Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop‐Based Microfluidics

Abstract Enzymes are highly specific catalysts delivering improved drugs and greener industrial processes. Naturally occurring enzymes must typically be optimized which is often accomplished through directed evolution; however, this is still a labor‐ and capital‐intensive process, due in part to multiple molecular biology steps including DNA extraction, in vitro library generation, transformation, and limited screening throughput. We present an effective and broadly applicable continuous evolution platform that enables controlled exploration of fitness landscape to evolve enzymes at ultrahigh throughput based on direct measurement of enzymatic activity. This drop‐based microfluidics platform cycles cells between growth and mutagenesis followed by screening with minimal human intervention, relying on the nCas9 chimera with mutagenesis polymerase to produce in vivo gene diversification using sgRNAs tiled along the gene. We evolve alditol oxidase to change its substrate specificity towards glycerol, turning a waste product into a valuable feedstock. We identify a variant with a 10.5‐fold catalytic efficiency.