Enzyme morphinan N-demethylase for more sustainable opiate processing

Abstract

Naturally occurring opiates, such as morphine and thebaine, are produced in poppy species. Thebaine is converted into painkillers and opiate addiction treatments, the latter requiring a chemical reaction called N-demethylation. Current opiate N-demethylation utilizes noxious reagents, resulting in copious amounts of harmful waste. One way to make opiate production more sustainable is to use enzymes rather than chemicals. Microorganisms provide a rich source of enzymes useful for metabolizing unique compounds in their environment. Therefore, an opium-processing waste stream was probed to identify an organism capable of catalysing opiate N-demethylation. A sludge sample was grown on minimal medium containing thebaine as the sole carbon source, to identify a biocatalyst. This led to the discovery of Thebainfresser, a Methylobacterium that metabolizes opiates by removing the N-methyl group. N-demethylation was induced following growth in minimal medium, a characteristic which led to discovery of the underlying gene MND (morphinan N-demethylase). The enzyme MND was found to be robust and versatile, N-demethylating structurally diverse substrates at varying temperatures and pH levels. In addition, MND tolerated selected organic solvents and maintained activity when immobilized. These properties make it an attractive candidate for further development for pharmaceutical manufacture.

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Fig. 1: Thebainfresser morphology and colonization on thebaine crystals.
Fig. 2: Thebainfresser N-demethylase induction.
Fig. 3: N-demethylase activity of morphinan N-demethylase.
Fig. 4: Compounds N-demethylated by morphinan N-demethylase.
Fig. 5: Morphinan N-demethylase activity in industrial solvents.
Fig. 6: Molecular phylogeny of morphinan N-demethylase amino acid sequence.

Data availability

The MND sequence reported herein has been deposited in GenBank with the accession no. MH258748. Additional data that support the findings of this study are available from the corresponding author upon request.

References

  1. 1.

    Summerton, L., Sneddon, H. F., Jones, L. C. & Clark, J. H. (eds) Green and Sustainable Medicinal Chemistry: Methods, Tools and Strategies for the 21st Century Pharmaceutical Industry (Royal Society for Chemistry, 2016).

  2. 2.

    Jimenez-Gonzalez, C. et al. Key green engineering research areas for sustainable manufacturing: a perspective from pharmaceutical and fine chemicals manufacturers. Org. Process Res. Dev. 15, 900–911 (2011).

  3. 3.

    Constable, D. J. C. et al. Key green chemistry research areas – a perspective from pharmaceutical manufacturers. Green Chem. 9, 411–420 (2007).

  4. 4.

    Challener, C. A. Going green with biocatalysis. Pharm. Technol. 40, 2 (2016).

  5. 5.

    Wells, A. S., Finch, G. L., Michels, P. C. & Wong, J. W. Use of enzymes in the manufacture of active pharmaceutical ingredients – a science and safety-based approach to ensure patient safety and drug quality. Org. Process Res. Dev. 16, 1986–1993 (2012).

  6. 6.

    Martinez, C. A. et al. Development of a chemoenzymatic manufacturing process for pregabalin. Org. Process Res. Dev. 12, 392–398 (2008).

  7. 7.

    Dunn, P. J., Wells, A. S. & Williams, M. T. Green Chemistry in the Pharmaceutical Industry (Wiley-VCH, 2010).

  8. 8.

    Savile, C. K. et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309 (2010).

  9. 9.

    Chien, A., Edgar, D. B. & Trela, J. M. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J. Bacteriol. 127, 1550–1557 (1976).

  10. 10.

    Copley, S. D. Evolution of efficient pathways for degradation of anthropogenic chemicals. Nat. Chem. Biol. 5, 559–566 (2009).

  11. 11.

    van der Meer, J. R., de Vos, W. M., Harayama, S. & Zehnder, A. J. Molecular mechanisms of genetic adaptation to xenobiotic compounds. Microbiol. Rev. 56, 677–694 (1992).

  12. 12.

    Comfort, D. A. et al. Strategic biocatalysis with hyperthermophilic enzymes. Green Chem. 6, 459–465 (2004).

  13. 13.

    Zhang, T. et al. Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance. Green Chem. 13, 2083–2090 (2011).

  14. 14.

    Allen, B. E. et al. Processes for the production of buprenorphine with reduced impurity formation. US patent 20100087647 (2010).

  15. 15.

    United Nations Office on Drugs and Crime & World Health Organization. Opioid Overdose: Preventing and Reducing Opioid Overdose and Mortality (United Nations, 2013).

  16. 16.

    Bonnie, R. J., Ford, M. A. & Phillips, J. K. (eds) Pain Management and the Opioid Epidemic: Balancing Societal and Individual Benefits and Risks of Prescription Opioid Use (National Academies Press, 2017).

  17. 17.

