Artemisinin Biosynthesis: Control of Transcription

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DESCRIPTION (provided by applicant): Artemisinin (AN), and its derivatives are proven effective antimalarial therapeutics that also been shown to have effective activity as broad spectrum antimicrobials, and anticancer therapeutics. AN is a complex compound and still best produced by the plant Artemisia annua. Little is known, however, about how AN biosynthesis is regulated, although the following is known about its production: more AN or its precursor, artemisinic acid (AA), is made when plants shift from vegetative growth to flowering, or grown on glucose instead of sucrose (our recent data). Since these conditions strongly affect AN production, we propose to use them to study the transcriptional regulation of AN biosynthetic genes. OUR HYPOTHESES: 1. Artemisinin biosynthesis is regulated at multiple steps in the pathway, both early (pre- FDP) and late (post-FDP). 2. Developmental stage and carbon source regulate the transcription of several genes in this pathway. 3. Coordinate control of artemisinin and sterol production occurs at the level of transcription. TECHNICAL OBJECTIVES: The long-term goal of our work is to develop a fundamental understanding of the regulatory control of terpene production in Artemisia annua so that metabolic manipulation of terpenoid biosynthesis can eventually be developed not only for artemisinin but other therapeutically important plant produced terpenoid drugs. This work has 3 objectives to be accomplished over 2.5 years: 1. To compare the relative expression of the terpenoid biosynthetic genes pre farnesyl diphosphate (FDP), and post FDP leading to AN with levels of both AA, DHAA, and AN production in A. annua seedlings as they are shifted from vegetative growth to conditions that induce flowering. 2. To compare the relative expression of the terpenoid biosynthetic genes pre farnesyl diphosphate (FDP), and post FDP leading to AN with levels of both AA, DHAA, and AN production in A. annua seedlings grown in glucose, fructose, and sucrose. 3. Include analysis of SQS expression in these studies to determine coordinate control of expression. Unfortunately little is still known about how important plant products like AN are produced in the plant and high production of terpenoids in low cost field-grown plants still remains challenging. This information is important because there has been little comprehensive effort to fully understand the regulation of not only this very important therapeutic sesquiterpene, but also other plant-derived terpenes that could have potentially very beneficial medicinal applications. The most potent therapeutic available for curing malaria, artemisinin, is produced in the plant, "Sweet Annie", but at levels too low to be able to provide adequate doses to annually treat the millions of cases that occur worldwide. The proposed study is a first step in helping us understand how to control artemisinin's biosynthesis to ultimately increase the production of this drug, and others that are chemically similar, in low cost field-grown plants.