In Vivo Assembly of Artificial Metalloenzymes and Application in Whole?Cell Biocatalysis**

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