Abstract
γ-aminobutyric acid (GABA) is an important metabolite used in neuroscience research and various industrial formulations, including those in the food, nutraceutical and mental health sectors. As environmentally responsible manufacturing practices continue to gain attention, the microbial production of GABA has become an attractive alternative to chemical synthesis. Specifically, microbial surface-display platforms provide a sustainable route for GABA production through the extracellular conversion of L-glutamate to GABA by the B isoform of the enzyme glutamate decarboxylase (GadB). Autotransporters can serve as secretion machinery for high-molecular weight passenger proteins, such as GadB. Here, we engineered the Bordetella pertussis BrkA autotransporter to display Lactobacillus plantarum GadB as the passenger protein on the surface of Escherichia coli UT5600. We successfully generated the recombinant pGADBLP plasmid via inverse PCR and Gibson Assembly, with proper construction confirmed by HindIII digestion and Nanopore sequencing. Western blotting, combined with a trypsin accessibility assay, further indicated that the BrkA-GadB construct was expressed at the cell surface. To evaluate GadB enzymatic activity, we optimized a thin-layer chromatography workflow detecting the conversion of extracellular L-glutamate to GABA. Preliminary results suggested low-level production of GABA due to BrkA-GadB. Altogether, these findings demonstrate that BrkA can export and display GadB at the E. coli surface and provide early evidence of catalytic function, supporting the future development of whole-cell biocatalysts for sustainable GABA production.