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.

There has been much debate about the extent to which mutational epistasis, that is, the dependence of the outcome of a mutation on the genetic background, constrains evolutionary trajectories. The degree of unpredictability introduced by epistasis, due to the non-additivity of functional effects, strongly hinders the strategies developed in protein design and engineering. While many studies have addressed this issue through systematic characterization of evolutionary trajectories within individual enzymes, the field lacks a consensus view on this matter. In this work, we performed a comprehensive analysis of epistasis by analyzing the mutational effects from nine adaptive trajectories toward new enzymatic functions. We quantified epistasis by comparing the effect of mutations occurring between two genetic backgrounds: the starting enzyme (for example, wild type) and the intermediate variant on which the mutation occurred during the trajectory. We found that most trajectories exhibit positive epistasis, in which the mutational effect is more beneficial when it occurs later in the evolutionary trajectory. Approximately half (49%) of functional mutations were neutral or negative on the wild-type background, but became beneficial at a later stage in the trajectory, indicating that these functional mutations were not predictable from the initial starting point. While some cases of strong epistasis were associated with direct interaction between residues, many others were caused by long-range indirect interactions between mutations. Our work highlights the prevalence of epistasis in enzyme adaptive evolution, in particular positive epistasis, and suggests the necessity of incorporating mutational epistasis in protein engineering and design to create highly efficient catalysts.Keywords: epistasis; enzyme evolution; adaptive mutations; evolutionary constraints; directed evolution; protein engineering; rational design Abbreviations: DHFR, dihydrofolate reductase; MIC, minimum inhibitory concentration Additional Supporting Information may be found in the online version of this article.Significance Statement: Epistasis, that is, non-additive mutational effects, constrains the adaptive evolution of proteins, yet we lack a consensus view of its extent. To address this need, we performed a systematic survey of mutational epistasis occurring within nine adaptive enzyme trajectories. Of particular importance to protein engineering, our results quantify the unpredictability of functional mutations and emphasize the need to incorporate epistasis for the accurate prediction of mutational effects.

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