“NAD‐display”: Ultrahigh‐Throughput in Vitro Screening of NAD(H) Dehydrogenases Using Bead Display and Flow Cytometry

NAD(H)‐utiliing enzymes have been the subject of directed evolution campaigns to improve their function. To enable access to a larger swath of sequence space, we demonstrate the utility of a cell‐free, ultrahigh‐throughput directed evolution platform for dehydrogenases. Microbeads (1.5 million per s...

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Veröffentlicht in:Angewandte Chemie International Edition Jg. 60; H. 16; S. 9015 - 9021
Hauptverfasser: Lindenburg, Laurens, Hollfelder, Florian
Format: Journal Article
Sprache:Englisch
Veröffentlicht: Germany Wiley Subscription Services, Inc 12.04.2021
John Wiley and Sons Inc
Ausgabe:International ed. in English
Schlagworte:
Beads
Biological evolution
cell-free expression
Dehydrogenases
Directed evolution
Flow cytometry
Fluorescence
Formate dehydrogenase
Libraries
Microspheres
Modular design
NAD
NADH
Nanoparticles
Nicotinamide adenine dinucleotide
Phenotypes
Saturation
saturation library
Screening
Substrates
water-in-oil emulsion droplets
cell-free expression
formate dehydrogenase
water-in-oil emulsion droplets
saturation library
directed evolution
ISSN:1433-7851, 1521-3773, 1521-3773
Online-Zugang:Volltext
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Zusammenfassung:NAD(H)‐utiliing enzymes have been the subject of directed evolution campaigns to improve their function. To enable access to a larger swath of sequence space, we demonstrate the utility of a cell‐free, ultrahigh‐throughput directed evolution platform for dehydrogenases. Microbeads (1.5 million per sample) carrying both variant DNA and an immobilised analogue of NAD+ were compartmentalised in water‐in‐oil emulsion droplets, together with cell‐free expression mixture and enzyme substrate, resulting in the recording of the phenotype on each bead. The beads’ phenotype could be read out and sorted for on a flow cytometer by using a highly sensitive fluorescent protein‐based sensor of the NAD+:NADH ratio. Integration of this “NAD‐display” approach with our previously described Split & Mix (SpliMLiB) method for generating large site‐saturation libraries allowed straightforward screening of fully balanced site saturation libraries of formate dehydrogenase, with diversities of 2×104. Based on modular design principles of synthetic biology NAD‐display offers access to sophisticated in vitro selections, avoiding complex technology platforms. A detection system was devised to screen dehydrogenase enzymes (DH) at ultrahigh‐throughput in in vitro droplet compartments. An NAD(H) analogue and a DH DNA library were co‐immobilised on beads. Water‐in‐oil emulsion droplets containing cell‐free expression mix and substrate allow compartmentalised protein production and catalysis. A fluorescent sensor of NAD(H) redox state was loaded onto beads, allowing bulk sorting of 2×104 DH variants in a day by flow cytometry.
Bibliographie:ObjectType-Article-1
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ISSN:1433-7851
1521-3773
1521-3773
DOI:10.1002/anie.202013486