Development of aVibrio natriegens-based plate-clearing assay for rapid screening of PET-hydrolyzing enzymes

Polyethylene terephthalate (PET) is a major contributor to plastic waste. Enzymatic PET degradation offers a sustainable recycling approach, but screening for effective enzymes remains challenging. Herein, we established an experimental plate-clearing assay withVibrio natriegens, exploiting its protein-secretion ability to rapidly identify catalytic activity without time-consuming downstream processing. To validate the assay’s robustness, we tested mutants ofFusarium solani pisicutinase (FsC) and discovered a highly active FsC mutant, T45P, with three-fold higher activity and TPA-to-MHET ratio compared to the wildtype. As a further test case, we designed novel protein structures by scaffolding functional sites from the PET-degrading enzyme LCC-ICCG using the machine-learning models RFdiffusion and ProteinMPNN. Although we successfully produced onede novoprotein, we did not detect catalytic activity on PET or BHET, nor did we detect substrate binding ability. This work provides a framework for experimental screening of PET-hydrolytic activity through a robustV. natriegens-based assay system.