Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Nature Chemical Biology volume 14, pages 738–743 (2018)Cite this article

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Ehrenworth, A. M. & Peralta-Yahya, P. Accelerating the semisynthesis of alkaloid-based drugs through metabolic engineering. Nat. Chem. Biol. 13, 249–258 (2017).

    Article  CAS  PubMed  Google Scholar 

  2. Tatsis, E. C. & O’Connor, S. E. New developments in engineering plant metabolic pathways. Curr. Opin. Biotechnol. 42, 126–132 (2016).

    Article  CAS  PubMed  Google Scholar 

  3. Galanie, S., Thodey, K., Trenchard, I. J., Filsinger Interrante, M. & Smolke, C. D. Complete biosynthesis of opioids in yeast. Science 349, 1095–1100 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Winzer, T. et al. Plant science. Morphinan biosynthesis in opium poppy requires a P450-oxidoreductase fusion protein. Science 349, 309–312 (2015).

    Article  CAS  PubMed  Google Scholar 

  5. Farrow, S. C., Hagel, J. M., Beaudoin, G. A. W., Burns, D. C. & Facchini, P. J. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Nat. Chem. Biol. 11, 728–732 (2015).

    Article  CAS  PubMed  Google Scholar 

  6. Service, R.F. Final step in sugar-to-morphine conversion deciphered. Science https://doi.org/10.1126/science.aac6897/ (2015).

  7. Keener, A.B. Yeast-based opioid production completed. The Scientist https://www.the-scientist.com/?articles.view/articleNo/43739/title/Yeast-Based-Opioid-Production-Completed/ (2015).

  8. Battersby, A. R. Tilden Lecture: The biosynthesis of alkaloids. Proc. Chem. Soc. 0, 189–228 (1963).

    Google Scholar 

  9. Lenz, R. & Zenk, M. H. Closure of the oxide bridge in morphine biosynthesis. Tetrahedr. Lett. 35, 3897–3900 (1994).

    Article  CAS  Google Scholar 

  10. Lenz, R. & Zenk, M. H. Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase from Papaver somniferum plant cell cultures. The enzyme catalyzing the formation of thebaine in morphine biosynthesis. J. Biol. Chem. 270, 31091–31096 (1995).

    Article  CAS  PubMed  Google Scholar 

  11. Barton, D. H. R., Bhakuni, D. S., James, R. & Kirby, G. W. Phenol oxidation and biosynthesis. Part XII. Stereochemical studies related to the biosynthesis of the morphine alkaloids. J. Chem. Soc. 0, 128–132 (1967).

    CAS  Google Scholar 

  12. Lotter, H., Gollwitzer, J. & Zenk, M. H. Revision of the configuration at C-7 of salutaridinol-I, the natural intermediate in morphine biosynthesis. Tetrahedr. Lett. 33, 2443–2446 (1992).

    Article  CAS  Google Scholar 

  13. Hagel, J. M. & Facchini, P. J. Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world. Plant Cell Physiol. 54, 647–672 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Ziegler, J. & Facchini, P. J. Alkaloid biosynthesis: metabolism and trafficking. Annu. Rev. Plant Biol. 59, 735–769 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Fisinger, U., Grobe, N. & Zenk, M. H. Thebaine synthase: a new enzyme in the morphine pathway in Papaver somniferum. Nat. Prod. Commun. 2, 249–253 (2007).

    CAS  Google Scholar 

  16. Barton, D. H. R. et al. Investigations on the biosynthesis of morphine alkaloids. J. Chem. Soc. 65, 2423–2438 (1965).

    Article  CAS  PubMed  Google Scholar 

  17. Raith, K. et al. Electrospray tandem mass spectrometric investigations of morphinans. J. Am. Soc. Mass Spectrom. 14, 1262–1269 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Pennanen, K., Kotiaho, T., Huikko, K. & Kostiainen, R. Identification of ozone-oxidation products of oxycodone by electrospray ion trap mass spectrometry. J. Mass Spectrom. 36, 791–797 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Sabarna, K. Approaches to isolating a cDNA encoding thebaine synthase of morphine biosynthesis from opium poppy Papaver somniferum. PhD thesis, Martin Luther University Halle-Wittenberg (2006).

  20. Fernandes, H., Michalska, K., Sikorski, M. & Jaskolski, M. Structural and functional aspects of PR-10 proteins. FEBS J. 280, 1169–1199 (2013).

