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.

Production of fine heterologus pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved and enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450 BM3 from Bacillus megaterium. Using a computer model, we illustrate how key P450 BM3 activ site mutations enable binding of non-native substrate amorphadiene, incorporating these mutations into P450 BM3 enabled the selective oxidation of amorphadiene arteminsinic-11s,12-epoxide, at titers of 250 mg L "1 in E. coli. We also demonstrate high-yeilding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high value anti-malarial drug artemisinin.

The quality of combinatorial libraries determines the success of biological screening in drug discovery programs. In this paper, we evaluate and compare various methods for measuring identity, purity, and quantity (yield) of combinatorial libraries. Determination of quantitative purity reveals the true library quality and often indicates potential quality problems before full-scale library production. The relative purity can be determined for every member in a large library in a high-throughput mode, but must be cautiously interpreted. In particular, many impurities are not observable by relative purity measurements using detectors such as UV(214), UV(254), and evaporative light-scattering detection. These "invisible" impurities may constitute a significant portion of the sample weight. We found that TFA, plastic extracts, inorganic compounds, and resin washout are among these impurities. With compelling evidence, we reach a conclusion that purification is the only way to remove "invisible" impurities and improve the quantitative purity of any compound even though some compounds may have a high relative purity before purification.

No abstract

No abstract