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 (...

Artemisinin, a sesquiterpene lactone endoperoxide derived from the plant Artemisia annua, forms the basis of the most important treatments of malaria in use today. In an effort to elucidate the biosynthesis of artemisinin, an expressed sequence tag approach to identifying the relevant biosynthetic genes was undertaken using isolated glandular trichomes as a source of mRNA. A cDNA clone encoding a cytochrome P450 designated CYP71AV1 was characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of the proposed biosynthetic intermediates amorpha-4,11-diene, artemisinic alcohol and artemisinic aldehyde. The identification of the CYP71AV1 gene should allow for the engineering of semi-synthetic production of artemisinin in appropriate plant or microbial hosts. Crown

In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) i n the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS1 1, which, in comparison to wild type (WT), has reduced levels of 20:l and 18:l and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Cenetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAC1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity t o elongate 18:l or to an increase in the relative A1 5 or A1 2 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS1 1 seed homogenates. Rather, the fatty acid phenotype of AS1 1 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:l biosynthesis during seed development, leaving more 18:l available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result i n dramatic changes i n fatty acid composition and, in particular, i n the accumulation of VLCFAs i n seed storage lipids.The fatty acyl composition of seed TAGs determines their physical and chemical properties and, thus, their use in edible oil or industrial applications. TAG composition depends on the interaction of several different groups of enzymes in the lipid biosynthesis pathway. The enzymes of the fatty acid synthase complex in the plastids of developing seeds are responsible for the biosynthesis of fatty acids up to and including oleic acid. Modifying enzymes, such as the extraplastidic A12 and A15 desaturases, elongases, hydroxylases, and epoxidases, yield polyunsaturated, very long-chain, hydroxy-, and epoxy-fatty acids, respectively. Acyltransferases insert specific acyl moieties onto the glyc-

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the ⌬11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde ⌬11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small ␣,␤-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.Since its discovery in the 1970s (1), the sesquiterpene lactone artemisinin (Fig. 1) from Artemisia annua has become a key factor in the drive to control malaria (2-4). Semi-synthetic derivatives of plant-derived artemisinin form the basis for artemisinin-based combination therapies, which are the treatment of choice for most forms of the disease. Given the pharmaceutical importance of artemisinin and its singular plant source, there is considerable interest in maintaining a reliable and low cost supply (5). A more complete knowledge of artemisinin biosynthesis and the genes involved is likely to provide ways of increasing production and lowering cost through crop improvement or microbial engineering (6, 7).As in other members of the Asteraceae, A. annua has 10-celled biseriate glandular trichomes that appear on the surfaces of aerial parts of the plant (8 -10). Along with other isoprenoids, the sesquiterpene lactone artemisinin accumulates to levels of 0.01-2% dry weight (11). Although progress is being made in understanding the biosynthesis of artemisinin, considerable gaps in our knowledge remain (6, 12). The formation of amorpha-4,11-diene by amorpha-4,11-diene synthase is the first committed step in the pathway. This is followed by oxidation at C-12 of amorpha-4,11-diene by the cytochrome P-450, Cyp71av1 to give artemisinic alcohol. These steps are well supported from biochemical studies and by the molecular cloning of genes encoding the relevant enzymes (12-14). The pathway beyond artemisinic alcohol is somewhat less well established (6, 7, 15). However, there is biochemical evidence supporting a route to dihydroartemsinic acid via artemisinic aldehyde and dihydroartemsinic aldehyde (12). This route includes the proposed reduction of the ⌬11(13) double bond of artemisinic aldeh...

Summary Biosynthesis of the sesquiterpene lactone and potent antimalarial drug artemisinin occurs in glandular trichomes of Artemisia annua plants and is subjected to a strict network of developmental and other regulatory cues. The effects of three hormones, jasmonate, gibberellin and cytokinin, were studied at the structural and molecular levels in two different A. annua chemotypes by microscopic analysis of gland development, and by targeted metabolite and transcript profiling. Furthermore, a genome‐wide cDNA‐amplified fragment length polymorphism (AFLP)‐based transcriptome profiling was carried out of jasmonate‐elicited leaves at different developmental stages. Although cytokinin and gibberellin positively affected at least one aspect of gland formation, these two hormones did not stimulate artemisinin biosynthesis. Only jasmonate simultaneously promoted gland formation and coordinated transcriptional activation of biosynthetic gene expression, which ultimately led to increased sesquiterpenoid accumulation with chemotype‐dependent effects on the distinct pathway branches. Transcriptome profiling revealed a trichome‐specific fatty acyl‐ coenzyme A reductase, trichome‐specific fatty acyl‐CoA reductase 1 (TFAR1), the expression of which correlates with trichome development and sesquiterpenoid biosynthesis. TFAR1 is potentially involved in cuticular wax formation during glandular trichome expansion in leaves and flowers of A. annua plants. Analysis of phytohormone‐modulated transcriptional regulons provides clues to dissect the concerted regulation of metabolism and development of plant trichomes.