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Abstract

The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.

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Acknowledgements

We are grateful to L. Brechenmacher and the Southern Alberta Mass Spectrometry Centre for assistance with the proteomics analysis, S. Yeaman for guidance with the genome assembly, and T. Back for advice on chemical reaction mechanisms. We acknowledge the expert services provided by the McGill University-Genome Québec Innovation Centre with respect to genome sequencing and preliminary assembly. We also thank A. Pigula, S. Muley, E. Eberhard, A. Kumar, K. Hetenyi, I. Esaid, H. Tang, H. Roth, M. Schmalisch, L. Hom, C. Savile, P. Seufer-Wasserthal, T. Noh, B. Walsh, and R. J. Kirk for technical assistance and project guidance. This work was supported by Genopaver, LLC., Epimeron Inc. and funds awarded through the Industrial Research Assistance Program (IRAP; Project 86155) operated by the National Research Council of Canada to Epimeron, Inc.

Author information

Affiliations

  1. Department of Biological Sciences, University of Calgary, Calgary, Canada

    • Xue Chen
    • , Jillian M. Hagel
    • , Limei Chang
    •  & Peter J. Facchini
  2. Epimeron Inc., Calgary, Canada

    • Xue Chen
    • , Jillian M. Hagel
    • , Limei Chang
    • , Joseph E. Tucker
    •  & Peter J. Facchini
  3. Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Canada

    • Joseph E. Tucker
  4. Intrexon Corp., Industrial Products Division, South San Francisco, CA, USA

    • Stacey A. Shiigi
    • , Yuora Yelpaala
    • , Hsiang-Yun Chen
    • , Rodrigo Estrada
    • , Jeffrey Colbeck
    • , Maria Enquist-Newman
    • , Ana B. Ibáñez
    • , Guillaume Cottarel
    •  & Genevieve M. Vidanes

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Contributions

X.C., J.M.H., J.E.T., G.C., G.M.V., and P.J.F. contributed to the project design; X.C. performed most of the enzymology, protein purification, molecular biology, gene silencing, and transcript profiling work; J.M.H. conducted the high-resolution LC-MS analysis and proposed the reaction mechanism; L.C., S.A.S., and M.E.-N. developed testing conditions and characterized gene candidates in yeast; R.E. and J.C. designed and built plasmids and yeast strains; Y.Y., H.-Y.C., and A.B.I. developed and performed LC-MS analysis for yeast fermentation experiments; X.C., J.M.H., and P.J.F. wrote the manuscript. P.J.F. supervised the project. All authors have read and approved the content of the manuscript.

Competing interests

Patent applications related to this work have been filed (PCT/CA2017/050779 and PCT/US2017/039589).

Corresponding author

Correspondence to Peter J. Facchini.

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DOI

https://doi.org/10.1038/s41589-018-0059-7