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submitted on 15.06.2020 and posted on 17.06.2020by Shreyans Chordia, Siddarth Narasimhan, Alessandra Lucini Paioni, Marc Baldus, Gerard Roelfes
Artificial metalloenzymes (ArMs), which are hybrids of catalytically active transition metal complexes
and proteins, have emerged as promising approach to the creation of biocatalysts for reactions that
have no equivalent in nature. Here we report the assembly and application in catalysis of ArMs in
the cytoplasm of E. coli cells based on the Lactococcal multidrug resistance regulator (LmrR) and an
exogeneously added copper(II)‐phenanthroline (Cu(II)‐phen) complex. The ArMs are spontaneously
assembled by addition of Cu(II)‐phen to E. coli cells that express LmrR and it is shown that the ArM
containing whole cells are active in the catalysis of the enantioselective vinylogous Friedel‐Crafts
alkylation 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 mutagenesis
study showed that the same trends in catalytic activity and enantioselectivity in response to
mutations of LmrR were observed for the ArM containing whole cells and the isolated ArMs. This
made it possible to perform a directed evolution study using ArMs in whole cells, which gave rise to
a mutant, LmrR_A92E_M8D that showed increased activity and enantioselectivity in the catalyzed
vinylogous Friedel‐Crafts alkylation of a variety of indoles. The unique aspect of this whole‐cell ArM
system is that no engineering of the microbial host, the protein scaffold or the cofactor is required to
achieve ArM assembly and catalysis. This makes this system attractive for applications in whole cell
biocatalysis and directed evolution, as demonstrated here. Moreover, our findings represent
important step forward towards achieving the challenging goal of a hybrid metabolism by integrating
artificial metalloenzymes in biosynthetic pathways.