Enantio- and Diastereodivergent Cyclopropanation of Allenes by Directed Evolution of an Iridium-Containing Cytochrome.

Alkylidene cyclopropanes (ACPs) are valuable synthetic intermediates because of their constrained structure and opportunities for further diversification. Although routes to ACPs are known, preparations of ACPs with control of both the configuration of the cyclopropyl (R vs S) group and the geometry of the alkene (E vs Z) are unknown. We describe enzymatic cyclopropanation of allenes with ethyl diazoacetate (EDA) catalyzed by an iridium-containing cytochrome (Ir(Me)-CYP119) that controls both stereochemical elements. Two mutants of Ir(Me)-CYP119 identified by 6-codon (6c, VILAFG) saturation mutagenesis catalyze the formation of (E)-ACPs with -93% to >99% ee and >99:1 E/Z ratio with just three rounds of 96 mutants. By four additional rounds of mutagenesis, an enzyme variant was identified that forms (Z)-ACPs with up to 94% ee and a 28:72 E/Z ratio. Computational studies show that the orientation of the carbene unit dictated by the mutated positions accounts for the stereoselectivity.

Progress, Challenges, and Opportunities with Artificial Metalloenzymes in Biosynthesis.

In this Perspective, we present progress, outstanding challenges, and opportunities for the incorporation of artificial metalloenzymes (ArMs) into biosynthetic pathways. We first explain discoveries within the field of ArMs that led to the potential inclusion of these enzymes in biosynthesis. We then describe the specific barriers that our laboratory, in collaboration with the laboratories of Keasling and Mukhopadhyay, addressed to establish a biosynthetic pathway containing an ArM. This biosynthesis produced an unnatural cyclopropyl terpenoid by combining heterologous production of the terpene with modification of its terminal alkene by an ArM built from a cytochrome P450. Finally, we describe the remaining challenges and opportunities related to the application of ArMs in synthetic biology.

Catalytic deconstruction of waste polyethylene with ethylene to form propylene.

The conversion of polyolefins to monomers would create a valuable carbon feedstock from the largest fraction of waste plastic. However, breakdown of the main chains in these polymers requires the cleavage of carbon-carbon bonds that tend to resist selective chemical transformations. Here, we report the production of propylene by partial dehydrogenation of polyethylene and tandem isomerizing ethenolysis of the desaturated chain. Dehydrogenation of high-density polyethylene with either an iridium-pincer complex or platinum/zinc supported on silica as catalysts yielded dehydrogenated material containing up to 3.2% internal olefins; the combination of a second-generation Hoveyda-Grubbs metathesis catalyst and [PdP(tBu)3(µ-Br)]2 as an isomerization catalyst selectively degraded this unsaturated polymer to propylene in yields exceeding 80%. These results show promise for the application of mild catalysis to deconstruct otherwise stable polyolefins.

Contra-thermodynamic Olefin Isomerization by Chain-Walking Hydroboration and Dehydroboration.

We report a dehydroboration process that can be coupled with chain-walking hydroboration to create a one-pot, contra-thermodynamic, short- or long-range isomerization of internal olefins to terminal olefins. This dehydroboration occurs by a sequence comprising activation with a nucleophile, iodination, and base-promoted elimination. The isomerization proceeds at room temperature without the need for a fluoride base, and the substrate scope of this isomerization is expanded over those of previous isomerizations we have reported with silanes.

Directed Evolution of Artificial Metalloenzymes in Whole Cells.

Artificial metalloenzymes (ArMs), created by introducing synthetic cofactors into protein scaffolds, are an emerging class of catalyst for non-natural reactions. Although many classes of ArMs are known, in vitro reconstitution of cofactors and proteins has been a limiting step in the high-throughput screening and directed evolution of ArMs because purification of individual host proteins is time-consuming. We describe the application of a platform to combine mutants of the P450 enzyme CYP119 and the cofactor Ir(Me)MPIX in vivo, by coexpression of the CYP119 mutants with the heme transporter encoded by the hug operon, to the directed evolution of ArMs containing Ir(Me)MPIX in whole cells. We applied this platform to the development an ArMs catalyzing the insertion of the acyclic carbene from α-diazopropanoate esters (Me-EDA) into the N-H bonds of N-alkyl anilines, a combination of carbene and amine classes for which mutant enzymes of natural hemoproteins previously reacted with low enantioselectivity. The mutants of the artificial metalloenzyme Ir(Me)CYP119 identified by an evolution campaign involving more than 4000 mutants are shown to catalyze the reaction of Me-EDA with N-methyl anilines to form chiral chiral amino esters with high TON and good enantioselectivity, thereby demonstrating that the directed evolution of ArMs can rival that of natural enzymes in vivo.

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

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.