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bioRxiv · 10.1101/2025.04.02.646774

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

Bolstad, I. M.·Trooyen, S. H.·Luciano, D.·Petersen, E.·Courtade, G.
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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 assays robustness, we tested mutants of Fusarium solani pisi cutinase (FsC) and found that one mutant, T45P, exhibited threefold higher activity and a TPA-to-MHET ratio than the wild-type. As a further test case, we screened an error-prone PCR library of FsC using the assay, obtaining a 25% hit rate after screening 150 colonies. This demonstrates the scalability of the method for screening PET-hydrolytic activity in a large number of enzymes.

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Bolstad, I. M., Trooyen, S. H., Luciano, D., Petersen, E., Courtade, G.. 2025-04-02. Development of a Vibrio natriegens-based plate-clearing assay for rapid screening of PET-hydrolyzing enzymes. https://doi.org/10.1101/2025.04.02.646774

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