title: 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 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.
authors.affiliation: California Institute of Quantitative Biomedical Research, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA.
authors.forename: Dae-Kyun
authors.initials: DK
authors.lastname: Ro
authors.forename: Eric M
authors.initials: EM
authors.lastname: Paradise
authors.forename: Mario
authors.initials: M
authors.lastname: Ouellet
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Fisher
authors.forename: Karyn L
authors.initials: KL
authors.lastname: Newman
authors.forename: John M
authors.initials: JM
authors.lastname: Ndungu
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Ho
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Eachus
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Ham
authors.forename: James
authors.initials: J
authors.lastname: Kirby
authors.forename: Michelle C Y
authors.initials: MC
authors.lastname: Chang
authors.forename: Sydnor T
authors.initials: ST
authors.lastname: Withers
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Shiba
authors.forename: Richmond
authors.initials: R
authors.lastname: Sarpong
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000737:chemistry / Q000191:economics / Q000378:metabolism*; D031172:Artemisia annua / Q000201:enzymology / Q000235:genetics; D037621:Artemisinins / Q000737:chemistry / Q000191:economics / Q000378:metabolism*; D019149:Bioreactors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D016527:Drug Costs / Q000639:trends; D005285:Fermentation; D008401:Gas Chromatography-Mass Spectrometry; D005818:Genetic Engineering; D016778:Malaria, Falciparum / Q000188:drug therapy* / Q000191:economics; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D012717:Sesquiterpenes / Q000737:chemistry / Q000191:economics / Q000378:metabolism*
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D012717:Sesquiterpenes; C047721:artemisic acid; D003577:Cytochrome P-450 Enzyme System; C031327:artemisinin; D008798:Mevalonic Acid