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 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 on; Escherichia coli; surface-displayed streptavidin (Sav; SD; hereafter). Through the binding of a ruthenium-pianostool cofactor to Sav; SD; , 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 of; in vivo; activity was observed for Sav; SD; S112T-K121G compared to the wild-type Sav; SD; (wt-Sav; SD; ). Finally, the best performing Sav isoforms were purified and tested; in vitro; (Sav; PP; hereafter). For Sav; PP; S112M-K121A, a total turnover number of 372 was achieved, corresponding to a 5.9-fold increase vs wt-Sav; PP; . To analyze the marked difference in activity observed between the surface-displayed and purified ArMs, the oligomeric state of Sav; SD; was determined. For this purpose, crosslinking experiments of; E. coli; cells overexpressing Sav; SD; were carried out, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The data suggest that Sav; SD; is most likely displayed as a monomer on the surface of; E. coli; . We hypothesize that the difference between the; in vivo; and; in vitro; screening results may reflect the difference in the oligomeric state of Sav; SD; vs soluble Sav; PP; (monomeric vs tetrameric). Accordingly, care should be applied when evolving oligomeric proteins using; E. coli; surface display.