Metabolic engineering Corynebacterium glutamicum for production of γ-aminobutyric acid by glutamate decarboxylase active at near-neutral pH and displaying at cell surface

Abstract

γ-Aminobutyric acid (GABA) is widely applied in pharmaceuticals, foods and feeds. Corynebacterium glutamicum that expresses exogenous glutamate decarboxylase (GAD) gene gad can produce GABA from glucose using self-produced L-glutamate. However, the incongruity between optimal pH for cell growth (7.0–7.5) and GAD (4.0–5.0) severely restricts the production of GABA. In this study, several GADs active at near-neutral pH were separately expressed in C. glutamicum by plasmid, LsGAD derived from Lactobacillus senmaizukei performed better and generated 10.9 g/L GABA. Subsequently, to perform the GAD reaction at the more acidic extracellular environment, LsGAD was displayed on cell surface by several anchoring motifs, and displaying by PorH and NCgl1307 motifs produced 9.9 g/L and 1.3 g/L GABA, respectively. To further improve GABA production, the metabolic pathways were modified and accompanied by integrating several gad genes in the chromosome, the best strain GSL-6 could produce 15.6 g/L GABA. Finally, the surface display plasmid of LsGAD was introduced into the chromosomally modified strain GSL-6 to catalyze GAD reaction both intracellularly and extracellularly, and 25.3 g/L and 42.3 g/L GABA was finally produced by shake flask and fed-batch fermentation, respectively. Thereby, this synergistic strategy is beneficial for GABA production in C. glutamicum.