Assembly and Evolution of Artificial Metalloenzymes within E. coli Nissle 1917 for Enantioselective and Site-Selective Functionalization of C─H and C═C Bonds
J. Am. Chem. Soc. 2022, 144, 883-890, 10.1021/jacs.1c10975
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.
Unnatural Biosynthesis by an Engineered Microorganism with Heterologously Expressed Natural Enzymes and an Artificial Metalloenzyme
Nat. Chem. 2021, 13, 1186-1191, 10.1038/s41557-021-00801-3
Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium–porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.
Metal: IrHost protein: CYP119Anchoring strategy: Metal substitutionOptimization: GeneticReaction: CyclopropanationMax TON: 2130ee: ---PDB: ---Notes: TON in vivo of (-)-carvone, WITHOUT limonene biosynthetic genes