    Article  CAS  PubMed  Google Scholar 

  21. Lee, E.-J. & Facchini, P. Norcoclaurine synthase is a member of the pathogenesis-related 10/Bet v1 protein family. Plant Cell 22, 3489–3503 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Mikkelsen, M. D. et al. Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform. Metab. Eng. 14, 104–111 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Desgagné-Penix, I., Farrow, S. C., Cram, D., Nowak, J. & Facchini, P. J. Integration of deep transcript and targeted metabolite profiles for eight cultivars of opium poppy. Plant Mol. Biol. 79, 295–313 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Dang, T. T., Onoyovwi, A., Farrow, S. C. & Facchini, P. J. Biochemical genomics for gene discovery in benzylisoquinoline alkaloid biosynthesis in opium poppy and related species. Methods Enzymol. 515, 231–266 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Boycheva, S., Daviet, L., Wolfender, J.-L. & Fitzpatrick, T. B. The rise of operon-like gene clusters in plants. Trends Plant Sci. 19, 447–459 (2014).

    Article  CAS  PubMed  Google Scholar 

  26. Nützmann, H.-W. & Osbourn, A. Gene clustering in plant specialized metabolism. Curr. Opin. Biotechnol. 26, 91–99 (2014).

    Article  CAS  PubMed  Google Scholar 

  27. Winzer, T. et al. A Papaver somniferum 10-gene cluster for synthesis of the anticancer alkaloid noscapine. Science 336, 1704–1708 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Ilari, A. et al. Structural basis of enzymatic (S)-norcoclaurine biosynthesis. J. Biol. Chem. 284, 897–904 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Robin, A. Y., Giustini, C., Graindorge, M., Matringe, M. & Dumas, R. Crystal structure of norcoclaurine-6-O-methyltransferase, a key rate-limiting step in the synthesis of benzylisoquinoline alkaloids. Plant J. 87, 641–653 (2016).

    Article  CAS  PubMed  Google Scholar 

  30. Löbermann, F., Mayer, P. & Trauner, D. Biomimetic synthesis of (-)-pycnanthuquinone C through the Diels-Alder reaction of a vinyl quinone. Angew. Chem. Int. Ed. Engl. 49, 6199–6202 (2010).

    Article  CAS  PubMed  Google Scholar 

  31. Fossati, E., Narcross, L., Ekins, A., Falgueyret, J. P. & Martin, V. J. Synthesis of morphinan alkaloids in Saccharomyces cerevisiae. PLoS One 10, e0124459 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Nessler, C. L., Allen, R. D. & Galewsky, S. Identification and characterization of latex-specific proteins in opium poppy. Plant Physiol. 79, 499–504 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bieniossek, C. et al. Automated unrestricted multigene recombineering for multiprotein complex production. Nat. Methods 6, 447–450 (2009).

    Article  CAS  PubMed  Google Scholar 

  34. Farrow, S. C., Hagel, J. M. & Facchini, P. J. Transcript and metabolite profiling in cell cultures of 18 plant species that produce benzylisoquinoline alkaloids. Phytochemistry 77, 79–88 (2012).

    Article  CAS  PubMed  Google Scholar 

  35. Chang, L., Hagel, J. M. & Facchini, P. J. Isolation and characterization of O-methyltransferases involved in the biosynthesis of glaucine in Glaucium flavum. Plant Physiol. 169, 1127–1140 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Morris, J. S. et al. Plug-and-play benzylisoquinoline alkaloid biosynthetic gene discovery in engineered yeast. Methods Enzymol. 575, 143–178 (2016).

    Article  CAS  PubMed  Google Scholar 

  37. Schmidt, J., Boettcher, C., Kuhnt, C., Kutchan, T. M. & Zenk, M. H. Poppy alkaloid profiling by electrospray tandem mass spectrometry and electrospray FT-ICR mass spectrometry after [ring-13C6]-tyramine feeding. Phytochemistry 68, 189–202 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Gambino, G., Perrone, I. & Gribaudo, I. A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem. Anal. 19, 520–525 (2008).

    Article  CAS  PubMed  Google Scholar 

Download references

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

Authors and 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

Authors
  1. Xue Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jillian M. Hagel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Limei Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joseph E. Tucker
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stacey A. Shiigi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yuora Yelpaala
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hsiang-Yun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rodrigo Estrada
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jeffrey Colbeck
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Maria Enquist-Newman
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ana B. Ibáñez
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Guillaume Cottarel
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Genevieve M. Vidanes
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Peter J. Facchini
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Peter J. Facchini.

Ethics declarations

Competing interests

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

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–22, Supplementary Tables 1–8, Supplementary Note 1–3

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing