Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme

Abstract

Synthetic biology enables microbial hosts to produce complex molecules that are otherwise produced by organisms that are rare or di cult to cultivate, but the structures of these molecules are limited to those formed by chemical reactions catalyzed by natural enzymes. The integration of arti cial metalloenzymes (ArMs) that catalyze unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically by microorganisms. We report an engineered microbial cell expressing a heterologous biosynthetic pathway, which contains both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. To create this hybrid biosynthetic organism, we engineered Escherichia coli (E. 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 titer 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, together with natural and arti cial enzymes in whole cells, molecules that were previously inaccessible to nature.

Intestinal Radiation Protection and Mitigation by Second-Generation Probiotic Lactobacillus-reuteri Engineered to Deliver Interleukin-22

“…The small intestine is known to be significantly radiosensitive compared to the large intestine [ 1 , 2 , 3 , 4 ], and, thus, has been studied as the target for delivery of oral/gavage administration of radiation protectors/mitigators. Specific functions of the microbiome are known to be critical for intestinal radiation protection by providing both metabolic functioning and detoxification of deleterious by-products of metabolism, which are important for intestinal radiation protection [ 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ].…”

Directed Evolution of Artificial Metalloenzymes in Whole Cells

“…Recently,w ed emonstrated the efficient transport of an iridium-porphyrin complex into E. coli by ah eterologous transport system to produce ArMs that form unnatural products by the combination of the reaction of the ArMs with natural enzymes in intact E. coli. [11] Theheme transport system encoded by the hug operon from Plesiomonas shigelloides [21] led to the efficient assembly of the ArMs. Thus,t oa ccelerate the directed evolution of ArMs for the insertion of carbenes into NÀHbonds,wesought to generate mutants of ArMs containing the Ir(Me)MPIX cofactor in E. coli expressing this transport system.…”

“…[10] Recently,c oexpression of transporters,s uch as ChuA and the Hug system, has been used to incorporate cofactors into heme proteins,resulting in awhole-cell catalyst for abiotic reactions and unnatural biosynthesis. [11] Chiral amines,o ne of the most important motifs in biomolecules and drugs,h ave been targets for catalytic processes for many years.B iocatalysis has become an approach for the enantioselective synthesis of chiral amines by transamination or reduction of imines. [12] In addition to these natural processes,repurposed hemoproteins and ArMs have generated chiral amines by nitrene insertions into CÀH bonds [13] and carbene insertions into NÀHbonds.…”

Directed Evolution of Artificial Metalloenzymes in Whole Cells

“…[10] Recently,c oexpression of transporters,s uch as ChuA and the Hug system, has been used to incorporate cofactors into heme proteins,resulting in awhole-cell catalyst for abiotic reactions and unnatural biosynthesis. [11] Chiral amines,o ne of the most important motifs in biomolecules and drugs,h ave been targets for catalytic processes for many years.B iocatalysis has become an approach for the enantioselective synthesis of chiral amines by transamination or reduction of imines. [12] In addition to these natural processes,repurposed hemoproteins and ArMs have generated chiral amines by nitrene insertions into CÀH bonds [13] and carbene insertions into NÀHbonds.…”