An ultrahigh‐throughput screening platform based on flow cytometric droplet sorting for mining novel enzymes from metagenomic libraries
2021
Ma, Fuqiang | Guo, Tianjie | Zhang, Yifan | Bai, Xue | Li, Changlong | Lu, Zelin | Deng, Xi | Li, Daixi | Kurabayashi, Katsuo | Yang, Guang‐yu
Uncultivable microbial communities provide enormous reservoirs of enzymes, but their experimental identification by functional metagenomics is challenging, mainly due to the difficulty of screening enormous metagenomic libraries. Here, we propose a reliable and convenient ultrahigh‐throughput screening platform based on flow cytometric droplet sorting (FCDS). The FCDS platform employs water‐in‐oil‐in‐water double emulsion droplets serving as single‐cell enzymatic micro‐reactors and a commercially available flow cytometer, and it can efficiently isolate novel biocatalysts from metagenomic libraries by processing single cells as many as 10⁸ per day. We demonstrated the power of this platform by screening a metagenomic library constructed from domestic running water samples. The FCDS assay screened 30 million micro‐reactors in only 1 h, yielding a collection of esterase genes. Among these positive hits, Est WY was identified as a novel esterase with high catalytic efficiency and distinct evolutionary origin from other lipolytic enzymes. Our study manifests that the FCDS platform is a robust tool for functional metagenomics, with the potential to significantly improve the efficiency of exploring novel enzymes from nature.
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