Assembly and Evolution of Artificial Metalloenzymes within <i>E. coli</i> Nissle 1917 for Enantioselective and Site-Selective Functionalization of C─H and C═C Bonds
Zhennan Liu, Jing Huang, Yang Gu, Douglas S. Clark, Aindrila Mukhopadhyay, Jay D. Keasling, John F. Hartwig
Abstract
The potential applications afforded by the generation and reactivity of artificial metalloenzymes (ArMs) in microorganisms are vast. We show that a non-pathogenic E. coli strain, Nissle 1917 (EcN), is a suitable host for the creation of ArMs from cytochrome P450s and artificial heme cofactors. An outer-membrane receptor in EcN transports an iridium porphyrin into the cell, and the Ir-CYP119 (CYP119 containing iridium porphyrin) assembled in vivo catalyzes carbene insertions into benzylic C–H bonds enantioselectively and site-selectively. The application of EcN as a whole-cell screening platform eliminates the need for laborious processing procedures, drastically increases the ease and throughput of screening, and accelerates the development of Ir-CYP119 with improved catalytic properties. Studies to identify the transport machinery suggest that a transporter different from the previously assumed ChuA receptor serves to usher the iridium porphyrin into the cytoplasm.