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, enabling reliable identification of improved mutants. Herein, 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 an alloc‐protected 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 (artificial) enzymes and will significantly expand the accessibility of ultrahigh‐throughput DE screening protocols. An artificial metalloenzyme was screened in vivo using a high‐throughput microfluidics‐based assay. Single E. coli cells expressing streptavidin were encapsulated in double emulsion droplets together with a fluorogenic substrate and a cofactor catalyzing the reaction to yield a fluorescent product. Using this method, a 400‐member library was screened, and the same hits found previously in a 96‐well plate assay were identified.