Development of a Vibrio natriegens -Based Plate-Clearing Assay for Rapid Screening of PET-Hydrolyzing Enzymes

Abstract

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 using Vibrio natriegens, exploiting its protein secretion system to rapidly identify catalytic activity without time-consuming downstream processing. To validate the assay’s robustness, we tested variants of Fusarium solanipisi cutinase (FsC) and found that one variant, T45P, exhibited 3-fold higher PET-film degradation yield than the wild type and favored the production of terephthalic acid. As a further test case, we screened an error-prone polymerase chain reaction (PCR) library of FsC using the assay, obtaining a 25% hit rate after screening 150 colonies. This demonstrates the scalability of the method to large enzyme libraries.