Engineering Escherichia coli for Surface Display of Helicobacter pylori α-Carbonic Anhydrase via the BrkA Autotransporter: A Strategy for CO2 Sequestration

Abstract

Reducing atmospheric carbon dioxide (CO₂) levels is critical to combat climate change. Biological catalysts such as carbonic anhydrases provide an environmentally friendly platform for carbon capture and sequestration. Surface display of carbonic anhydrase in Escherichia coli to produce whole-cell biocatalysts eliminates the need for enzyme purification, presenting a promising strategy for efficient mineralization of CO₂. In this study, we investigated the surface expression of Helicobacter pylori ɑ-carbonic anhydrase (HPCA) in E. coli UT5600 using the Bordetella resistant to killing (BrkA) autotransporter. We engineered a recombinant BrkA-HPCA plasmid (pHPCA1I) by substituting a portion of the passenger domain with the HPCA encoding domain and confirmed the expression of HPCA via western blot analysis. The use of β-mercaptoethanol in the trypsin accessibility assay validated HPCA secretion and display. These findings highlighted the BrkA system's ability to export HPCA and suggested the potential for HPCA multimerization. Additionally, the functionality of BrkA-HPCA was evaluated with a pH assay in CO₂-saturated water, and suggested potential catalytic activity. Overall, our findings demonstrate the potential for BrkA-mediated export of HPCA and contribute to the diverse repertoire of heterologous proteins that can be expressed by the BrkA surface display system.