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

Abstract

Artificial metalloenzymes (ArMs) combine characteristics of both homogeneous catalysts and enzymes. Merging abiotic and biotic features allows for the implementation of new-to-nature reactions in living organisms. Here, we present the directed evolution of an artificial metalloenzyme based onEscherichia colisurface-displayed streptavidin (SavSDhereafter). Through the binding of a ruthenium-pianostool cofactor to SavSD, an artificial allylic deallylase (ADAse hereafter) is assembled, which displays catalytic activity toward the deprotection of alloc-protected 3-hydroxyaniline. The uncaged aminophenol acts as a gene switch and triggers the overexpression of a fluorescent green fluorescent protein (GFP) reporter protein. This straightforward readout of ADAse activity allowed the simultaneous saturation mutagenesis of two amino acid residues in Sav near the ruthenium cofactor, expediting the screening of 2762 individual clones. A 1.7-fold increase ofin vivoactivity was observed for SavSDS112T-K121G compared to the wild-type SavSD(wt-SavSD). Finally, the best performing Sav isoforms were purified and testedin vitro(SavPPhereafter). For SavPPS112M-K121A, a total turnover number of 372 was achieved, corresponding to a 5.9-fold increase vs wt-SavPP. To analyze the marked difference in activity observed between the surface-displayed and purified ArMs, the oligomeric state of SavSDwas determined. For this purpose, crosslinking experiments ofE. colicells overexpressing SavSDwere carried out, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The data suggest that SavSDis most likely displayed as a monomer on the surface ofE. coli. We hypothesize that the difference between thein vivoandin vitroscreening results may reflect the difference in the oligomeric state of SavSDvs soluble SavPP(monomeric vs tetrameric). Accordingly, care should be applied when evolving oligomeric proteins usingE. colisurface display.