Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics.

Pierre-Yves Colin,Charlotte M Miton,Dick B Janssen,Fabrice Gielen,Gerhard Fischer,Diego P Morgavi,Marko Hyvönen,Florian Hollfelder,Mark F Mohamed,Balint Kintses

doi:10.1038/ncomms10008

Abstract

Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions.

Highlights

Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor
Functional annotation is further complicated by increasing evidence for catalytic promiscuity
Library members were expressed in Escherichia coli and single cells were compartmentalized into monodisperse 2 pl droplets

Summary

Introduction

Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Microfluidic droplets have been successfully used for directed evolution[25,26,27] strain selections[28,29] or bioprospecting[30] We employ such droplets as vessels to miniaturize cell lysate assays27—the most frequently used screening formats in metagenomics19,31—to the single-cell level. A microfluidic screening platform is used to recover and identify various hydrolases from metagenomic sources by screening for the hydrolysis of two substrates: sulfate monoesters, containing one of the most unreactive functional groups in biology[32], and phosphate triesters The latter are non-natural environmental pollutants historically used as pesticides[33] that we use to probe the catalytic potential of microbial communities for degradation of xenobiotics. The high sensitivity of our platform enabled us to isolate enzymes with strong and weak activities, including promiscuous side activities that have not been selected in the course of evolution and could not be predicted by sequence homology

Methods

Results

Conclusion