Cell-free Directed Evolution of a Protease in Microdroplets at Ultrahigh Throughput

[Image: see text] Compartmentalization of single genes in water-in-oil emulsion droplets is a powerful approach to create millions of reactors for enzyme library selections. When these droplets are formed at ultrahigh throughput in microfluidic devices, their perfect monodispersity allows quantitative enzyme assays with a high precision readout. However, despite its potential for high quality cell-free screening experiments, previous demonstrations of enrichment have never been successfully followed up by actual enzyme library selections in monodisperse microfluidic droplets. Here we develop a three-step workflow separating three previously incompatible steps that thus far could not be carried out at once: first droplet-compartmentalized DNA is amplified by rolling circle amplification; only after completion of this step are reagents for in vitro protein expression and, finally, substrate added via picoinjection. The segmented workflow is robust enough to allow the first in vitro evolution in droplets, improving the protease Savinase that is toxic to E. coli for higher activity and identifying a 5-fold faster enzyme.