Ultrahigh-Throughput Screening of an Artificial Metalloenzyme using Double Emulsions

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage throughout optimization, enabling reliable identification of improved mutants. The full potential of droplet microfluidics remains unexplored, however, as droplet sorting often relies on low-throughput, custom-made devices that typically only allow simultaneous analysis of two parameters. Here, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially-available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of allocprotected aminocoumarin. The most active variants, identified by next generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of enzymes and will significantly expand the accessibility of ultrahighthroughput DE screening protocols.