An ultrahigh-throughput screening platform based on flow cytometric droplet sorting for mining novel enzymes from metagenomic libraries
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:23 |
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| Enthalten in: |
Environmental microbiology - 23(2021), 2 vom: 02. Feb., Seite 996-1008 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Ma, Fuqiang [VerfasserIn] |
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| Links: |
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| Themen: |
Emulsions |
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| Anmerkungen: |
Date Completed 21.05.2021 Date Revised 21.05.2021 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1111/1462-2920.15257 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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NLM31556461X |
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| 245 | 1 | 3 | |a An ultrahigh-throughput screening platform based on flow cytometric droplet sorting for mining novel enzymes from metagenomic libraries |
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| 500 | |a Date Revised 21.05.2021 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © 2020 Society for Applied Microbiology and John Wiley & Sons Ltd. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a Emulsions |2 NLM | |
| 650 | 7 | |a Enzymes |2 NLM | |
| 700 | 1 | |a Guo, Tianjie |e verfasserin |4 aut | |
| 700 | 1 | |a Zhang, Yifan |e verfasserin |4 aut | |
| 700 | 1 | |a Bai, Xue |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Changlong |e verfasserin |4 aut | |
| 700 | 1 | |a Lu, Zelin |e verfasserin |4 aut | |
| 700 | 1 | |a Deng, Xi |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Daixi |e verfasserin |4 aut | |
| 700 | 1 | |a Kurabayashi, Katsuo |e verfasserin |4 aut | |
| 700 | 1 | |a Yang, Guang-Yu |e verfasserin |4 aut | |
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