A microbial biomanufacturing platform for natural and semisynthetic opioids


Opiates and related molecules are medically essential, but their production via field cultivation of opium poppy Papaver somniferum leads to supply inefficiencies and insecurity. As an alternative production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the transformation of opiates. Yeast strains engineered to express heterologous genes from P. somniferum and bacterium Pseudomonas putida M10 convert thebaine to codeine, morphine, hydromorphone, hydrocodone and oxycodone. We discovered a new biosynthetic branch to neopine and neomorphine, which diverted pathway flux from morphine and other target products. We optimized strain titer and specificity by titrating gene copy number, enhancing cosubstrate supply, applying a spatial engineering strategy and performing high-density fermentation, which resulted in total opioid titers up to 131 mg/l. This work is an important step toward total biosynthesis of valuable benzylisoquinoline alkaloid drug molecules and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for opioids.

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Figure 1: Engineering a heterologous morphine biosynthesis pathway in yeast.
Figure 2: Altering gene copy number and localizing COR1.3 to the endoplasmic reticulum increases pathway specificity for morphine.
Figure 3: Incorporating bacterial enzymes allows for the biological synthesis of semisynthetic opioids.
Figure 4: Optimized yeast strains for the production of diverse opioids.

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  1. 1

    Bernáth, J. Poppy: the genus Papaver. (Harwood Academic Publishers, Amsterdam, the Netherlands, 1998).

  2. 2

    International Narcotics Control Board. Narcotic Drugs: Estimated World Requirements for 2013, Statistics for 2011 (2012).

  3. 3

    PDR Network Physicians' Desk Reference. Edn. 66. (Medical Economics Co., Montvale, NJ, 2012).

  4. 4

    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).

    CAS  Article  Google Scholar 

  5. 5

    Rinner, U. & Hudlicky, T. Synthesis of morphine alkaloids and derivatives. Top. Curr. Chem. 309, 33–66 (2012).

    CAS  Article  Google Scholar 

  6. 6

    Hagel, J.M. & Facchini, P.J. Dioxygenases catalyze the O-demethylation steps of morphine biosynthesis in opium poppy. Nat. Chem. Biol. 6, 273–275 (2010).

    CAS  Article  Google Scholar 

  7. 7

    Larkin, P.J. et al. Increasing morphinan alkaloid production by over-expressing codeinone reductase in transgenic Papaver somniferum. Plant Biotechnol. J. 5, 26–37 (2007).

    CAS  Article  Google Scholar 

  8. 8

    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 

  9. 9

    Wijekoon, C.P. & Facchini, P.J. Systematic knockdown of morphine pathway enzymes in opium poppy using virus-induced gene silencing. Plant J. 69, 1052–1063 (2012).

    Article  Google Scholar 

  10. 10

    Becker, J.V. et al. Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol. FEMS Yeast Res. 4, 79–85 (2003).

    CAS  Article  Google Scholar 

  11. 11

    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 

  12. 12

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

    CAS  Article  Google Scholar 

  13. 13

    Kim, J.S. et al. Improvement of reticuline productivity from dopamine by using engineered Escherichia coli. Biosci. Biotechnol. Biochem. 77, 2166–2168 (2013).

    CAS  Article  Google Scholar 

  14. 14

    Nakagawa, A. et al. A bacterial platform for fermentative production of plant alkaloids. Nat. Commun. 2, 326 (2011).

    Article  Google Scholar 

  15. 15

    Nakagawa, A. et al. Bench-top fermentative production of plant benzylisoquinoline alkaloids using a bacterial platform. Bioeng. Bugs 3, 49–53 (2012).

    PubMed  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

    Siddiqui, M.S., Thodey, K., Trenchard, I. & Smolke, C.D. Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools. FEMS Yeast Res. 12, 144–170 (2012).

    CAS  Article  Google Scholar 

  18. 18

    Alcantara, J., Bird, D.A., Franceschi, V.R. & Facchini, P.J. Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment. Plant Physiol. 138, 173–183 (2005).

    CAS  Article  Google Scholar 

  19. 19

    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 

  20. 20

    Onoyovwe, A. et al. Morphine biosynthesis in opium poppy involves two cell types: sieve elements and laticifers. Plant Cell 25, 4110–4122 (2013).

    CAS  Article  Google Scholar 

  21. 21

    Conrado, R.J. et al. DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic Acids Res. 40, 1879–1889 (2012).

