Abstract
β-hexachlorocyclohexane (β-HCH) is a toxic byproduct of the organochlorine pesticide industry that poses severe risks to human and animal health, inducing developmental, hepatic, and neurological effects. A potential bioremediation strategy lies in bacteria from the genus Sphingobium that express LinB, a haloalkane dehalogenase that hydrolytically cleaves chlorine groups from HCH isomers, funneling them into degradation pathways and significantly reducing recalcitrance. Our study makes use of the Bordetella-resistant-to-killing autotransporter (BrkA) type Va secretion system from Bordetella pertussis, which can be harnessed to surface display foreign proteins in Gram-negative bacteria such as Escherichia coli. The use of the BrkA autotransporter to surface express LinB on the outer membrane of E. coli is explored as a potential future bioremediation strategy to break down β-HCH. To this end, surface localization of LinB could allow these E. coli cells to be introduced into contaminated environments, where extracellularly exposed LinB may directly interact with and degrade β-HCH. We designed a recombinant plasmid, pITAS, by inserting the linB gene into the BrkA passenger domain of the pOCS plasmid backbone, a vector expressing a 6x-Histidine (6x-His) tag for visualization on western blot and containing an OmpT cut site for future secretion experiments. The engineered construct was introduced into E. coli UT5600 cells, where LinB was confirmed to be displayed on the cell surface. This work establishes a foundation for future bioremediation strategies by employing the BrkA autotransporter for LinB surface expression to target β-HCH contamination.