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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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.
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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.
The authors declare the filing of the international patent application PCT/US2018/014271.
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Supplementary Methods, Supplementary References 1–7, Supplementary Figs. 1–11, Supplementary Tables 1–5
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Augustin, M.M., Augustin, J.M., Brock, J.R. et al. Enzyme morphinan N-demethylase for more sustainable opiate processing. Nat Sustain 2, 465–474 (2019). https://doi.org/10.1038/s41893-019-0302-6