Engineering a Recombinant Plasmid for Prospective IsPETase Expression in Escherichia coli Via the BrkA Autotransporter for Biocatalytic PET Recycling

Abstract

 Global accumulation of polyethylene terephthalate (PET) poses a significant environmental threat due to its resistance to degradation. Traditional recycling approaches remain inefficient, driving interest in enzymatic bioremediation as a sustainable alternative. Surface expression of PET-hydrolyzing enzymes on the outer membrane of Escherichia coli may serve as a promising bioremediation strategy to reduce the impact of plastic pollution on the environment. By utilizing bacterial surface display systems, the need for enzyme purification can be mitigated. This study explores the potential for utilizing the Bordetella pertussis BrkA autotransporter to display IsPETase on the outer membrane of E. coli. We engineered a BrkA-PETase recombinant plasmid, pFIJI, by inserting an IsPETase sequence into the passenger domain of BrkA. Our approach involved linearizing a BrkA-containing plasmid via inverse PCR, amplifying an IsPETase gene block, and assembling the two fragments using Gibson assembly. The construct was successfully validated by restriction digest and whole plasmid sequencing. This study provides the genetic framework for downstream PET degradation functional analyses utilizing the pFIJI recombinant plasmid to confirm enzymatic activity. Our findings highlight the feasibility of using BrkA-mediated surface display to express complex enzymes that can be used in the development of autotransporter systems for bioremediation of PET plastic pollution.