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"NAD-display": Ultrahigh-Throughput in Vitro Screening of NAD(H) Dehydrogenases Using Bead Display and Flow Cytometry.

Accepted version
Peer-reviewed

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Type

Article

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Authors

Lindenburg, Laurens 
Hollfelder, Florian  ORCID logo  https://orcid.org/0000-0002-1367-6312

Abstract

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.

Description

Keywords

cell-free expression, directed evolution, formate dehydrogenase, saturation library, water-in-oil emulsion droplets, Flow Cytometry, Formate Dehydrogenases, High-Throughput Screening Assays, NAD

Journal Title

Angew Chem Int Ed Engl

Conference Name

Journal ISSN

1433-7851
1521-3773

Volume Title

60

Publisher

Wiley

Rights

All rights reserved
Sponsorship
Engineering and Physical Sciences Research Council (EP/H046593/1)
Engineering and Physical Sciences Research Council (EP/D048664/1)
Engineering and Physical Sciences Research Council (EP/C013174/1)
European Commission (659029)
EPSRC, EU H2020