Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae


The benzylisoquinoline alkaloids (BIAs) are a diverse class of metabolites that exhibit a broad range of pharmacological activities and are synthesized through plant biosynthetic pathways comprised of complex enzyme activities and regulatory strategies. We have engineered yeast to produce the key intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants from different plants and determined optimal expression levels. By synthesizing both stereoisomer forms of reticuline and integrating enzyme activities from three plant sources and humans, we demonstrated the synthesis of metabolites in the sanguinarine/berberine and morphinan branches. We also demonstrated that a human P450 enzyme exhibits a novel activity in the conversion of (R)-reticuline to the morphinan alkaloid salutaridine. Our engineered microbial hosts offer access to a rich group of BIA molecules and associated activities that will be further expanded through synthetic chemistry and biology approaches.

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Figure 1: Microbial production of (R,S)-reticuline.
Figure 2: Effects of enzyme levels, substrate levels and culture time on reticuline production levels.
Figure 3: A new strategy for tuning enzyme expression levels.
Figure 4: Microbial production of BIA metabolites along the sanguinarine and berberine branches.
Figure 5: Microbial production of morphinan alkaloids.


  1. 1

    Endy, D. Foundations for engineering biology. Nature 438, 449–453 (2005).

    CAS  Article  Google Scholar 

  2. 2

    McDaniel, R. & Weiss, R. Advances in synthetic biology: on the path from prototypes to applications. Curr. Opin. Biotechnol. 16, 476–483 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Ro, D.K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006).

    CAS  Article  Google Scholar 

  4. 4

    Kealey, J.T., Liu, L., Santi, D.V., Betlach, M.C. & Barr, P.J. Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc. Natl. Acad. Sci. USA 95, 505–509 (1998).

    CAS  Article  Google Scholar 

  5. 5

    Szczebara, F.M. et al. Total biosynthesis of hydrocortisone from a simple carbon source in yeast. Nat. Biotechnol. 21, 143–149 (2003).

    CAS  Article  Google Scholar 

  6. 6

    Martin, M.L. et al. Antispasmodic activity of benzylisoquinoline alkaloids analogous to papaverine. Planta Med. 59, 63–67 (1993).

    CAS  Article  Google Scholar 

  7. 7

    Morais, L.C., Barbosa-Filho, J.M. & Almeida, R.N. Central depressant effects of reticuline extracted from Ocotea duckei in rats and mice. J. Ethnopharmacol. 62, 57–61 (1998).

    CAS  Article  Google Scholar 

  8. 8

    Nakaoji, K., Nayeshiro, H. & Tanahashi, T. Norreticuline and reticuline as possible new agents for hair growth acceleration. Biol. Pharm. Bull. 20, 586–588 (1997).

    CAS  Article  Google Scholar 

  9. 9

    Kemeny-Beke, A. et al. Apoptotic response of uveal melanoma cells upon treatment with chelidonine, sanguinarine and chelerythrine. Cancer Lett. 237, 67–75 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Cassels, B.K. et al. Structure-antioxidative activity relationships in benzylisoquinoline alkaloids. Pharmacol. Res. 31, 103–107 (1995).

    CAS  Article  Google Scholar 

  11. 11

    Exley, R. et al. Evaluation of benzyltetrahydroisoquinolines as ligands for neuronal nicotinic acetylcholine receptors. Br. J. Pharmacol. 146, 15–24 (2005).

    CAS  Article  Google Scholar 

  12. 12

    Kashiwada, Y. et al. Anti-HIV benzylisoquinoline alkaloids and flavonoids from the leaves of Nelumbo nucifera, and structure-activity correlations with related alkaloids. Bioorg. Med. Chem. 13, 443–448 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Lai, J.H. Immunomodulatory effects and mechanisms of plant alkaloid tetrandrine in autoimmune diseases. Acta Pharmacol. Sin. 23, 1093–1101 (2002).

