SMART platform for enzyme engineering: A novel single-molecule display approach for evolution and functional studies of D-amino acid oxidase

This study introduces SMART (Single Molecule Assay on Ribonucleic Acid by Translated Product), an innovative in vitro platform integrating mRNA display, next-generation sequencing, and bioinformatics to address several key challenges in enzyme engineering. The characteristic feature of SMART is its auxiliary unit, attached to the mRNA-displayed enzyme library to mediate the biotinylation of reactive enzyme variants and enable their enrichment by streptavidin beads pull-down. The unit comprises a hairpin single-stranded DNA that hybridizes with the mRNA and anchors a DNA-binding protein, single-chain Cro, which carries functional auxiliary molecules specific to the enzyme of interest. Here, we report on the establishment of SMART for the engineering of oxidases, specifically D-amino acid oxidase (DAAO) from Schizosaccharomyces pombe, with engineered ascorbate peroxidase 2 as its auxiliary enzyme. In a single selection round of a DAAO library with randomized catalytic residue Y232 and D-Alanine as the substrate, we identified several catalytically active enzyme variants with altered substrate specificity in a matter of days. This surpasses traditional enzyme engineering methods in speed, cost, library capacity, and precision. This research underscores the potential of SMART for the engineering of various oxidases, and other enzymes after the corresponding adjustment of the auxiliary unit.