Abstract
Heavy metal contamination in aquatic environments is a global concern, disrupting marine ecosystems and posing severe risks to human health. Consequently, bioremediation strategies to sequester heavy metals using microbial constructs have gained increasing interest. One approach involves genetically engineered microorganisms to surface display metal-binding peptides. In this study, we investigated if Escherichia coli can express the BrkA autotransporter protein to surface display a hexahistidine (6xHis) tag for metal sequestering. Two constructs, UT5600_pENS and UT5600_pPALMC1, were generated, each encoding BrkA with a 6xHis tag in differing locations. The expression of 6xHis on the cell surface was validated through a trypsin accessibility assay, SDS-PAGE, and western blot analysis. A nickel bead-binding assay and zinc strip concentration test were then developed to assess metal-binding capacity. However, although previous research has indicated the ability of 6xHis to sequester cadmium ions (Cd²⁺), our constructs did not appear to bind to nickel resin or zinc ions (Zn²⁺). To explore this outcome, the zeta potential of nickel resin was measured and revealed a slight negative charge, suggesting that there could be weak electrostatic repulsion between the resin and the negatively charged cell surface. Additionally, AlphaFold was used to predict the three-dimensional (3D) structures of the BrkA-6xHis constructs encoded by pENS and pPALMC1. Structural visualizations and western blot analysis suggested that repositioning of the 6xHis insert could improve its accessibility, however further research is required to optimize the metal-binding ability of histidine based autotransporter constructs.