Droplet-based microfluidics for high-throughput screening of a metagenomic library for isolation of microbial enzymes

Masahito Hosokawa, Yuri Hoshino, Yohei Nishikawa, Tomotada Hirose, Donghyun Yoon, Tetsushi Mori, Tetsushi Sekiguchi, Shuichi Shoji, Haruko Takeyama

Research output: Contribution to journalArticle

44 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)379-385
Number of pages7
JournalBiosensors and Bioelectronics
Volume67
DOIs
Publication statusPublished - 2015 May 5

Fingerprint

Metagenomics
Microfluidics
Screening
Enzymes
Throughput
Gels
Clone Cells
Quercus
Periplasm
Workflow
Substrates
Esterases
Protein Sorting Signals
Gene Library
Fluorescent Dyes
Japan
Oils
Soil
Genes
Cells

Keywords

  • Droplet
  • Lipolytic enzyme
  • Metagenomic library
  • Microfluidics
  • Single-cell analysis

ASJC Scopus subject areas

  • Biophysics
  • Biomedical Engineering
  • Biotechnology
  • Electrochemistry

Cite this

Droplet-based microfluidics for high-throughput screening of a metagenomic library for isolation of microbial enzymes. / Hosokawa, Masahito; Hoshino, Yuri; Nishikawa, Yohei; Hirose, Tomotada; Yoon, Donghyun; Mori, Tetsushi; Sekiguchi, Tetsushi; Shoji, Shuichi; Takeyama, Haruko.

In: Biosensors and Bioelectronics, Vol. 67, 05.05.2015, p. 379-385.

Research output: Contribution to journalArticle

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