Ultrahigh-Throughput Screening of an Artificial Metalloenzyme using Double Emulsions.

Autor: Vallapurackal J; Department of Chemistry, University of Basel, Mattenstrasse 24a, 4058, Basel, Switzerland.; National Competence Center in Research (NCCR) Molecular Systems Engineering, Basel, Switzerland., Stucki A; Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058, Basel, Switzerland.; National Competence Center in Research (NCCR) Molecular Systems Engineering, Basel, Switzerland., Liang AD; Department of Chemistry, University of Basel, Mattenstrasse 24a, 4058, Basel, Switzerland.; National Competence Center in Research (NCCR) Molecular Systems Engineering, Basel, Switzerland., Klehr J; Department of Chemistry, University of Basel, Mattenstrasse 24a, 4058, Basel, Switzerland.; National Competence Center in Research (NCCR) Molecular Systems Engineering, Basel, Switzerland., Dittrich PS; Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058, Basel, Switzerland.; National Competence Center in Research (NCCR) Molecular Systems Engineering, Basel, Switzerland., Ward TR; Department of Chemistry, University of Basel, Mattenstrasse 24a, 4058, Basel, Switzerland.; National Competence Center in Research (NCCR) Molecular Systems Engineering, Basel, Switzerland.
Jazyk: angličtina
Zdroj: Angewandte Chemie (International ed. in English) [Angew Chem Int Ed Engl] 2022 Nov 25; Vol. 61 (48), pp. e202207328. Date of Electronic Publication: 2022 Oct 27.
DOI: 10.1002/anie.202207328
Abstrakt: 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.
(© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)
Databáze: MEDLINE
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