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

Although many efforts have been devoted to the modification of polyethylene terephthalate (PET) hydrolases for improving the efficiency of PET degradation, the catalytic performance of these enzymes at near-ambient temperatures remains a challenge. Herein, a multi-enzyme cascade system (PT-EC) was developed and validated by assembling three well-developed PETases, PETase^EHA, Fast-PETase, and Z1-PETase, respectively, together with carboxylesterase TfCa, and hydrophobic binding module CBM3a using scaffold proteins. The resulting PT-EC^EHA, PT-EC^FPE, PT-EC^ZPE all demonstrated outstanding PET degradation efficacy. Notably, PT-EC^EHA exhibited a 16.5-fold increase in product release compared to PETase^EHA, and PT-EC^ZPE yielded the highest amount of product. Subsequently, PT-ECs were displayed on the surface of Escherichia coli, respectively, and their degradation efficiency toward three PET types was investigated. The displayed PT-EC^EHA exhibited a 20-fold increase in degradation efficiency with PET film compared to the surface-displayed PETase^EHA. Remarkably, an almost linear increase in product release was observed for the displayed PT-EC^ZPE over a one-week degradation period, reaching 11.56 ± 0.64 mM after 7 days. TfCa^I69W/L281Y evolved using a docking-based virtual screening strategy showed a further 2.5-fold increase in the product release of PET degradation. Collectively, these advantages of PT-EC demonstrated the potential of a multi-enzyme cascade system for PET bio-cycling.