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 (...

Background: The uptake of nutrients, expulsion of metabolic wastes and maintenance of ion homeostasis by the intraerythrocytic malaria parasite is mediated by membrane transport proteins. Proteins of this type are also implicated in the phenomenon of antimalarial drug resistance. However, the initial annotation of the genome of the human malaria parasite Plasmodium falciparum identified only a limited number of transporters, and no channels. In this study we have used a combination of bioinformatic approaches to identify and attribute putative functions to transporters and channels encoded by the malaria parasite, as well as comparing expression patterns for a subset of these.

As the malaria parasite, Plasmodium falciparum, grows within its host erythrocyte it induces an increase in the permeability of the erythrocyte membrane to a range of low-molecular-mass solutes, including Na+ and K+ (ref. 1). This results in a progressive increase in the concentration of Na+ in the erythrocyte cytosol. The parasite cytosol has a relatively low Na+ concentration and there is therefore a large inward Na+ gradient across the parasite plasma membrane. Here we show that the parasite exploits the Na+ electrochemical gradient to energize the uptake of inorganic phosphate (P(i)), an essential nutrient. P(i) was taken up into the intracellular parasite by a Na+-dependent transporter, with a stoichiometry of 2Na+:1P(i) and with an apparent preference for the monovalent over the divalent form of P(i). A P(i) transporter (PfPiT) belonging to the PiT family was cloned from the parasite and localized to the parasite surface. Expression of PfPiT in Xenopus oocytes resulted in Na+-dependent P(i) uptake with characteristics similar to those observed for P(i) uptake in the parasite. This study provides new insight into the significance of the malaria-parasite-induced alteration of the ionic composition of its host cell.

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study,

PfSulP, encoded by the human malaria parasite Plasmodium falciparum, is a member of the sulphate permease family of anion transporter proteins. By transfecting the parasite with an epitope-tagged version of PfSulP, and detecting via western blot and indirect immunofluorescent assay microscopy, we show that PfSulP is localised to the surface of the intraerythrocytic parasite, where it is postulated to play a role in the flux of anions across the parasite plasma membrane.