Understanding final steps of artemisinin synthesis in Artemisia annua

Abstract: Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for malaria currently available.  The CNAP Artemisia project developed A. annua F1 hybrids with increased artemisinin yields of 1.4% dry leaf biomass, which are now being used for commercial production of Artemisinin Combination Therapies.

Previously, the biochemical pathways for artemisinin biosynthesis had been fully elucidated; this enabled synthetic biologists, led by Jay Keasling, to transfer the pathway into yeast paving the way to semi-synthetic production of artemisinic acid at remarkable yields of 25g/L.  However, artemisinic acid has to be converted into artemisinin via a series of costly photochemical reactions. Despite its initial success, semi-synthetic artemisinin has not been successful commercially, principally due to cost of production. There is therefore an urgent need to understand the final steps of artemisinin synthesis in A. annua, which might enable transferring its production to microbial systems. 

We have identified a mutation that disrupts the CYP71AV1 enzyme, responsible for a series of oxidation reactions in the artemisinin biosynthetic pathway. Detailed metabolic studies of cyp71av1-1 revealed that the consequence of blocking the artemisinin biosynthetic pathway is the redirection of sesquiterpene metabolism to a novel sesquiterpene epoxide, which we designate arteannuin X.  This sesquiterpene approaches half the concentration observed for artemisinin in wild type plants, demonstrating high-flux plasticity in A. annua glandular trichomes and their potential as factories for the production of novel alternate sesquiterpenes at commercially viable levels. 

Detailed metabolite profiling of leaves and precursor-feeding experiments revealed that non-enzymatic conversion steps are central to both artemisinin and arteannuin X biosynthesis. In particular, feeding studies using 13C-labelled dihydroartemisinic acid (DHAA) provided strong evidence that the final steps of artemisinin synthesis are non-enzymatic and light-dependant in vivo. Our findings also suggest that the specialised sub-apical cavity of glandular secretory trichomes functions as a location for both the chemical conversion and storage of phytotoxic compounds, including artemisinin. 

We conclude that metabolic engineering to produce high yields of novel secondary compounds such as sesquiterpenes is feasible in glandular trichomes. Such systems offer advantages over single cell microbial hosts for production of toxic natural products. Furthermore, our work highlights the potential for improving plant-based artemisinin production.