1. California Institute of Quantitative Biomedical Research,
2. Department of Chemical Engineering,
3. Department of Chemistry,
4. Department of Bioengineering, and
5. Berkeley Center for Synthetic Biology, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA
6. Amyris Biotechnologies Inc., Emeryville, California 94608, USA
7. *These authors contributed equally to this work

Correspondence to: Jay D. Keasling^1,2,4,5 Correspondence and requests for materials should be addressed to J.D.K. (Email: keasling@berkeley.edu). Artemisia annua CYP71AV1 and CPR gene sequence information has been deposited in GenBank under accession numbers DQ268763 and DQ318192, respectively.

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 falciparum^{2, 3}. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing^{4, 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 artemisinin^{8, 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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