A microbial biomanufacturing platform for natural and semisynthetic opioids

Abstract

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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Acknowledgements

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.).

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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.

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Correspondence to Christina D Smolke.

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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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