g L⁻¹, and an inoculum density of 1.5-2.0% (v/v) acted synergistically to maximize GABA production. Under these optimized conditions, the GABA titer reached 1.40 ± 0.05 g L⁻¹, closely matching the model-predicted value of 1.52 g L⁻¹, while the volumetric productivity, biomass-specific yield, and specific product formation rate increased by up to 1.6-fold. Integrated kinetic and metabolic interpretation indicated that the optimized fermentation conditions preferentially channelled MSG-derived carbon and nitrogen through the glutamate-GABA-glutamine network toward GABA formation. Overall, these findings provide a mechanistic basis and process-level framework for scaling up GABA biomanufacturing using E. faecium as a tunable fermentation platform.