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 (SavSD hereafter). 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 of in vivo activity was observed for SavSD S112T-K121G compared to the wild-type SavSD (wt-SavSD). Finally, the best performing Sav isoforms were purified and tested in vitro (SavPP hereafter). For SavPP S112M-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 SavSD was determined. For this purpose, crosslinking experiments of E. coli cells overexpressing SavSD were carried out, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The data suggest that SavSD 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 SavSD vs soluble SavPP (monomeric vs tetrameric). Accordingly, care should be applied when evolving oligomeric proteins using E. coli surface display.

Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high in vivo efficacy.

There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 µg mL-1, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 µg mL-1). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.