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A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis

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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Fig. 1: Production of thebaine from salutaridine in opium poppy.
Fig. 2: SalAT-coupled assay conducted in the presence of opium poppy latex protein yields more thebaine (m/z 312) and reduced levels of hydroxylated byproduct (m/z 330).
Fig. 3: In vitro SalAT-coupled assay performed on six candidates identifies Bet v1-1 as THS.
Fig. 4: Virus-induced gene silencing of THS in opium poppy reduced thebaine levels and increased the accumulation of upstream pathway intermediates.
Fig. 5: Expression of THS2 in S. cerevisiae engineered with a multistep, opiate production pathway substantially increased thebaine production.

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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.

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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.

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Correspondence to Peter J. Facchini.

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

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Chen, X., Hagel, J.M., Chang, L. et al. A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis. Nat Chem Biol 14, 738–743 (2018). https://doi.org/10.1038/s41589-018-0059-7

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