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

Artificial metalloenzymes (ArMs), which are hybrids of catalytically active transition metal complexesand proteins, have emerged as promising approach to the creation of biocatalysts for reactions thathave no equivalent in nature. Here we report the assembly and application in catalysis of ArMs inthe cytoplasm of E. coli cells based on the Lactococcal multidrug resistance regulator (LmrR) and anexogeneously added copper(II)‐phenanthroline (Cu(II)‐phen) complex. The ArMs are spontaneouslyassembled by addition of Cu(II)‐phen to E. coli cells that express LmrR and it is shown that the ArMcontaining whole cells are active in the catalysis of the enantioselective vinylogous Friedel‐Craftsalkylation of indoles. The ArM assembly in E. coli is further supported by a combination of cell‐fractionation and inhibitor experiments and confirmed by in‐cell solid‐state NMR. A mutagenesisstudy showed that the same trends in catalytic activity and enantioselectivity in response tomutations of LmrR were observed for the ArM containing whole cells and the isolated ArMs. Thismade it possible to perform a directed evolution study using ArMs in whole cells, which gave rise toa mutant, LmrR_A92E_M8D that showed increased activity and enantioselectivity in the catalyzedvinylogous Friedel‐Crafts alkylation of a variety of indoles. The unique aspect of this whole‐cell ArMsystem is that no engineering of the microbial host, the protein scaffold or the cofactor is required toachieve ArM assembly and catalysis. This makes this system attractive for applications in whole cellbiocatalysis and directed evolution, as demonstrated here. Moreover, our findings representimportant step forward towards achieving the challenging goal of a hybrid metabolism by integratingartificial metalloenzymes in biosynthetic pathways.