Abstract

Culture-Independent Method for Identification of Microbial Enzyme-Encoding Genes by Single-Cell Sequencing Using a Water-in-Oil Microdroplet Platform

Takashi Funatsu, Professor, University of Tokyo

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.