基于荧光纳米探针的聚对苯二甲酸乙二醇酯水解酶高通量液滴微流控筛选

High-throughput fluorescence-activated droplet sorting of polyethylene terephthalate hydrolases based on fluorescent nanoparticle biosensors

摘要:

聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)是全球产量最大的合成聚酯之一，其废弃物难以降解，导致了严重的环境污染。酶法降解被认为是实现PET绿色循环利用的理想途径之一，但天然PET水解酶催化活性有限，且传统筛选方法存在通量低、耗时长等瓶颈。为突破传统筛选方法在通量和效率上的限制，本研究构建了一种兼具底物真实性与高检测灵敏度的荧光纳米探针技术，并在超高通量荧光激活液滴分选(fluorescence-activated droplet sorting, FADS)平台实现整合，用于PET水解酶的单细胞水平筛选与定向进化。通过将荧光素二月桂酸酯(fluorescein dilaurate, FDL)包埋于PET基质中，制备了PET-FDL NPs荧光纳米探针。结合大肠杆菌表面展示技术，构建了mScarletI-叶枝堆肥角质酶(leaf-branch compost cutinase, LCC)融合蛋白表达体系，并建立了基于荧光素/mScarletI双荧光比值的定量评价方法。利用FADS技术对易错PCR (error-prone PCR, epPCR)构建的LCC突变库进行了筛选，筛选通量可达10⁷个液滴/d。微孔板复筛结果表明，该策略将阳性克隆的命中率由传统平板筛选的约5%提升至44%，形成了面向PET水解酶定向进化的稳健、可扩展筛选流程。

Abstract:

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.