Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

Nature volume 440, pages 940–943 (2006)Cite this article

Abstract

Malaria is a global health problem that threatens 300–500 million people and kills more than one million people annually1. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum2,3. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing4,5. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers6. Although total synthesis of artemisinin is difficult and costly7, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin8,9. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l-1) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic representation of the engineered artemisinic acid biosynthetic pathway in S. cerevisiae strain EPY224 expressing CYP71AV1 and CPR.
Figure 2: Production of amorphadiene by S. cerevisiae strains.
Figure 3: GC–MS analysis of artemisinic acid produced from A. annua and transgenic yeast.
Figure 4: In vitro enzyme assays for CYP71AV1 activities.

Similar content being viewed by others

References

  1. World Health Organization. World Malaria Report 2005. (WHO, Geneva, 2005)

    Book  Google Scholar 

  2. Korenromp, E. L., Williams, B. G., Gouws, E., Dye, C. & Snow, R. W. Measurement of trends in childhood malaria mortality in Africa: An assessment of progress toward targets based on verbal autopsy. Lancet Infect. Dis. 3, 349–358 (2003)

    Article  Google Scholar 

  3. Marsh, K. Malaria disaster in Africa. Lancet 352, 924 (1998)

    Article  CAS  Google Scholar 

  4. Hoffman, S. Save the children. Nature 430, 940–941 (2004)

    Article  ADS  CAS  Google Scholar 

  5. Vennerstrom, J. L. et al. Identification of an antimalarial synthetic trioxolane drug development candidate. Nature 430, 900–904 (2004)

    Article  ADS  CAS  Google Scholar 

  6. Enserink, M. Infectious diseases: Source of new hope against malaria is in short supply. Science 307, 33 (2005)

    Article  CAS  Google Scholar 

  7. Schmid, G. & Hofheinz, W. Total synthesis of Qinghaosu. J. Am. Chem. Soc. 105, 624–625 (1983)

    Article  CAS  Google Scholar 

  8. Acton, N. & Roth, R. J. On the conversion of dihydroartemisinic acid into artemisinin. J. Org. Chem. 57, 3610–3614 (1992)

    Article  CAS  Google Scholar 

  9. Haynes, R. K. & Vonwiller, S. C. Cyclic peroxyacetal lactone, lactol and ether compounds. US patent 5,310,946 (1994).

  10. Mercke, P., Bengtsson, M., Bouwmeester, H. J., Posthumus, M. A. & Brodelius, P. E. Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L. Arch. Biochem. Biophys. 381, 173–180 (2000)

    Article  CAS  Google Scholar 

  11. Martin, V. J., Pitera, D. J., Withers, S. T., Newman, J. D. & Keasling, J. D. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotechnol. 21, 796–802 (2003)

    Article  CAS  Google Scholar 

  12. Donald, K., Hampton, R. & Fritz, I. Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 63, 3341–3344 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Gardner, R. G. & Hampton, R. Y. A highly conserved signal controls degradation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in eukaryotes. J. Biol. Chem. 274, 31671–31678 (1999)

    Article  CAS  Google Scholar 

  14. Davies, B. S. J., Wang, H. S. & Rine, J. Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms. Mol. Cell. Biol. 25, 7375–7385 (2005)

    Article  CAS  Google Scholar 

  15. Jackson, B. E., Hart-Wells, E. A. & Matsuda, S. P. T. Metabolic engineering to produce sesquiterpenes in yeast. Org. Lett. 5, 1629–1632 (2003)

    Article  CAS  Google Scholar 

  16. Seaman, F. C. Sesquiterpene lactones as taxonomic characters in the Asteraceae. Bot. Rev. 48, 121–592 (1982)

    Article  CAS  Google Scholar 

  17. Bertea, C. M. et al. Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–47 (2005)

    Article  CAS  Google Scholar 

  18. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990)

    Article  CAS  Google Scholar 

  19. Lupien, S., Karp, F., Wildung, M. & Croteau, R. Regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha) species: cDNA isolation, characterization, and functional expression of (- )-4S-limonene-3-hydroxylase and (- )-4S-limonene-6-hydroxylase. Arch. Biochem. Biophys. 368, 181–192 (1999)

    Article  CAS  Google Scholar 

  20. Ralston, L. et al. Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum). Arch. Biochem. Biophys. 393, 222–235 (2001)

    Article  CAS  Google Scholar 

  21. Wang, E. et al. Suppression of a P450 hydroxylase gene in plant trichome glands enhances natural-product-based aphid resistance. Nature Biotechnol. 19, 371–374 (2001)

