Abstract
Gamma-aminobutyric acid is an important metabolite used in neuroscience research. It serves as the primary inhibitory neurotransmitter in the brain, regulating various neurological processes. It is also utilized widely in industrial applications, including food and nutraceutical production. Engineered microbial biosynthesis has emerged as a sustainable alternative to chemical synthesis of this metabolite, with bacterial autotransporter-based surface display platforms enabling extracellular conversion of glutamate to gamma-aminobutyric acid by glutamate decarboxylase. Autotransporters are a promising secretion strategy because they allow proteins to fold after translocation across the outer membrane, making them suitable for transporting large enzymes to the outer surface of bacteria, where they can be exposed to extracellular proteases or other activating processes. Previous work has shown that the Bordetella resistance to killing A protein autotransporter can display glutamate decarboxylase on the cell surface, but limited enzymatic activity suggests constraints in functionality. In this study, we aimed to improve gamma-aminobutyric acid production by engineering an autotransporter system containing a protease cleavage site to enable the release of glutamate decarboxylase into the extracellular environment. The recombinant construct was expressed in bacterial strains containing or lacking protease activity to compare surface-bound and secreted forms of the enzyme. Protein expression, localization, and cleavage were assessed using Western blot analysis of whole cell lysates, cytoplasmic and membrane fractions, and concentrated extracellular proteins. The recombinant protein was detected only at low intracellular levels in OmpT-positive cells, and neither surface display nor extracellular secretion of glutamate decarboxylase was observed, likely due to a deletion within the construct’s N-terminus signal peptide that impaired Sec-dependent translocation across the inner membrane. Although functional conversion of glutamate to gamma-aminobutyric acid was not evaluated, this study establishes a framework for evaluating enzyme localization and extracellular secretion in autotransporter systems and supports future efforts to optimize microbial production of gamma-aminobutyric acid.