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Single-Cell Biophysics: Measurement, Modulation, and Modeling
Poster Abstracts
56
23-POS
Board 12
Culture-Independent Method for Identification of Microbial Enzyme-Encoding Genes by
Single-Cell Sequencing Using a Water-in-oil Microdroplet Platform
Kazuki Nakamura
1
, Ryo Iizuka
1
, Shinro Nishi
2
, Takao Yoshida
2
, Yuji Hatada
2
, Yoshihiro
Takaki
2
, Ayaka Iguchi
3
, Dong H. Yoon
3
, Tetsushi Sekiguchi
3
, Shuichi Shoji
3
,
Takashi
Funatsu
1
.
1
The University of Tokyo, Tokyo, Japan,
2
Japan Agency for Marine-Earth Science and
Technology, Kanagawa, Japan,
3
Waseda University, Tokyo, Japan.
Environmental microbes are a major source of industrially valuable enzymes with potent and
unique catalytic activities. Unfortunately, the majority of microbes remain unculturable or
difficult to cultivate 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 exhibiting specific enzymatic activities. First, 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 at the single cell level.
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.
This method was successfully used to identify 14 novel beta-glucosidase genes from uncultured
bacterial cells in marine samples. Our method contributes to the screening and identification of
genes encoding industrially valuable enzymes.