Cloning, Prokaryotic Expression, and Functional Verification of Whole-Cell GABA Synthesis by the MoGAD from Moringa oleifera

Moringa oleifera is rich in γ-aminobutyric acid (GABA), a functional non-protein amino acid with significant antihypertensive and neuroprotective activities. However, the key enzymes responsible for catalyzing the conversion of L-glutamate (L-Glu) to GABA—glutamate decarboxylases (GADs)—have not been functionally characterized in M. oleifera, which limits its metabolic engineering applications. In this study, the previously obtained MoGAD1 (PZ458702) and MoGAD2 (PZ458703) genes were heterologously expressed in Escherichia coli Rosetta (DE3) to produce recombinant proteins. SDS-PAGE and Western blot analyses showed that both MoGAD1 and MoGAD2 were solubly expressed at 20 °C and 37 °C. Their catalytic functions were verified via whole-cell biocatalysis, and high-performance liquid chromatography (HPLC) analysis confirmed that both MoGAD1 and MoGAD2 could convert L-Glu to GABA. The GABA yields of the engineered strains harboring MoGAD1 and MoGAD2 reached 3.67 ± 0.1833 g/L and 0.648 ± 0.002 g/L, with conversion rates of 61.2% and 10.8%, respectively. Both MoGAD1 and MoGAD2 exhibited favorable docking with PLP, with binding energies of −5.489 kcal/mol and −5.297 kcal/mol, respectively; they also showed good docking with L-Glu, with binding energies of −4.207 kcal/mol and −4.49 kcal/mol, respectively. This study provides the first experimental evidence for the activity of the MoGAD protein encoded by the GAD gene from M. oleifera, elucidates the molecular mechanism underlying GABA accumulation, and offers candidate genes for biotechnological production of GABA.