Production of the antimalarial drug precursor artemisinic acid in engineered yeast.
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.
Ro
Dae-Kyun
DK
California Institute of Quantitative Biomedical Research, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA.
Paradise
Eric M
EM
Ouellet
Mario
M
Fisher
Karl J
KJ
Newman
Karyn L
KL
Ndungu
John M
JM
Ho
Kimberly A
KA
Eachus
Rachel A
RA
Ham
Timothy S
TS
Kirby
James
J
Chang
Michelle C Y
MC
Withers
Sydnor T
ST
Shiba
Yoichiro
Y
Sarpong
Richmond
R
Keasling
Jay D
JD
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*
D000962:Antimalarials; D037621:Artemisinins; D012717:Sesquiterpenes; C047721:artemisic acid; D003577:Cytochrome P-450 Enzyme System; C031327:artemisinin; D008798:Mevalonic Acid