Stepwise metabolic engineering of a plasmid-free Corynebacterium glutamicum for efficient production of γ-aminobutyric acid (GABA) by co-utilizing lignocellulosic feedstock-derived sugars.

γ-aminobutyric acid (GABA) can be synthesized through plasmid-based expression of glutamate decarboxylase in L-glutamic acid producing Corynebacterium glutamicum strain. However, the addition of antibiotic to maintain the expression plasmid during the fermentation not only increases production and recovery costs, but also poses potential food safety hazards. In this study, a plasmid-free GABA producing C. glutamicum strain was constructed from C. glutamicum GJ04 chassis, which can produce L-glutamate by co-utilizing lignocellulose-derived glucose and xylose. Secretory glutamate decarboxylase was integrated into the genome of C. glutamicum GJ04 in three copies by replacing ldh, gabT, gabD genes. The metabolic flux in engineered C. glutamicum was further fine-tuned by knocking out aceA and gabP genes to enhance GABA production. The recombinant strain C. glutamicum GJ09 can produce 44.3 ± 3.8 g/L GABA from 15 % (w/w) solids loading corncob residues hydrolysate with the yield and productivity of 0.45 g/g and 0.74 g/L/h. The highest GABA titer reached 63.4 g/L by fed-batch fermentation using corncob residues-derived syrup. This study provided a robust and plasmid-free C. glutamicum strain by stepwise metabolic engineering for industrial production of GABA from lignocellulosic feedstocks.