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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.

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Fig. 1: A schematic representation of our envisioned reaction sequence to produce an unnatural product by a heterologously expressed natural biosynthetic pathway and reaction catalysed by an ArM in E. coli.
Fig. 2: The whole cells containing Ir–CYP119 catalyse the cyclopropanation of (−)-carvone with high diastereoselectivity.
Fig. 3: Combining limonene biosynthesis with ArM.
Fig. 4: Increasing the diastereoselectivity and titre of cyclopropyl limonene by directed evolution and process optimization.

Data availability

All data are available in the main text or the Supplementary Information. Source Data for Figs. 24 are provided with the paper. Source data are provided with this paper.


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We thank D.P. Henderson for kindly sharing pHug21 plasmid. This work was supported by the Department of Energy (DOE), Laboratory Directed Research and Development funding, Joint BioEnergy Institute (, which is supported by the DOE, Office of Science, Office of Biological and Environmental Research under contract DE-AC02-05CH11231 and National Science Foundation grant 2027943. Z.L. is an A*Star predoctoral fellow. We thank the College of Chemistry’s NMR facility for resources. Instruments in CoC-NMR are supported in part by the NIH (S10OD024998). Inductively coupled plasma mass spectrometry measurements were performed in the OHSU Elemental Analysis Core with partial support from NIH (S10RR025512).

Author information




J.F.H., D.S.C., A.M. and J.D.K. conceived the project and all authors participated in designing the experiments. J.H., Z.L. and B.L.B. conducted all the experiments and collected data for the project. All authors interpreted the data and wrote the manuscript.

Corresponding authors

Correspondence to Douglas S. Clark, Aindrila Mukhopadhyay, Jay D. Keasling or John F. Hartwig.

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Competing interests

J.D.K. has a financial interest in Amyris, Lygos, Demetrix, Maple Bio, Napigen, Apertor Pharma, Ansa Biotechnologies, Berkeley Yeast and Zero Acre Farms. A provisional patent application (US application number 17/200,715; inventors: J.H., Z.L., D.S.C., J.D.K., A.M. and J.F.H.) has been filed through the Lawrence Berkeley National Laboratory based on the results presented here.

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Peer review information Nature Chemistry thanks the anonymous reviewers for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–19 and Tables 1–4.

Source data

Source Data Fig. 2

Statistical Source Data

Source Data Fig. 3

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Source Data Fig. 4

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Huang, J., Liu, Z., Bloomer, B.J. et al. Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme. Nat. Chem. 13, 1186–1191 (2021).

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