title: Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform.
abstract: Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped genetic resources. Here we present a methodological approach for the identification of genes that encode metabolically active enzymes in environmental microbes in a culture-independent manner. Our method is based on activity-based single-cell sequencing, which focuses on microbial cells showing specific enzymatic activities. First, at the single-cell level, environmental microbes were encapsulated in water-in-oil microdroplets with a fluorogenic substrate for the target enzyme to screen for microdroplets that contain microbially active cells. Second, the microbial cells were recovered and subjected to whole genome amplification. Finally, the amplified genomes were sequenced to identify the genes encoding target enzymes. Employing this method, we successfully identified 14 novel β-glucosidase genes from uncultured bacterial cells in marine samples. Our method contributes to the screening and identification of genes encoding industrially valuable enzymes.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nakamura
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Iizuka
authors.affiliation: Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka-shi, Kanagawa 237-0061, Japan.
authors.forename: Shinro
authors.initials: S
authors.lastname: Nishi
authors.affiliation: Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka-shi, Kanagawa 237-0061, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Yoshida
authors.affiliation: Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka-shi, Kanagawa 237-0061, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hatada
authors.affiliation: Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka-shi, Kanagawa 237-0061, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Takaki
authors.affiliation: Department of NanoscieWnce and Nanoengineering (ASE Graduate School), Waseda University, 3-4-1 Okubo, Shinju-ku, Tokyo 169-8555, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Iguchi
authors.affiliation: Department of NanoscieWnce and Nanoengineering (ASE Graduate School), Waseda University, 3-4-1 Okubo, Shinju-ku, Tokyo 169-8555, Japan.
authors.forename: Dong Hyun
authors.initials: DH
authors.lastname: Yoon
authors.affiliation: Research Organization for Nano &Life Innovation, Waseda University, 513, Waseda-tsurumaki-cho, Shinjuku-ku, Tokyo, 162-0041, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sekiguchi
authors.affiliation: Department of NanoscieWnce and Nanoengineering (ASE Graduate School), Waseda University, 3-4-1 Okubo, Shinju-ku, Tokyo 169-8555, Japan.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Shoji
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Funatsu
mesh_terms: D001419:Bacteria / Q000145:classification / Q000166:cytology / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001431:Bacteriological Techniques; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009821:Oils / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012623:Seawater / Q000382:microbiology; D017422:Sequence Analysis, DNA / Q000379:methods; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*; D014867:Water / Q000737:chemistry; D014871:Water Microbiology / Q000592:standards; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D009821:Oils; D012336:RNA, Ribosomal, 16S; D014867:Water; D001617:beta-Glucosidase