    CAS  Article  Google Scholar 

  22. 22

    Delebecque, C.J., Lindner, A.B., Silver, P.A. & Aldaye, F.A. Organization of intracellular reactions with rationally designed RNA assemblies. Science 333, 470–474 (2011).

    CAS  Article  Google Scholar 

  23. 23

    Dueber, J.E. et al. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759 (2009).

    CAS  Article  Google Scholar 

  24. 24

    Moon, T.S., Dueber, J.E., Shiue, E. & Prather, K.L. Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli. Metab. Eng. 12, 298–305 (2010).

    CAS  Article  Google Scholar 

  25. 25

    Dumas, B. et al. 11 beta-hydroxylase activity in recombinant yeast mitochondria. In vivo conversion of 11-deoxycortisol to hydrocortisone. Eur. J. Biochem. 238, 495–504 (1996).

    CAS  Article  Google Scholar 

  26. 26

    Avalos, J.L., Fink, G.R. & Stephanopoulos, G. Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nat. Biotechnol. 31, 335–341 (2013).

    CAS  Article  Google Scholar 

  27. 27

    Bayer, T.S. et al. Synthesis of methyl halides from biomass using engineered microbes. J. Am. Chem. Soc. 131, 6508–6515 (2009).

    CAS  Article  Google Scholar 

  28. 28

    Farhi, M. et al. Harnessing yeast subcellular compartments for the production of plant terpenoids. Metab. Eng. 13, 474–481 (2011).

    CAS  Article  Google Scholar 

  29. 29

    Unterlinner, B., Lenz, R. & Kutchan, T.M. Molecular cloning and functional expression of codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum. Plant J. 18, 465–475 (1999).

    CAS  Article  Google Scholar 

  30. 30

    Nielsen, B., Roe, J. & Brochmann-Hanssen, E. Oripavine - a new opium alkaloid. Planta Med. 48, 205–206 (1983).

    CAS  Article  Google Scholar 

  31. 31

    Parker, H.I., Blaschke, G. & Rapoport, H. Biosynthetic conversion of thebaine to codeine. J. Am. Chem. Soc. 94, 1276–1282 (1972).

    CAS  Article  Google Scholar 

  32. 32

    Bruce, N.C. et al. Microbial degradation of the morphine alkaloids: identification of morphine as an intermediate in the metabolism of morphine by Pseudomonas putida M10. Arch. Microbiol. 154, 465–470 (1990).

    CAS  Article  Google Scholar 

  33. 33

    Bruce, N.C., Wilmot, C.J., Jordan, K.N., Stephens, L.D. & Lowe, C.R. Microbial degradation of the morphine alkaloids. Purification and characterization of morphine dehydrogenase from Pseudomonas putida M10. Biochem. J. 274, 875–880 (1991).

    CAS  Article  Google Scholar 

  34. 34

    French, C.E. & Bruce, N.C. Purification and characterization of morphinone reductase from Pseudomonas putida M10. Biochem. J. 301, 97–103 (1994).

    CAS  Article  Google Scholar 

  35. 35

    French, C.E. et al. Biological production of semisynthetic opiates using genetically engineered bacteria. Bio/Technology 13, 674–676 (1995).

    CAS  PubMed  Google Scholar 

  36. 36

    Zhang, Q., Rich, J.O., Cotterill, I.C., Pantaleone, D.P. & Michels, P.C. 14-Hydroxylation of opiates: catalytic direct autoxidation of codeinone to 14-hydroxycodeinone. J. Am. Chem. Soc. 127, 7286–7287 (2005).

    CAS  Article  Google Scholar 

  37. 37

    Lister, D.L., Kanungo, G., Rathbone, D.A. & Bruce, N.C. Transformations of codeine to important semisynthetic opiate derivatives by Pseudomonas putida m10. FEMS Microbiol. Lett. 181, 137–144 (1999).

    CAS  Article  Google Scholar 

  38. 38

    Gollwitzer, J., Lenz, R., Hampp, N. & Zenk, M.H. The transformation of neopinone to codeinone in morphine biosynthesis proceeds non-enzymatically. Tetrahedr. Lett. 34, 5703–5706 (1993).

    CAS  Article  Google Scholar 

  39. 39

    Lenz, R. & Zenk, M.H. Purification and properties of codeinone reductase (NADPH) from Papaver somniferum cell cultures and differentiated plants. Eur. J. Biochem. 233, 132–139 (1995).

