Abstract
The prevalence of global environmental and health-related issues stemming from historically high levels of heavy metal pollution in our natural surroundings has emphasized the need for more robust microbial biotechnology-based methods to remove these pollutants. As a result, bioremediation of heavy metal contaminants in environmental ecosystems using engineered microbial strains has become a significant area of academic interest. Recent studies have explored the use of the Type V autotransporter BrkA to display EC10, a phytochelator domain, on the surface of Escherichia coli as a viable strategy for microbially driven bioremediation. However, their findings reported reduced secretion of the BrkA–EC10 recombinant protein which was attributed to disulfide bond formation within the cysteine-rich EC10 domain. Subsequent experiments aimed to minimize disulfide bond formation using β-mercaptoethanol (βME) treatment and ∆dsbA strains. However, neither approaches improved BrkA–EC10 surface expression. The secretion pathway of BrkA-EC10 remains poorly understood, and no effective chemical or biological strategies have been identified to enhance BrkA–EC10 secretion in E. coli. Investigation of other periplasmic proteins interacting with BrkA-EC10 has identified the DegP protease as a potential contributor to BrkA-EC10 degradation. We hypothesize that E. coli cells lacking DegP exhibit enhanced cell surface expression of BrkA-EC10. Trypsin accessibility assay and Western blot analysis showed no significant improvement of BrkA-EC10 surface expression in ∆degP background E. coli strain JW0157. The liquid growth assay did show that the ∆degP background and intracellular expression of BrkA-EC10 does not significantly reduce cell viability or growth rate. However, as expected, the Cu2+ colorimetric assay shows that intracellularly expressed BrkA-EC10 does not cause a significant increase in Cu2+ heavy metal ion sequestration. These findings suggest that the removal of DegP protease alone is insufficient to significantly improve BrkA-EC10 surface expression and secretion, and that effective heavy metal ion sequestration may require high-efficiency surface expression of the BrkA-EC10 protein as a strategy for improved microbial bioremediation.