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.

Figure 1. Schematic workflow for identifying microbial enzyme-encoding genes by activity-based single-cell sequencing using microdroplets.

(a) The workflow includes single microbial cell isolation in W/O microdroplets (step 1), activity-based single-cell screening and recovery of target cells (step 2), whole genome amplification (step 3) and genome sequencing (step 4). Genes encoding the target enzymes are identified based on genomic information (step 5). The entire process could be completed in 4–5 days. (b) Schematic representation of single microbial cell isolation in W/O microdroplets using a microfluidic device. (c) Schematic representation of activity-based single-cell screening and recovery of target cells.

Figure 2. Isolation and genome amplification of bacteria exhibiting BGL activities from surface and deep seawater.

(a) Bright-field (left) and fluorescence (right) images of W/O microdroplets encapsulating environmental bacteria with FDGlu. The white arrowhead shows a fluorescent bacterial cell in a W/O microdroplet. Scale bar represents 20 μm. (b) PCR amplification of 16S rRNA genes from MDA products. The amplicons were analysed with 1% agarose gel electrophoresis and stained with SYBR Safe. The estimated amplicon size is approximately 1,466 bp. Lane M, DNA marker (λ-EcoT14 I digest); lane 1–4, MDA products from surface seawater (Droplet No. 1–4), lane 5–9, MDA products from deep seawater (Droplet No. 5–9).