Polyethylene terephthalate (PET) is a plastic widely used in industry and daily life (1). However, its high durability results in the accumulation of discarded PET in the environment for hundreds of years. This is a concern because of its negative impact on ecosystems and human health (2–4).
Several recycling methods have been developed for the recovery of PET. Mechanical and chemical recycling are representative methods (5,6). Mechanical recycling is a method in which PET is crushed, dissolved, and remolded. It is considered to be cheaper than chemical recycling (7). However, the drawback is that the product properties deteriorate with each cycle (8). In chemical recycling, PET is chemically depolymerized into monomers, which are then repolymerized. Therefore, PET can be recycled with minimal quality loss (9). However, chemical recycling is more expensive than mechanical recycling and thus, it offers fewer economic benefits (10). In addition, the chemical decomposition of PET requires high-temperature, high-pressure conditions, and large amounts of energy (11).
PET degradation by microorganism-derived enzymes proceeds under mild conditions, such as 30°C to 70°C and normal pressure. Therefore, this strategy has attracted attention as a new option for environment-friendly PET recycling. To date, several enzymes have been reported to be involved in the degradation of PET, such as the cutinase HiC from Humilica insolens (12), the cutinase LCC from leaf and branch compost (13), and hydrolase TfH from Thermobifida fusca (14).
In 2016, the bacterium Ideonella sakaiensis 201-F6 was shown to grow on PET as a major energy and carbon source in recycling plants in Japan (15). PETase, a PET-degrading enzyme secreted by this bacterium, shows higher PET degradation efficiency and substrate specificity than other PET-degrading enzymes at room temperature. PETase hydrolyzes PET and releases mono(hydroxyethyl)terephthalate (MHET) and terephthalate (TPA). In recent years, protein engineering modifications of this enzyme have been widely used to improve the enzymatic activity and thermal stability of PET (16,17). Notable mutant enzymes include DuraPETase (18) and FAST-PETase (19). Both mutant PETases have extremely high PET degradation activity and thermal stability compared to the wild-type PETase, and therefore, the implementation of enzymatic PET degradation is becoming a reality.
When such enzymes are used industrially, they are generally prepared using microorganisms such as recombinant E. coli. The desired enzyme can be obtained through cultivation of recombinant Escherichia coli, cell disruption, and enzyme purification. However, the cost of enzyme purification is known to be very high (20). In addition, because the enzyme is water-soluble, it can only be used once for the required reaction and then it becomes waste. To implement PET degradation using enzymes, it is desirable to address these issues.
Techniques have been developed to display target enzymes on the cell surface using membrane anchors to eliminate the enzyme purification process (21,22). When fused with the target enzyme, membrane anchors display the target enzyme and express enzyme activity at the cell surface (23). Therefore, these cells can be used as immobilized catalysts. Cells can be easily separated from the generated monomers by centrifugation or filtration, without the need for cell disruption. Furthermore, the separated cells can be reused (24).
Various membrane-anchor-based surface display systems have been developed for E. coli. Outer membrane proteins (25), ice nucleation proteins (26) and autotransporters (27) are used as anchors. In this study, we used the PgsA protein from Bacillus subtilis as the anchor protein. This protein is part of the enzyme complex that synthesizes poly-γ-glutamic acid (PGA) in B. subtilis (28). Narita et al. successfully fused PgsA with α-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica to display these enzymes in an active form on the cell surface of E. coli (29). Gallus et al. developed a new cell surface display system using the post-translational fusion of target proteins and membrane anchors using the SpyCatcher/SpyTag system (30). This system has been reported to successfully display heme- and diflavin-containing cytochrome P450 BM3 monooxygenase from Bacillus megaterium in E. coli, with higher levels of presentation than conventional genetic fusion using a plasmid (31).
We heterologously expressed PETase and PgsA in E. coli via genetic and post-translational fusion. In both cases, we successfully expressed the fusion protein and confirmed that the active form of the PETase was present on the cell surface of E. coli. E. coli expressing PETase by genetic fusion was able to degrade the PET intermediate bis(2-Hydroxyethyl) terephthalate (BHET) more efficiently than E. coli expressing PETase intracellularly or in a crude enzyme solution. It was also confirmed that E. coli can degrade PET films, albeit in small amounts, indicating that this is a promising new approach to PET degradation, although further improvement of the degradation efficiency is necessary.