Optimization of polyethylene terephthalate biodegradation using a self-assembled multi-enzyme cascade strategy

Area of Science:

• Biotechnology
• Enzyme Engineering
• Polymer Science

Background:

• Improving polyethylene terephthalate (PET) degradation efficiency at near-ambient temperatures remains a significant challenge.
• Existing PET hydrolases often require optimization for industrial applications.
• Enzyme engineering efforts have focused on enhancing catalytic activity and stability.

Purpose of the Study:

• To develop a multi-enzyme cascade system (PT-EC) for efficient PET degradation.
• To enhance the catalytic performance of PET hydrolases using a scaffold protein system.
• To investigate the efficacy of engineered enzymes and displayed systems for PET bio-cycling.

Main Methods:

• Assembled three PETases (PETase^EHA, Fast-PETase, Z1-PETase) with carboxylesterase TfCa and CBM3a on scaffold proteins to create PT-ECs.
• Displayed PT-ECs on the surface of Escherichia coli for enhanced PET degradation.
• Evolved carboxylesterase TfCa using docking-based virtual screening to further improve product release.

Main Results:

• PT-ECs demonstrated outstanding PET degradation efficacy, with PT-EC^EHA showing a 16.5-fold increase in product release compared to PETase^EHA.
• Surface-displayed PT-EC^EHA achieved a 20-fold increase in degradation efficiency with PET film.
• Engineered TfCa^I69W/L281Y further increased product release by 2.5-fold, highlighting the system's potential.

Conclusions:

• The developed multi-enzyme cascade system (PT-EC) significantly enhances PET degradation.
• Surface display of PT-ECs on E. coli offers a promising strategy for efficient PET bio-recycling.
• Enzyme engineering and cascade system assembly are effective approaches for advancing plastic biodegradation.