title: Droplet-based microfluidics for high-throughput screening of a metagenomic library for isolation of microbial enzymes.
abstract: This paper proposes a high-throughput, function-based screening approach of a metagenomic library for isolating novel microbial enzymes by droplet-based microfluidics. We used gel microdroplets (GMDs) dispersed in oil as picoliter-volume reaction vessels for lipolytic enzyme by encapsulating cells in individual GMDs. Using this approach, we monitored the growth of individual cells encapsulated in GMDs and assessed the enzyme reaction activities at the level of an individual GMD. We then applied this method to screen lipolytic enzyme genes from the metagenomic library constructed from soil collected from a quercus serrate forest of Mount Tsukuba, Ibaraki, Japan. In the workflow presented in this study, metagenomic library clones were encapsulated in 100-pL GMDs with a fluorogenic reporter substrate. A total of 67,000 metagenomic library clones can be screened in only 24 h with reduced consumption of reagents (i.e., <10 μL). As a result, we identified a novel lipolytic enzyme, EstT1, belonging to the EstD2 family of esterases and containing a putative signal peptide, which facilitates enzyme export and catalyzation of substrates in the periplasm. Our study demonstrates the potential of microfluidic GMDs as an efficient tool for metagenomic library screening of industrially relevant enzymes with the potential of significantly reducing the cost and time factors involved in successful practical application of microbial enzymes.
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; Institute for Nanoscience and Nanotechnology, Waseda University, 513, Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan; Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Hosokawa
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Hoshino
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Nishikawa
authors.affiliation: Faculty of Science and Engineering, Waseda University, 3-4-1, Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Tomotada
authors.initials: T
authors.lastname: Hirose
authors.affiliation: Faculty of Science and Engineering, Waseda University, 3-4-1, Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Dong Hyun
authors.initials: DH
authors.lastname: Yoon
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; Institute for Nanoscience and Nanotechnology, Waseda University, 513, Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan; Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Mori
authors.affiliation: Institute for Nanoscience and Nanotechnology, Waseda University, 513, Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sekiguchi
authors.affiliation: Institute for Nanoscience and Nanotechnology, Waseda University, 513, Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan; Faculty of Science and Engineering, Waseda University, 3-4-1, Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Shoji
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; Institute for Nanoscience and Nanotechnology, Waseda University, 513, Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan; Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan. Electronic address: haruko-takeyama@waseda.jp.
authors.forename: Haruko
authors.initials: H
authors.lastname: Takeyama
mesh_terms: D015374:Biosensing Techniques; D057166:High-Throughput Screening Assays; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification*; D056186:Metagenomics; D044085:Microfluidics; D012988:Soil Microbiology; D013379:Substrate Specificity
chemical_list: D008049:Lipase
keywords: Droplet; Lipolytic enzyme; Metagenomic library; Microfluidics; Single-cell analysis