    CAS  PubMed  Google Scholar 

  14. 14

    Kwan, C.Y. & Achike, F.I. Tetrandrine and related bis-benzylisoquinoline alkaloids from medicinal herbs: cardiovascular effects and mechanisms of action. Acta Pharmacol. Sin. 23, 1057–1068 (2002).

    CAS  PubMed  Google Scholar 

  15. 15

    Chulia, S. et al. Relationships between structure and vascular activity in a series of benzylisoquinolines. Br. J. Pharmacol. 122, 409–416 (1997).

    CAS  Article  Google Scholar 

  16. 16

    Bentley, K.W. β-Phenylethylamines and the isoquinoline alkaloids. Nat. Prod. Rep. 23, 444–463 (2006).

    CAS  Article  Google Scholar 

  17. 17

    Facchini, P.J. Alkaloid biosynthesis in plants: biochemistry, cell biology, molecular regulation, and metabolic engineering applications. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 29–66 (2001).

    CAS  Article  Google Scholar 

  18. 18

    Sato, F., Inui, T. & Takemura, T. Metabolic engineering in isoquinoline alkaloid biosynthesis. Curr. Pharm. Biotechnol. 8, 211–218 (2007).

    CAS  Article  Google Scholar 

  19. 19

    Allen, R.S. et al. Metabolic engineering of morphinan alkaloids by over-expression and RNAi suppression of salutaridinol 7-O-acetyltransferase in opium poppy. Plant Biotechnol. J. 6, 22–30 (2008).

    CAS  PubMed  Google Scholar 

  20. 20

    Allen, R.S. et al. RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat. Biotechnol. 22, 1559–1566 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Sato, F. et al. Metabolic engineering of plant alkaloid biosynthesis. Proc. Natl. Acad. Sci. USA 98, 367–372 (2001).

    CAS  Article  Google Scholar 

  22. 22

    Zulak, K.G. et al. Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism. Planta 225, 1085–1106 (2007).

    CAS  Article  Google Scholar 

  23. 23

    Minami, H. et al. Microbial production of plant benzylisoquinoline alkaloids. Proc. Natl. Acad. Sci. USA 105, 7393–7398 (2008).

    CAS  Article  Google Scholar 

  24. 24

    Ziegler, J. et al. Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis. Plant J. 48, 177–192 (2006).

    CAS  Article  Google Scholar 

  25. 25

    Sato, F., Tsujita, T., Katagiri, Y., Yoshida, S. & Yamada, Y. Purification and characterization of S-adenosyl-L-methionine: norcoclaurine 6-O-methyltransferase from cultured Coptis japonica cells. Eur. J. Biochem. 225, 125–131 (1994).

    CAS  Article  Google Scholar 

  26. 26

    Ounaroon, A., Decker, G., Schmidt, J., Lottspeich, F. & Kutchan, T.M. (R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferase of Papaver somniferum - cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant J. 36, 808–819 (2003).

    CAS  Article  Google Scholar 

  27. 27

    Hawkins, K.M. & Smolke, C.D. The regulatory roles of the galactose permease and kinase in the induction response of the GAL network in Saccharomyces cerevisiae. J. Biol. Chem. 281, 13485–13492 (2006).

    CAS  Article  Google Scholar 

  28. 28

    Nevoigt, E. et al. Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 72, 5266–5273 (2006).

    CAS  Article  Google Scholar 

  29. 29

    Kutchan, T.M. & Dittrich, H. Characterization and mechanism of the berberine bridge enzyme, a covalently flavinylated oxidase of benzophenanthridine alkaloid biosynthesis in plants. J. Biol. Chem. 270, 24475–24481 (1995).

    CAS  Article  Google Scholar 

  30. 30

    Bird, D.A. & Facchini, P.J. Berberine bridge enzyme, a key branch-point enzyme in benzylisoquinoline alkaloid biosynthesis, contains a vacuolar sorting determinant. Planta 213, 888–897 (2001).

