[High-throughput fluorescence-activated droplet sorting of polyethylene terephthalate hydrolases based on fluorescent...

Polyethylene terephthalate (PET) is one of the most widely produced synthetic plastics globally, posing serious environmental challenges due to its resistance to natural degradation. Enzymatic degradation offers a sustainable solution for PET recycling. However, natural PET hydrolases often suffer from limited catalytic efficiency, and existing screening methods are labor-intensive with low throughput. To overcome the limitations of conventional methods in throughput and efficiency, this study developed a fluorescent nanoprobe technology combining substrate authenticity with high detection sensitivity, which was integrated into an ultra-high-throughput fluorescence-activated droplet sorting (FADS) platform for the single-cell screening and directed evolution of PET hydrolases. Fluorescein dilaurate (FDL)-loaded PET nanoparticles (PET-FDL NPs) were synthesized as specific fluorogenic probes. By combining Escherichia coli surface display of mScarletI-leaf-branch compost cutinase (LCC) fusion proteins with a dual-fluorescence ratiometric assay (fluorescein/mScarletI), we achieved precise normalization of enzymatic activity against expression variations. An LCC mutant library generated by error-prone PCR (epPCR) was screened by FADS at a throughput of 10⁷ droplets per day. The results showed that FADS enrichment significantly increased the proportion of positive droplets. Subsequent microplate rescreening revealed that this strategy improved the positive hit rate from ~5% (plate screening) to 44%, establishing a robust and scalable workflow for directed evolution of PET hydrolases.