    CAS  Article  Google Scholar 

  40. 40

    Homeyer, A.H. & Shilling, W.L. Isolation and purification of neopine. J. Org. Chem. 12, 356–358 (1947).

    CAS  Article  Google Scholar 

  41. 41

    Al-Amri, A.M., Smith, R.M. & El-Haj, B.M. The GC-MS detection and characterization of neopine resulting from opium use and codeine metabolism and its potential as an opiate-product-use marker. Anal. Bioanal. Chem. 382, 830–835 (2005).

    CAS  Article  Google Scholar 

  42. 42

    Farrow, S.C. & Facchini, P.J. Dioxygenases catalyze O-demethylation and O,O-demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy. J. Biol. Chem. 288, 28997–29012 (2013).

    CAS  Article  Google Scholar 

  43. 43

    Liscombe, D.K., Ziegler, J., Schmidt, J., Ammer, C. & Facchini, P.J. Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species. Plant J. 60, 729–743 (2009).

    CAS  Article  Google Scholar 

  44. 44

    Gueldener, U., Heinisch, J., Koehler, G.J., Voss, D. & Hegemann, J.H. A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res. 30, e23 (2002).

    CAS  Article  Google Scholar 

  45. 45

    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 

  46. 46

    Alberti, S., Gitler, A.D. & Lindquist, S. A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24, 913–919 (2007).

    CAS  Article  Google Scholar 

  47. 47

    Parlati, F., Dominguez, M., Bergeron, J.J. & Thomas, D.Y. Saccharomyces cerevisiae CNE1 encodes an endoplasmic reticulum (ER) membrane protein with sequence similarity to calnexin and calreticulin and functions as a constituent of the ER quality control apparatus. J. Biol. Chem. 270, 244–253 (1995).

    CAS  Article  Google Scholar 

  48. 48

    Bevis, B.J., Hammond, A.T., Reinke, C.A. & Glick, B.S. De novo formation of transitional ER sites and Golgi structures in Pichia pastoris. Nat. Cell Biol. 4, 750–756 (2002).

    CAS  Article  Google Scholar 

  49. 49

    Meuzelaar, G.J., Woudenberg, R.H., Sinnema, A. & Maat, L. Synthesis of neopine and its 5β- and 7-substituted derivatives (chemistry of opium alkaloids, part XL). Recl. Trav. Chim. Pays Bas 112, 573–577 (1993).

    CAS  Article  Google Scholar 

  50. 50

    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 F.J. Lu for contributions to strain construction and testing; Noramco for the gift of standards codeinone, dihydrocodeine, oripavine, morphinone and 14-hydroxycodeine; members of the Stanford Cell Sciences Imaging Facility for providng fluorescence microscopy access (Leica SP5 NIH grant SIG number: 1S10RR02557401) and training; members of the Stanford Chemistry NMR Facility and S. Lynch for instrument access and training; J. Li for helpful discussions and sharing equipment for compound isolation; D. Endy, M. McKeague, I. Trenchard, A.L. Chang and Y.-H. Wang for valuable feedback in the preparation of the manuscript. This work was supported by US National Institutes of Health (grant to C.D.S., DP1OD009329), the National Science Foundation (grant to C.D.S., CBET-1066100; fellowship to S.G.), the Bill and Melinda Gates Foundation (grant to C.D.S., OPP1058690), the New Zealand Foundation for Research, Science and Technology (fellowship to K.T., SFRD0901) and Stanford University (fellowship to S.G.).

Author information




K.T. and C.D.S. conceived of the project, designed the experiments, analyzed the results and wrote the manuscript. K.T. constructed and tested the strains. S.G. performed NMR spectroscopy and in vitro assays, and prepared parts of the manuscript.

Corresponding author

Correspondence to Christina D Smolke.

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Competing interests

C.D.S., K.T. and S.G. have filed a patent application (US application no. 14/211,611) covering yeast strains developed for the production of opioids and other benzylisoquinoline alkaloids.

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Supplementary Results, Supplementary Figures 1–9 and Supplementary Tables 1–5. (PDF 2153 kb)

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Thodey, K., Galanie, S. & Smolke, C. A microbial biomanufacturing platform for natural and semisynthetic opioids. Nat Chem Biol 10, 837–844 (2014). https://doi.org/10.1038/nchembio.1613

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