Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin

<jats:p> Malaria, caused by <jats:italic>Plasmodium sp</jats:italic> , 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 <jats:italic>Artemisia annua</jats:italic> . 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 <jats:italic>Saccharomyces cerevisiae</jats:italic> , and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used <jats:italic>S. cerevisiae</jats:italic> S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) <jats:italic>Nature</jats:italic> 440:940–943]. We have now overexpressed every enzyme of the mevalonate pathway to <jats:italic>ERG20</jats:italic> in <jats:italic>S. cerevisiae</jats:italic> 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. </jats:p>