Utilizing BrkA Passenger Domain to Surface Display Lead Remediator EC10 on Escherichia coli

According to the WHO, lead contamination poses significant environmental and health challenges globally and is responsible for 1.5 million deaths worldwide in 2021. One natural bioremediation strategy involves the use of phytochelatins, a cysteine-rich peptide naturally synthesized in plants to chelate heavy metals via thiolate binding. Engineered bacteria have also emerged as a promising bioremediation solution by leveraging Type V autotransporters, a bacterial system that surface displays peptides, such as BrkA from Bordetella pertussis, to display non-native peptides on the bacterial surface. However, disulfide-rich peptides have previously been found to hinder secretion in Type V autotransporters. Our study investigates if BrkA is able to secrete a synthetic phytochelatin, EC10, in Escherichia coli as a lead bioremediator and whether β-mercaptoethanol (βME) treatment is necessary for its secretion. We designed a recombinant BrkA-EC10 plasmid (pNARS) by inserting the EC10 gene block into the BrkA passenger domain of pENS, a backbone plasmid vector with a 6xHistidine-tag within its BrkA passenger domain. We then amplified pNARS in E. coli TOP10, validated the construct by double restriction digest and nanopore sequencing, and transformed the plasmid into E. coli BW25113. Western blot analysis and trypsin accessibility assays indicated EC10 expression and secretion, although at lower levels compared to wildtype BrkA. βME treatment had little effect on EC10 secretion, while a growth assay revealed that the EC10 insert had minimal impact on bacterial viability. These findings highlight the potential for BrkA to secrete disulfide-rich peptides under optimized conditions and expand on lead bioremediation strategies.