"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 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.