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


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Authors

Colin, Pierre-Yves 
Kintses, Balint 
Gielen, Fabrice 
Miton, Charlotte M 
Fischer, Gerhard 

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.

Description

Keywords

Bacteria, Bacterial Proteins, Enzymes, Gene Library, Metagenomics, Protein Conformation, Soil Microbiology

Journal Title

Nat Commun

Conference Name

Journal ISSN

2041-1723
2041-1723

Volume Title

6

Publisher

Springer Science and Business Media LLC
Sponsorship
Biotechnology and Biological Sciences Research Council (BB/L002469/1)
Engineering and Physical Sciences Research Council (EP/H046593/1)
Biotechnology and Biological Sciences Research Council (BB/I004327/1)
This research was funded by the Engineering and Physical Sciences Research Council (EPSRC) and the Biological and Biotechnological Research Council (BBSRC). FH is an ERC Starting Investigator. PYC holds a fellowship of the EU ITN PhosChemRec, CM of the ITN ProSA and ENEFP. BK and MM were supported by postdoctoral Marie-Curie fellowships. The authors thank Sean Devenish and Nobuhiko Tokuriki for useful comments on the manuscript and Gabrielle Potocki-Veronese and Stephane Emond for help with the design of the library strategy. We thank Raindance for the gift of EA surfactant.