Directed Evolution of a Surface-Displayed Artificial Allylic Deallylase Relying on a GFP Reporter Protein

Artificial metalloenzymes (ArMs) combine characteristics of both homogeneous catalysts and enzymes. Merging abiotic biotic features allows for the implementation new-to-nature reactions in living organisms. Here, we present directed evolution an artificial metalloenzyme based on Escherichia coli surface-displayed streptavidin (SavSD hereafter). Through binding a ruthenium-pianostool cofactor to SavSD, allylic deallylase (ADAse hereafter) is assembled, which displays catalytic activity toward deprotection alloc-protected 3-hydroxyaniline. The uncaged aminophenol acts as gene switch triggers overexpression fluorescent green protein (GFP) reporter protein. This straightforward readout ADAse allowed simultaneous saturation mutagenesis two amino acid residues Sav near ruthenium cofactor, expediting screening 2762 individual clones. A 1.7-fold increase vivo was observed SavSD S112T-K121G compared wild-type (wt-SavSD). Finally, best performing isoforms were purified tested vitro (SavPP For SavPP S112M-K121A, total turnover number 372 achieved, corresponding 5.9-fold vs wt-SavPP. To analyze marked difference between ArMs, oligomeric state determined. this purpose, crosslinking experiments E. cells overexpressing carried out, followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) Western blot. data suggest that most likely displayed monomer surface coli. We hypothesize results may reflect soluble (monomeric tetrameric). Accordingly, care should be applied when evolving proteins using display.