“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 al arger swath of sequence space,w e demonstrate the utility of ac ell-free,u ltrahigh-throughput directed evolution platform for dehydrogenases.M icrobeads (1.5 million per sample) carrying both variant DNAa nd an immobilised analogue of NAD + were compartmentalised in water-in-oil emulsion droplets,t ogether with cell-free expression mixture and enzyme substrate,r esulting in the recording of the phenotype on each bead. The beads phenotype could be read out and sorted for on aflowc ytometer by using ahighly sensitive fluorescent protein-based sensor of the NAD + :NADH ratio.I ntegration of this "NAD-display" approach with our previously described Split &M ix (SpliMLiB) method for generating large site-saturation libraries allowed straightforward screening of fully balanced site saturation libraries of formate dehydrogenase,w ith diversities of 2 10 4 .B ased on modular design principles of synthetic biology NAD-display offers access to sophisticated in vitro selections,a voiding complex technology platforms.

Ultrahigh Throughput Evolution of Tryptophan Synthase in Droplets via an Aptamer Sensor

“…Short oligonucleotides, or aptamers, can be evolved through SELEX to bind any small molecule of interest. − Numerous methods exist to engineer the resulting binder into a sensor suitable for uHTS: RNA-based aptamers can be engineered into a sensor through fusion with the “Spinach” probe, which adapts its secondary structure when the original aptamer binds the target analyte, rendering it fluorescent. , For DNA-based aptamers, a sensor can be engineered by fluorescently labeling the aptamer while designing a complementary strand with a quencher . When the target analyte is present, the complementary strand is displaced in favor of the target analyte, resulting in a fluorescence increase.…”

“…Protein sensors require engineering a conditional inactive state that can be reversed by binding a molecule of interest . For example, a protein sensor was engineered to detect the redox state of NAD, thus enabling the screening of dehydrogenases on nonfluorogenic substrates in droplets by fluorescent activated cell sorting (FACS) …”

“… 13 For example, a protein sensor was engineered to detect the redox state of NAD, thus enabling the screening of dehydrogenases on nonfluorogenic substrates in droplets by fluorescent activated cell sorting (FACS). 14 …”

Ultrahigh throughput evolution of tryptophan synthase in droplets via an aptamer-biosensor

“…11 For example, a protein sensor was engineered to detect the redox state of NAD, thus enabling screening of dehydrogenases on non-fluorogenic substrates in droplets by fluorescent activated cell sorting (FACS). 12 Short oligonucleotides, or aptamers, can be evolved through SELEX to bind any small molecule of interest. [13][14][15] .…”

“…11 For example, a protein sensor was engineered to detect the redox state of NAD, thus enabling screening of dehydrogenases on non-fluorogenic substrates in droplets by fluorescent activated cell sorting (FACS). 12…”