In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths 1 . The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers 2 . A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid 3 . Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.Before the discovery of the enzymes that complete the biosynthetic pathway of artemisinin production (see Supplementary Fig. 1 for a complete overview), several improvements were made to the original amorphadiene-producing strain Y337 (ref. 3). We replaced the MET3 promoter with the copper-regulated CTR3 promoter (Fig. 1a), enabling restriction of ERG9 expression (ERG9 encodes squalene synthase, which catalyses the competing reaction of joining two farnesyl diphosphate moieties to form squalene) by addition of the inexpensive repressor CuSO 4 to the medium rather than the more expensive methionine 4-6 . Strains Y1516 (P CTR3 -ERG9) and Y337 (P MET3 -ERG9) (Supplementary Table 1) both produced similar amounts of amorphadiene ( Supplementary Fig. 2), demonstrating the equivalence of the MET3 and CTR3 promoters for repression of ERG9 expression. We compared the production of amorphadiene from Y337 with the production of artemisinic acid from Y285, a variant of Y337 that also expressed the amorphadiene oxidase CYP71AV1 (a cytochrome P450) and A. annua CPR1 (...

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