Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments

Novel and improved biocatalysts are increasingly sourced from libraries via experimental screening. The success of such campaigns is crucially dependent on the number of candidates tested. Water-in-oil emulsion droplets can replace the classical test tube, to provide in vitro compartments as an alternative screening format, containing genotype and phenotype and enabling a readout of function. The scale-down to micrometer droplet diameters and picoliter volumes brings about a >107-fold volume reduction compared to 96-well-plate screening. Droplets made in automated microfluidic devices can be integrated into modular workflows to set up multistep screening protocols involving various detection modes to sort >107 variants a day with kHz frequencies. The repertoire of assays available for droplet screening covers all seven enzyme commission (EC) number classes, setting the stage for widespread use of droplet microfluidics in everyday biochemical experiments. We review the practicalities of adapting droplet screening for enzyme discovery and for detailed kinetic characterization. These new ways of working will not just accelerate discovery experiments currently limited by screening capacity but profoundly change the paradigms we can probe. By interfacing the results of ultrahigh-throughput droplet screening with next-generation sequencing and deep learning, strategies for directed evolution can be implemented, examined, and evaluated.