    Article  CAS  Google Scholar 

  22. Elmarakby, S. A., El-Feraly, F. S., Elsohly, H. N., Croom, E. M. & Hufford, C. D. Microbiological transformations of artemisinic acid. Phytochemistry 27, 3089–3091 (1988)

    Article  CAS  Google Scholar 

  23. Kim, S., Han, J. & Lim, Y. Revised assignment of 1H-NMR signals of artemisinic acid. Planta Med. 62, 480–481 (1996)

    Article  CAS  Google Scholar 

  24. Helliwell, C. A., Poole, A., Peacock, W. J. & Dennis, E. S. Arabidopsis ent-kaurene oxidase catalyzes three steps of gibberellin biosynthesis. Plant Physiol. 119, 507–510 (1999)

    Article  CAS  Google Scholar 

  25. Helliwell, C. A., Chandler, P. M., Poole, A., Dennis, E. S. & Peacock, W. J. The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway. Proc. Natl Acad. Sci. USA 98, 2065–2070 (2001)

    Article  ADS  CAS  Google Scholar 

  26. Pompon, D., Louerat, B., Bronine, A. & Urban, P. Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol. 272, 51–64 (1996)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Nelson for assigning an official CYP number, and D. Ockey and D. McPhee for artemisinic acid isolation from the A. annua plant. We are also grateful to N. A. Da Silva for pÎŽ-UB, R. Y. Hampton for pRH127-3 and pRH973, and J. Rine for pBD33 and pBD36. We thank R. Michelmore and other members in the Compositae Genomics Project for the support of this project. This research was conducted under the sponsorship of the Institute for OneWorld Health, through the generous support of The Bill and Melinda Gates Foundation, and through funding from the Akibene Foundation, the United States Department of Agriculture, a University of California Discovery Grant, the Diversa Corporation and the National Science Foundation. Author Contributions D.-K.R., E.M.P. and J.D.K. designed the project and experiments. D.-K.R., E.M.P., Y.S., M.C.Y.C., S.T.W. and J.K. performed experiments. K.J.F. conducted NMR analysis of artemisinic acid. J.M.N. and R.S. semi-synthesized artemisinic alcohol and artemisinic aldehyde. T.S.H. performed bioinformatics analysis of the Compositae EST-database. M.O., R.A.E. and K.A.H. provided technical assistance. D.-K.R., E.M.P., K.L.N. and J.D.K. wrote the paper. All authors discussed the results and commented on the manuscript.

Author information

Author notes

  1. Dae-Kyun Ro and Eric M. Paradise: *These authors contributed equally to this work

Authors and Affiliations

  1. California Institute of Quantitative Biomedical Research,

    Dae-Kyun Ro, Mario Ouellet, Karyn L. Newman, Kimberly A. Ho, Rachel A. Eachus, Michelle C. Y. Chang & Jay D. Keasling

  2. Department of Chemical Engineering,

    Eric M. Paradise, James Kirby, Sydnor T. Withers, Yoichiro Shiba & Jay D. Keasling

  3. Department of Chemistry,

    John M. Ndungu & Richmond Sarpong

  4. Department of Bioengineering,

    Timothy S. Ham & Jay D. Keasling

  5. Berkeley Center for Synthetic Biology, Lawrence Berkeley National Laboratory, University of California, Berkeley, California, 94720, USA

    Jay D. Keasling

  6. Amyris Biotechnologies Inc., Emeryville, California, 94608, USA

    Karl J. Fisher

Authors
  1. Dae-Kyun Ro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eric M. Paradise
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mario Ouellet
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karl J. Fisher
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Karyn L. Newman
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John M. Ndungu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kimberly A. Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rachel A. Eachus
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Timothy S. Ham
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. James Kirby
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Michelle C. Y. Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Sydnor T. Withers
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yoichiro Shiba
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Richmond Sarpong
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Jay D. Keasling
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jay D. Keasling.

Ethics declarations

Competing interests

J.D.K. is a founder of Amyris Biotechnologies, a company that may eventually use the genes and yeast strains described in this paper for the production of artemisinin. However, neither Amyris Biotechnologies nor the University of California will make any profit from the production and sale of the antimalarial drug artemisinin.

Supplementary information

Supplementary Data

This file contains Supplementary Figure 1, Supplementary Methods, Supplementary Discussion, and Supplementary Tables I, II and III. (DOC 98 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ro, DK., Paradise, E., Ouellet, M. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006). https://doi.org/10.1038/nature04640

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature04640

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing