Production of the antimalarial drug precursor artemisinic acid in engineered yeast

Ro, Dae-Kyun; Paradise, Eric M.; Ouellet, Mario; Fisher, Karl J.; Newman, Karyn L.; Ndungu, John M.; Ho, Kimberly A.; Eachus, Rachel A.; Ham, Timothy S.; Kirby, James; Chang, Michelle C. Y.; Withers, Sydnor T.; Shiba, Yoichiro; Sarpong, Richmond; Keasling, Jay D.

doi:10.1038/nature04640

Cited by 2,805Plant biochemistry and biosynthesisMicrobial Metabolic Engineering and BioproductionFungal and yeast genetics research

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

Ro, Dae-Kyun, Paradise, Eric M., Ouellet, Mario, Fisher, Karl J., Newman, Karyn L., Ndungu, John M., Ho, Kimberly A., Eachus, Rachel A. +7 more·2006·Nature

DOI 10.1038/nature04640

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Abstract

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. 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 sufferers. Although total synthesis of artemisinin is difficult and costly, 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 artemisinin. 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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CYP71AV1 catalyses the three-step conversion of amorpha-4,11-diene to artemisinic acid.
“We have chosen to target CYP71AV1, which catalyses the three-step conversion of amorpha-4,11-diene to artemisinic acid (13, 26, 27).”
From Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism (2016) · cites this paper as [26]
Compared to WT and heterozygous material, the juvenile leaves of cyp71av1-1 contain significantly elevated levels of amorpha-4,11-diene (A-4,11-D).
“Compared to WT and heterozygous material, the juvenile leaves of cyp71av1-1 contain significantly elevated levels of the first committed metabolite in the artemisinin pathway, amorpha4,11-diene (A-4,11-D; Fig. 1A i), Supplemental T ables 2 and 4), consistent with the reported in vitro activity of CYP71AV1 (13, 26, 27).”
From Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism (2016) · cites this paper as [26]
The approach outlined in Scheme 13 has succeeded in producing high levels of amorpha-4,11-diene from E. coli and artemisinic acid from S. cerevisiae in quantities exceeding those from A. annua itself (100 mg-1 g L-1).
“The approach outlined in Scheme 13 has succeded in producing high levels of amorpha-4,11-diene (451) from E. coli [384] and artemisinic acid (473) from S. cerevisiae in quantities which exceed those from A. annua itself (100 mg–1·g·L-1) [385,386].”
From The Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao) (2010) · cites this paper as [384]

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Authors: Ro, D.-K., Paradise, E. M., Ouellet, M., Fisher, K. J., Newman, K. L., Ndungu, J. M., Ho, K. A., Eachus, R. A., Ham, T. S., Kirby, J., Chang, M. C. Y., Withers, S. T., Shiba, Y., Sarpong, R., & Keasling, J. D.
Affiliations: QB3 (US)
Amyris (United States) (US)
Corresponding author: Keasling, Jay D.
ORCID: Ro, Dae-Kyun
Newman, Karyn L.
Ndungu, John M.
Kirby, James
Chang, Michelle C. Y.
Sarpong, Richmond
Keasling, Jay D.
Venue: Nature
Publisher: Springer Science and Business Media LLC
Published: 2006-04-13
Volume: 440
Issue: 7086
Pages: 940-943
ISSN: 0028-0836 (ISSN-L)
1476-4687
DOI: 10.1038/nature04640
OpenAlex: W2149202606
Cited by: 2,805
References: 25
Citations by year: 2026: 86 · 2025: 94 · 2024: 132 · 2023: 115 · 2022: 133 · 2021: 120 · 2020: 151 · 2019: 131 · 2018: 155 · 2017: 153 · 2016: 165 · 2015: 165 · 2014: 190 · 2013: 191 · 2012: 230
FWCI: 40.83
Type: journal article
Language: en
Open access: closed
Licence: www.springer.com/tdm (tdm)
www.springer.com/tdm (vor)
Locations: Nature (publishedVersion)
PubMed (publishedVersion)
RePEc: Research Papers in Economics (submittedVersion)
Research Explorer (The University of Manchester) (submittedVersion)
Funders: National Science Foundation
U.S. Department of Agriculture
Bill and Melinda Gates Foundation
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
Keywords: artemisinic acid, artemisinin, cytochrome P450 monooxygenase, CYP71AV1, Artemisia annua, Saccharomyces cerevisiae, microbial production, mevalonate pathway, sesquiterpene lactone biosynthesis, malaria
MeSH terms: Animals, Antimalarials, Cytochrome P-450 Enzyme System, Fermentation, Genetic Engineering, Gas Chromatography-Mass Spectrometry, Mevalonic Acid, Molecular Sequence Data, Plasmodium falciparum, Saccharomyces cerevisiae, Sesquiterpenes, Drug Costs, Malaria, Falciparum, Bioreactors, Artemisia annua, Artemisinins

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Production of the antimalarial drug precursor artemisinic acid in engineered yeast

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