Autodisplay and Functional Characterization of the Burkholderia cepacia Lipase and Its Chaperone Foldase Using the BrkA Autotransporter System in Escherichia coli

Abstract

Bacterial display of functional lipases has promising applications for biofuel production and wastewater treatment. Gram-negative bacteria use autotransporter systems to display proteins on their outer membranes. The Bordetella resistance to killing (BrkA) autotransporter from Bordetella pertussis is one such system that can be harnessed to display recombinant proteins in other gram-negative bacteria like Escherichia coli. BrkA has recently been shown to support the surface display of functional homodimeric enzymes, demonstrating that BrkA can be used to display proteins that interact with each other. However, there is a gap in understanding whether BrkA can support the display of enzymes that require a specific chaperone for proper folding and activity, acting as heterodimeric enzymes. This project investigated whether the BrkA system can co-display the Burkholderia cepacia lipase (LipA) and its necessary chaperone foldase (LipB) on the surface of E. coli to produce active enzymes. In this study, we replaced the BrkA passenger domain in the pPALMC1 plasmid with either LipA or LipB, constructing an ampicillin-resistant plasmid containing LipA and a chloramphenicol-resistant plasmid containing LipB. Both plasmids were co-transformed into UT5600 E. coli cells, and western blotting and flow cytometry confirmed expression of each protein individually on the surface of E. coli. Lipolytic activity was evaluated through a p-NPP lipase assay and an olive oil and phenol red plate assay, however the results were inconclusive. Demonstrating successful co-display and lipase activity would expand the utility of BrkA for displaying complex, chaperone-dependent enzymes, and support its broader application in industrial and environmental biotechnology.