    United Nations Office on Drugs and Crime. World Drug Report 2016 (United Nations, 2016).

  18. 18.

    Bart, H.-J. R. & Pilz, S. Industrial Scale Natural Products Extraction (Wiley-VCH, 2011).

  19. 19.

    Kok, G. B., Pye, C. C., Singer, R. D. & Scammells, P. J. Two-step iron(0)-mediated N-demethylation of N-methyl alkaloids. J. Org. Chem. 75, 4806–4811 (2010).

  20. 20.

    Kok, G. B. & Scammells, P. J. N-demethylation of N-methyl alkaloids with ferrocene. Bioorg. Med. Chem. Lett. 20, 4499–4502 (2010).

  21. 21.

    McCamley, K., Ripper, J. A., Singer, R. D. & Scammells, P. J. Efficient N-demethylation of opiate alkaloids using a modified nonclassical Polonovski reaction. J. Org. Chem. 68, 9847–9850 (2003).

  22. 22.

    Pham, D. D. D., Kelso, G. F., Yang, Y. Z. & Hearn, M. T. W. Studies on the oxidative N-demethylation of atropine, thebaine and oxycodone using a Fe-III-TAML catalyst. Green Chem. 16, 1399–1409 (2014).

  23. 23.

    Anastas, P. T. & Warner, J. C. Green Chemistry: Theory and Practice (Oxford Univ. Press, 1998).

  24. 24.

    Roschangar, F., Sheldon, R. A. & Senanayake, C. H. Overcoming barriers to green chemistry in the pharmaceutical industry – the Green Aspiration Level (TM) concept. Green Chem. 17, 752–768 (2015).

  25. 25.

    Augustin, M. M. et al. Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells. Plant J. 82, 991–1003 (2015).

  26. 26.

    Kilgore, M. B. et al. Cloning and characterization of a norbelladine 4’-O-methyltransferase involved in the biosynthesis of the Alzheimer’s drug galanthamine in Narcissus sp. aff. pseudonarcissus. PLoS ONE 9, e103223 (2014).

  27. 27.

    Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinformatics 10, 421 (2009).

  28. 28.

    Boratyn, G. M. et al. BLAST: a more efficient report with usability improvements. Nucleic Acids Res. 41, W29–W33 (2013).

  29. 29.

    Mockler, T. C. et al. The DIURNAL project: DIURNAL and circadian expression profiling, model-based pattern matching, and promoter analysis. Cold Spring Harb. Symp. Quant. Biol. 72, 353–363 (2007).

  30. 30.

    Finn, R. D. et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44, D279–D285 (2016).

  31. 31.

    Chistoserdova, L., Chen, S. W., Lapidus, A. & Lidstrom, M. E. Methylotrophy in Methylobacterium extorquens AM1 from a genomic point of view. J. Bacteriol. 185, 2980–2987 (2003).

  32. 32.

    Galli, R. & Leisinger, T. Specialized bacterial strains for the removal of dichloromethane from industrial-waste. Conserv. Recycl. 8, 91–100 (1985).

  33. 33.

    Kato, Y., Asahara, M., Arai, D., Goto, K. & Yokota, A. Reclassification of Methylobacterium chloromethanicum and Methylobacterium dichloromethanicum as later subjective synonyms of Methylobacterium extorquens and of Methylobacterium lusitanum as a later subjective synonym of Methylobacterium rhodesianum. J. Gen. Appl. Microbiol. 51, 287–299 (2005).

  34. 34.

    McDonald, I. R., Doronina, N. V., Trotsenko, Y. A., McAnulla, C. & Murrell, J. C. Hyphomicrobium chloromethanicum sp. nov. and Methylobacterium chloromethanicum sp. nov., chloromethane-utilizing bacteria isolated from a polluted environment. Int. J. Syst. Evol. Microbiol. 51, 119–122 (2001).

  35. 35.

    Guo, X. F. & Lidstrom, M. E. Physiological analysis of Methylobacterium extorquens AM1 grown in continuous and batch cultures. Arch. Microbiol. 186, 139–149 (2006).

  36. 36.

    Zanger, U. M. & Schwab, M. Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharm. Ther. 138, 103–141 (2013).

  37. 37.

    Summers, R. M. et al. Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids. J. Bacteriol. 194, 2041–2049 (2012).

  38. 38.

    Richter, N., Zepeck, F. & Kroutil, W. Cobalamin-dependent enzymatic O-, N-, and S-demethylation. Trends Biotechnol. 33, 371–373 (2015).

  39. 39.

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

  40. 40.

    Kunz, D. A., Reddy, G. S. & Vatvars, A. Identification of transformation products arising from bacterial oxidation of codeine by Streptomyces griseus. Appl. Environ. Microbiol. 50, 831–836 (1985).

  41. 41.

