Harnessing the Power of SMART Single-Molecule Display for Enzyme Evolution: A Focus on Oxidase.
For a rapid and cost-effective evolution of tailor-made enzymes, we established a high-throughput  in vitro  selection platform named SMART (Single-Molecule Assay on Ribonucleic acid by Translated product), integrating mRNA display, next-generation sequencing, and bioinformatics. SMART represents a versatile system where a module termed an auxiliary unit allows enzyme-specific selection under various experimental conditions. Here, we report on the establishment of SMART for oxidases using a model enzyme,  Schizosaccharomyces pombe  d-amino acid oxidase (SpDAAO), and ascorbate peroxidase 2 as the auxiliary enzyme to detect hydrogen peroxide produced by the oxidase, and mediate biotinylation of active single-molecule display complexes. As a proof-of-concept, a library including site-saturation mutagenesis at the catalytic residue Y232 of SpDAAO was subjected to a single SMART selection round, yielding enrichment of the active enzyme variant. The results demonstrate the utility of SMART as a fast, robust, and efficient platform with the potential of customization for other enzyme chemistries through appropriate modifications of the auxiliary unit. Using SMART, desired enzyme variants can be selected in just a few hours by a single person without the need for costly equipment or any bias or limitations.
Munaweera K, Odake N, Halim HP, Ikeda K, Zhu B, Camagna M, Ito T, Kitaguchi T, Nemoto N, Nakano H, Damnjanović J