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.
