Integrating Enzyme Evolution and Metabolic Engineering to Improve the Productivity of Γ-Aminobutyric Acid by Whole-Cell Biosynthesis in Escherichia Coli

gamma-Aminobutyric acid (GABA) is used widely in various fields, such as agriculture, food, pharmaceuticals, and biobased chemicals. Based on glutamate decarboxylase (GadBM4) derived from our previous work, three mutants, GadM4-2, GadM4-8, and GadM4-31, were obtained by integrating enzyme evolution and high-throughput screening methods. The GABA productivity obtained through whole-cell bioconversion using recombinant Escherichia coli cells harboring mutant GadBM4-2 was enhanced by 20.27% compared to that of the original GadBM4. Further introduction of the central regulator GadE of the acid resistance system and the enzymes from the deoxyxylulose-5-phosphate-independent pyridoxal 5 '-phosphate biosynthesis pathway resulted in a 24.92% improvement in GABA productivity, reaching 76.70 g/L/h without any cofactor addition with a greater than 99% conversion ratio. Finally, when one-step bioconversion was applied for the whole-cell catalysis in a 5 L bioreactor, the titer of GABA reached 307.5 +/- 5.94 g/L with a productivity of 61.49 g/L/h by using crude L-glutamic acid (L-Glu) as the substrate. Thus, the biocatalyst constructed above combined with the whole-cell bioconversion method represents an effective approach for industrial GABA production.