    Madyastha, K. M., Reddy, G. V. B., Nagarajappa, H. & Sridhar, G. R. N-Demethylation and N-oxidation of thebaine, an isoquinoline alkaloid by Mucor piriformis. Indian J. Chem. B 39, 377–381 (2000).

  42. 42.

    Hudlicky, T. Recent advances in process development for opiate-derived pharmaceutical agents. Can. J. Chem. 93, 492–501 (2015).

  43. 43.

    Kok, G., Ashton, T. D. & Scammells, P. J. An improved process for the N-demethylation of opiate alkaloids using an iron(II) catalyst in acetate buffer. Adv. Synth. Catal. 351, 283–286 (2009).

  44. 44.

    Kok, G. B. & Scammells, P. J. Efficient iron-catalyzed N-demethylation of tertiary amine-N-oxides under oxidative conditions. Aust. J. Chem. 64, 1515–1521 (2011).

  45. 45.

    Carrea, G., Colombi, F., Mazzola, G., Cremonesi, P. & Antonini, E. Immobilized hydroxysteroid dehydrogenases for the transformation of steroids in water—organic solvent systems. Biotechnol. Bioeng. 21, 39–48 (1979).

  46. 46.

    Fukui, S., Ikeda, S., Fujimura, M., Yamada, H. & Kumagai, H. Comparative studies on the properties of tryptophanase and tyrosine phenol-lyase immobilized directly on Sepharose or by use of Sepharose-bound pyridoxal 5’-phosphate. Eur. J. Biochem. 51, 155–164 (1975).

  47. 47.

    Schaaf, P. M., Heide, L. E., Leistner, E. W., Tani, Y. & el-Olemy, M. M. Immobilization of isochorismate hydroxymutase. J. Nat. Prod. 56, 1304–1312 (1993).

  48. 48.

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

  49. 49.

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

  50. 50.

    Nakagawa, A. et al. Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli. Nat. Commun. 7, 10390 (2016).

  51. 51.

    Chen, X. et al. A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis. Nat. Chem. Biol. 14, 738–743 (2018).

  52. 52.

    Vojkovsky, T. Detection of secondary amines on solid phase. Pept. Res 8, 236–237 (1995).

  53. 53.

    Bertani, G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62, 293–300 (1951).

  54. 54.

    Giannoukos, G. et al. Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes. Genome Biol. 13, R23 (2012).

  55. 55.

    Burriesci, M. S., Lehnert, E. M. & Pringle, J. R. Fulcrum: condensing redundant reads from high-throughput sequencing studies. Bioinformatics 28, 1324–1327 (2012).

  56. 56.

    Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

  57. 57.

    Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011).

  58. 58.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

  59. 59.

    Paoni, N. F. & Koshland, D. E. Jr. Permeabilization of cells for studies on the biochemistry of bacterial chemotaxis. Proc. Natl Acad. Sci. USA 76, 3693–3697 (1979).

  60. 60.

    Tartoff, K. D. & Hobbs, C. A. Improved media for growing plasmid and cosmid clones. Focus 9, 12 (1987).

  61. 61.

    Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729 (2013).

  62. 62.

    Jones, D. T., Taylor, W. R. & Thornton, J. M. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282 (1992).

  63. 63.

    Kumar, S., Stecher, G. & Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).

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Acknowledgements

The authors would like to acknowledge support from The Proteomics and Mass Spectrometry Facility at The Donald Danforth Plant Science Center regarding assistance with the QTRAP 6500, which was acquired by funding from the National Science Foundation (grant No. DBI-1427621), and for amino acid sequence analysis. We would also like to acknowledge support from the William H. Danforth Fellowship in Plant Sciences granted to J.R.B. We would like to thank H. Berg of the Integrated Microscopy Facility at the Donald Danforth Plant Science Center for images. The authors would also like to thank N. Grobe for culture stock maintenance. We would like to dedicate this manuscript to the late Professor Dr M. H. Zenk, whose contributions to the field of plant biochemistry and natural products remain unparallelled, whose scientific enquiry was an inspiration to all who worked with him and without whom this work would not have been possible.

Author information

Sludge material was obtained by T.M.K. Bacterial and RNA isolation was performed by M.M.A. The Thebainfresser transcriptome was assembled and annotated by J.M.A. Gene expression was compiled by J.M.A. Phylogenetic analysis of Thebainfresser 16 S rRNA was completed by J.R.B. HAYSTACK analysis, candidate gene curation and cloning were performed by M.M.A. Experiments for morphinan N-demethylase characterization were planned by T.M.K. and M.M.A. and carried out by M.M.A. Figures were prepared by M.M.A. and J.R.B. The manuscript was prepared by T.M.K. and M.M.A.

Correspondence to M. M. Augustin or T. M. Kutchan.

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The authors declare the filing of the international patent application PCT/US2018/014271.

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