State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; State Key Laboratory of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
J Hazard Mater. 2022 Feb 15;424(Pt B):127417. doi: 10.1016/j.jhazmat.2021.127417. Epub 2021 Oct 7.
Enzymes that can decompose synthetic plastics such as polyethylene terephthalate (PET) are urgently needed. Still, a bottleneck remains due to a lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation and incubation of droplets encapsulating single cells, picoinjection of fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with this method, including specificity and sensitivity for discriminating PETase from other enzymes. We then optimized its performance and compatibility with environmental samples. The system was used to screen a wastewater sample from a PET textile mill. We successfully obtained PET-degrading species from nine different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate that the FADS pipeline can be widely adopted to discover new plastic-degrading microorganisms and enzymes in various environments and may be utilized in the directed evolution of degrading enzymes using synthetic biology.
我们急需能够分解聚对苯二甲酸乙二醇酯（PET）等合成塑料的酶。然而，由于缺乏检测和分类具有广泛多样性和丰度的环境微生物的技术，这仍然是一个瓶颈。在这里，我们开发了一种荧光激活液滴分选（FADS）管道，用于高通量筛选 PET 降解微生物或酶（PETases）。该管道包括三个步骤：生成和孵育包裹单个细胞的液滴、皮考啉注射荧光素二苯甲酸酯（FDBz）作为荧光探针，以及筛选获得 PET 降解细胞的液滴。我们对与该方法相关的关键因素进行了表征，包括区分 PETase 和其他酶的特异性和灵敏度。然后，我们优化了其性能和与环境样本的兼容性。该系统用于筛选来自 PET 纺织厂的废水样本。我们成功地从九个不同的属中获得了具有 PET 降解能力的物种。此外，从内生菌 Kinococcus endophyticus Un-5 和表皮葡萄球菌 Un-C2-8 的分离物中获得了两种可能的 PETases，并对其进行了基因衍生、异源表达，并初步验证了其 PET 降解活性。我们推测，FADS 管道可以广泛应用于在各种环境中发现新的塑料降解微生物和酶，并可能用于使用合成生物学对降解酶进行定向进化。
