A Dual Fluorescence Assay Enables High‐Throughput Screening for Poly(ethylene terephthalate) Hydrolases

Abstract The drastically increasing consumption of petroleum‐derived plastics hasserious environmental impacts and raises public concerns. Poly(ethylene terephthalate) (PET) is amongst the most extensively produced synthetic polymers. Enzymatic hydrolysis of PET recently emerged as an enticing path for plastic degradation and recycling. In‐lab directed evolution has revealed the great potential of PET hydrolases (PETases). However, the time‐consuming and laborious PETase assays hinder the identification of effective variants in large mutant libraries. Herein, we devise and validate a dual fluorescence‐based high‐throughput screening (HTS) assay for a representative Is PETase. The two‐round HTS of a pilot library consisting of 2850 Is PETase variants yields six mutant Is PETases with 1.3–4.9 folds improved activities. Compared to the currently used structure‐ or computational redesign‐based PETase engineering, this HTS approach provides a new strategy for discovery of new beneficial mutation patterns of PETases.