Malaria currently affects one half of the world's population with an estimated 100-200 million cases and about 1 million deaths annually. Incidence of the disease is increasing due to drug resistance of Plasmodium falciparum. A promising therapeutic, artemisinin, is a terpenoid found at low levels (1% or less) in the herb, Artemisia annua. In tissue cultured plants it occurs at even lower levels. Higher production levels, in either native plants or their cultured tissues, could make therapeutic use of artemisinin and its derivatives feasible on a large scale. Our long-range goal is to complete the bench-scale process studies needed for pilot- scale demonstration of production of artemisinin from transformed Artemisia roots. This work is divided into 4 major objectives to be accomplished over 3 years: 1.) 0ptimize culture conditions to maximize biomass and artemisinin (and related compounds) productivity. Factors to be optimized include culture nutrients (e.g. phosphate, nitrogen, sugar, CO2 etc.), environmental parameters (light, pH, temperature, etc.), and other factors (phytohormones, inoculum age, elicitors etc.) using methods of fractional factorial experimental design. 2.) Minimize artemisinin degradation by peroxidase. Both biomass and artemisinin productivity will be measured and compared with peroxidase activity in the presence of POD inhibitors. 3.) Compare artemisinin recovery processes. Methods using solvents and adsorbents for the nonlethal recovery of artemisinin from root cultures will be studied to maximize product recovery. 4.) Evaluate potential culture systems. Several promising types of bench-scale bioreactors will be used to determine which is most suitable for culture and production of artemisinin from transformed roots using measurements of productivity (g 1-1 day-1). Using the results obtained by these studies, conditions for growth and AN production will be defined. This fundamental information will represent a major advance towards establishing the potential of using root cultures for the production of the antimalarial therapeutic, artemisinin, as well as other plant derived therapeutics.
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