    CAS  Article  Google Scholar 

  31. 31

    Ikezawa, N. et al. Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family, from cultured Coptis japonica cells. J. Biol. Chem. 278, 38557–38565 (2003).

    CAS  Article  Google Scholar 

  32. 32

    Hirata, K., Poeaknapo, C., Schmidt, J. & Zenk, M.H. 1,2-Dehydroreticuline synthase, the branch point enzyme opening the morphinan biosynthetic pathway. Phytochemistry 65, 1039–1046 (2004).

    CAS  Article  Google Scholar 

  33. 33

    Zhu, W., Cadet, P., Baggerman, G., Mantione, K.J. & Stefano, G.B. Human white blood cells synthesize morphine: CYP2D6 modulation. J. Immunol. 175, 7357–7362 (2005).

    CAS  Article  Google Scholar 

  34. 34

    Liscombe, D.K. & Facchini, P.J. Evolutionary and cellular webs in benzylisoquinoline alkaloid biosynthesis. Curr. Opin. Biotechnol. 19, 173–180 (2008).

    CAS  Article  Google Scholar 

  35. 35

    Winkel, B.S. Metabolic channeling in plants. Annu. Rev. Plant Biol. 55, 85–107 (2004).

    CAS  Article  Google Scholar 

  36. 36

    Sambrook, J. & Russell, D.W. Molecular Cloning 3rd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001).

    Google Scholar 

  37. 37

    Gillam, E.M., Guo, Z., Martin, M.V., Jenkins, C.M. & Guengerich, F.P. Expression of cytochrome P450 2D6 in Escherichia coli, purification, and spectral and catalytic characterization. Arch. Biochem. Biophys. 319, 540–550 (1995).

    CAS  Article  Google Scholar 

  38. 38

    Urban, P., Mignotte, C., Kazmaier, M., Delorme, F. & Pompon, D. Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J. Biol. Chem. 272, 19176–19186 (1997).

    CAS  Article  Google Scholar 

  39. 39

    Mapoles, J., Berthou, F., Alexander, A., Simon, F. & Menez, J.F. Mammalian PC-12 cell genetically engineered for human cytochrome P450 2E1 expression. Eur. J. Biochem. 214, 735–745 (1993).

    CAS  Article  Google Scholar 

  40. 40

    Gietz, R.D. & Woods, R.A. Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Methods Mol. Biol. 313, 107–120 (2006).

    CAS  PubMed  Google Scholar 

  41. 41

    Thomas, B.J. & Rothstein, R. Elevated recombination rates in transcriptionally active DNA. Cell 56, 619–630 (1989).

    CAS  Article  Google Scholar 

  42. 42

    Guldener, U., Heck, S., Fielder, T., Beinhauer, J. & Hegemann, J.H. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24, 2519–2524 (1996).

    CAS  Article  Google Scholar 

  43. 43

    Chen, J. et al. Analysis of major alkaloids in Rhizoma coptidis by capillary electrophoresis-electrospray-time of flight mass spectrometry with different background electrolytes. Electrophoresis 29, 2135–2147 (2008).

    CAS  Article  Google Scholar 

  44. 44

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

    CAS  Article  Google Scholar 

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We thank P. Facchini (University of Calgary), F.P. Guengerich (Vanderbilt University) and D. Pompon (Centre de Génétique Moléculaire, CNRS) for generously providing cDNAs and yeast strains used in this work. This work was supported by the Center for Biological Circuit Design at Caltech (fellowship to K.M.H.) and the US National Institutes of Health (grant to C.D.S.).

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K.M.H. designed and performed research, analyzed data and wrote the paper; C.D.S. designed research, analyzed data and wrote the paper.

Corresponding author

Correspondence to Christina D Smolke.

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The authors declare competing financial interests in the form of a pending patent application.

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Hawkins, K., Smolke, C. Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat Chem Biol 4, 564–573 (2008).

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