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Production of the antimalarial drug precursor artemisinic acid in engineered yeast

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.

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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.

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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 pδ-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.

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Correspondence to Jay D. Keasling.

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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.

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

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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

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