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Abstract
Directed evolution has long been a key strategy to generate enzymes with desired properties like high selectivity, but experimental barriers and analytical costs of screening enormous mutant libraries have limited such efforts. Here, we describe an ultrahigh-throughput dual-channel microfluidic droplet screening system that can be used to screen up to ~107 enzyme variants per day. As an example case, we use the system to engineer the enantioselectivity of an esterase to preferentially produce desired enantiomers of profens, an important class of anti-inflammatory drugs. Using two types of screening working modes over the course of five rounds of directed evolution, we identify (from among 5 million mutants) a variant with 700-fold improved enantioselectivity for the desired (S)-profens. We thus demonstrate that this screening platform can be used to rapidly generate enzymes with desired enzymatic properties like enantiospecificity, chemospecificity, and regiospecificity.
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Details
1 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
2 Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA
3 MIIT Key Laboratory of Critical Materials Technology for New Energy Conversion and Storage, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, China
4 Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA
5 Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA; Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, MI, USA