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.

Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms

“…respectively used optoelectronic or optical tweezers to push human ovarian cancer cells or red blood cells out of the microfluidic device and into a collection tube; Nakamura et al . used a micromanipulator (CellTram Vario, Eppendorf) to pick up droplets encapsulating single environmental microbes and transfer them into PCR tubes28. Expensive commercial equipment played a key role for single cell export in these studies.…”

“…Cells were recovered from the droplets by votex and centrifugation at approximately 4,000 rpm as described in literature44. All tubes were cultivated at 30 °C aerobically while shaking at 180 rpm.…”

Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside

“…Using the metagenomics technique microbial genes from diverse environments without cultivable technology can be directly obtained [16,17]. Recently, β-glucosidase genes from environmental samples have been obtained via metagenomics technology [18][19][20]. The sources of uncultured microbes from which β-glucosidase genes have been isolated are mainly populated in the sea [20], compost [21], rumen and cecum [22,23], soil [19], spring water [24], and sewage [25].…”

“…Recently, β-glucosidase genes from environmental samples have been obtained via metagenomics technology [18][19][20]. The sources of uncultured microbes from which β-glucosidase genes have been isolated are mainly populated in the sea [20], compost [21], rumen and cecum [22,23], soil [19], spring water [24], and sewage [25]. A few glycosidase hydrolase family 1 (GH1) β-glucosidases from extreme thermal environments have shown the highest activity at 90 • C [24,26].…”

Metaproteogenomics-guided enzyme discovery: Targeted identification of novel proteases in microbial communities

“…To find these possible ideal enzymes in the uncultivated microbial biomass there has been increased interest for more pure-culture independent methods for enzyme discovery [80].…”