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

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 descr

SUBMITTER: Lindenburg L 

PROVIDER: S-EPMC8048591 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature