title: Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics.
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.
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: Pierre-Yves
authors.initials: PY
authors.lastname: Colin
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: Balint
authors.initials: B
authors.lastname: Kintses
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Gielen
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: Charlotte M
authors.initials: CM
authors.lastname: Miton
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Fischer
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: Mark F
authors.initials: MF
authors.lastname: Mohamed
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: Marko
authors.initials: M
authors.lastname: Hyvönen
authors.affiliation: INRA, UMR1213 Herbivores, F-63122 Saint-Genès-Champanelle, France.|Clermont Université, VetAgro Sup, UMR Herbivores, BP 10448, F-63000 Clermont-Ferrand, France.
authors.forename: Diego P
authors.identifier: http://orcid.org/0000-0002-3883-0937
authors.initials: DP
authors.lastname: Morgavi
authors.affiliation: Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: Florian
authors.identifier: http://orcid.org/0000-0002-1367-6312
authors.initials: F
authors.lastname: Hollfelder
mesh_terms: D001419:Bacteria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004798:Enzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D056186:Metagenomics; D011487:Protein Conformation; D012988:Soil Microbiology
chemical_list: D001426:Bacterial Proteins; D004798:Enzymes