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 spectroscopy, 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 required 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 with biosynthesis to generate a hybrid metabolism.