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Abstract Directed evolution generates novel biomolecules with desired functions by iteratively diversifying the genetic sequence of wildtype biomolecules, relaying the genetic information to the molecule with function, and selecting the variants that progresses towards the properties of interest. While traditional directed evolution consumes significant labor and time for each step, continuous evolution seeks to automate all steps so directed evolution can proceed with minimum human intervention and dramatically shortened time. A major application of continuous evolution is the generation of novel enzymes, which catalyze reactions under conditions that are not favorable to their wildtype counterparts, or on altered substrates. The challenge to continuously evolve enzymes lies in automating sufficient, unbiased gene diversification, providing selection for a wide array of reaction types, and linking the genetic information to the phenotypic function. Over years of development, continuous evolution has accumulated versatile strategies to address these challenges, enabling its use as a general tool for enzyme engineering. As the capability of continuous evolution continues to expand, its impact will increase across various industries. In this review, we summarize the working mechanisms of recently developed continuous evolution strategies, discuss examples of their applications focusing on enzyme evolution, and point out their limitations and future directions. 

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.