In vivo Assembly of Artificial Metalloenzymes and Application in Whole-Cell Biocatalysis

We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)-phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation and inhibitor experiments, supplemented with in-cell solid-state NMR, confirmed the in-cell assembly. The ArM containing whole cells were active in the catalysis of the enantioselective Friedel-Crafts alkylation of indoles and the Diels-Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system requires no engineering of the microbial host, the protein scaffold or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis in biosynthesis, achieving a hybrid metabolism.