Expression of Fusarium Solani Cutinase via the BrkA Autotransporter in Escherichia coli for Polyethylene Terephthalate Degradation

Abstract

Polyethylene terephthalate (PET) accumulation has led to significant environmental pollution, which takes approximately 450 years to decompose naturally. The utilization of genetically modified microorganisms as an alternative to conventional recycling methods is a promising solution to the accumulation of PET waste. Cutinase from Fusarium solani (FsC) has been shown to be capable of degrading PET. Engineering cutinase into the passenger domain of the BrkA autotransporter allows protein to be presented extracellularly. A BrkA-FsC plasmid (pGRGS) was constructed using Gibson Assembly by engineering FsC into the passenger domain of pDO6935-H-CS BrKA plasmid, which contains an additional OmpT recognition cut site. This allows FsC protein to be translocated, cleaved and released into the extracellular space, and ultimately secreted to the environment. Direct surface localization of the enzyme on Escherichia coli can bypass the periplasmic space and enhance secretion efficiency. Successful generation of pGRGS was confirmed through positive colony PCR for FsC amplification and 100% sequencing match to SnapGene Gibson Assembly construct. Challenges are still observed for the expression of FsC and BrkA-FsC recombinant protein, thus further research needs to be conducted to resolve this issue.