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

Abstract

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.

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

“…It is interesting to note that the AD yield (18 mg/L/OD) using the DXP pathway was among some of the highest yields reported to date Zhou et al 2013). However, as compared to the highest AD yield using the mevalonate (MVA) pathway in Saccharomyces cerevisiae (242 mg/L/ OD) (Westfall et al 2012), our yield was~13-fold lower. Preliminary study of optimizing the MVA pathway using EDASPO resulted in a comparable AD yield of more than 200 mg/L/OD in E. coli (unpublished observation), demonstrating the broader utility of this approach.…”

Carnosic acid biosynthesis elucidated by a synthetic biology platform

“…1A) have strong antiparasitic, antifungal, and antibacterial activities (17)(18)(19). These compounds belong to the group of labdane-type diterpenes (20), which also includes several other members with bioactive properties, such as tanshinones (2)(3)(4), which exhibit strong antiinflammatory activity (21) and are effective against various cardiovascular and cerebrovascular disorders (22,23), and forskolin (5), which activates adenylyl cyclase and increases the intracellular levels of cAMP (24)(25)(26) (Fig. 1A).…”

Inhibitory cross-talk upon introduction of a new metabolic pathway into an existing metabolic network

“…Synthetic biologists have generated metabolic pathways that do not exist in nature for synthesis of platform chemicals such as isobutyric acid (4), specialty chemicals such as vanillin (5), and a variety of secondary metabolites that have potential uses as therapeutic agents (6,7). Notable successes include the microbial syntheses of amorphadiene (8), a precursor of the antimalarial drug artemisinin, and taxadiene, the precursor of the potent anticancer drug Taxol (9). Assembly of a pathway that degrades the toxic insecticide paraoxon has also been reported (10).…”