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 (...

Isoprenoids are the most numerous and structurally diverse family of natural products. Terpenoids, a class of isoprenoids often isolated from plants, are used as commercial flavor and fragrance compounds and antimalarial or anticancer drugs. Because plant tissue extractions typically yield low terpenoid concentrations, we sought an alternative method to produce high-value terpenoid compounds, such as the antimalarial drug artemisinin, in a microbial host. We engineered the expression of a synthetic amorpha-4,11-diene synthase gene and the mevalonate isoprenoid pathway from Saccharomyces cerevisiae in Escherichia coli. Concentrations of amorphadiene, the sesquiterpene olefin precursor to artemisinin, reached 24 microg caryophyllene equivalent/ml. Because isopentenyl and dimethylallyl pyrophosphates are the universal precursors to all isoprenoids, the strains developed in this study can serve as platform hosts for the production of any terpenoid compound for which a terpene synthase gene is available.

Malaria, caused by Plasmodium sp , results in almost one million deaths and over 200 million new infections annually. The World Health Organization has recommended that artemisinin-based combination therapies be used for treatment of malaria. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua . However, the supply and price of artemisinin fluctuate greatly, and an alternative production method would be valuable to increase availability. We describe progress toward the goal of developing a supply of semisynthetic artemisinin based on production of the artemisinin precursor amorpha-4,11-diene by fermentation from engineered Saccharomyces cerevisiae , and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used S. cerevisiae S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) Nature 440:940–943]. We have now overexpressed every enzyme of the mevalonate pathway to ERG20 in S. cerevisiae CEN.PK2, and compared production to CEN.PK2 engineered identically to the previously engineered S288C strain. Overexpressing every enzyme of the mevalonate pathway doubled artemisinic acid production, however, amorpha-4,11-diene production was 10-fold higher than artemisinic acid. We therefore focused on amorpha-4,11-diene production. Development of fermentation processes for the reengineered CEN.PK2 amorpha-4,11-diene strain led to production of > 40 g/L product. A chemical process was developed to convert amorpha-4,11-diene to dihydroartemisinic acid, which could subsequently be converted to artemisinin. The strains and procedures described represent a complete process for production of semisynthetic artemisinin.

Many applications of synthetic biology require the balanced expression of multiple genes. Although operons facilitate coordinated expression of multiple genes in prokaryotes and eukaryotes, coordinating the many post-transcriptional processes that determine the relative levels of gene expression in operons by a priori design remains a challenge. We describe a method for tuning the expression of multiple genes within operons by generating libraries of tunable intergenic regions (TIGRs), recombining various post-transcriptional control elements and screening for the desired relative expression levels. TIGRs can vary the relative expression of two reporter genes over a 100-fold range and balance expression of three genes in an operon that encodes a heterologous mevalonate biosynthetic pathway, resulting in a sevenfold increase in mevalonate production. This technology should be useful for optimizing the expression of multiple genes in synthetic operons, both in prokaryotes and eukaryotes.

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