Ultrahigh-Throughput Screening of an Artificial Metalloenzyme using Double Emulsions
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Date
2022-11-25
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Journal Article
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yes
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Abstract
The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage, enabling reliable identification of improved mutants. Herein, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of an alloc-protected aminocoumarin. The most active variants, identified by next-generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of (artificial) enzymes and will significantly expand the accessibility of ultrahigh-throughput DE screening protocols.
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published
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Volume
61 (48)
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Publisher
Wiley-VCH
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Subject
Allylic Deallylation; Artificial Metalloenzymes; Directed Evolution; Droplet Microfluidics; High-Throughput Screening
Organisational unit
03807 - Dittrich, Petra / Dittrich, Petra
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Funding
681587 - Engineering of hybrid cells using lab-on-chip technology (EC)