Plant & Animal Genomes XIV Conference Plant & Animal Genomes XIV ConferenceJanuary 14-18, 2006
Town & Country Convention Center
San Diego, CA
Dae-Kyun Ro , Eric M. Paradise , Mario Ouellet , Karyn L. Newman , Jay D. Keasling
The sesquiterpene lactone is one of the major classes of plant natural products with an enormous structural diversity. These sesquiterpene lactones and their derivatives are mostly isolated from plant family, Asteraceae (or Compositae). Undoubtedly artemisinin, a sesquiterpene lactone endoperoxide, is the most valuable natural product at this moment that is extracted from Artemisia annua L. (Asteraceae), due to its potent anti-malarial property against multi-drug resistant malaria parasite, Plasmodium falciparum. However, this natural product is in short supply and unaffordable to most malaria sufferers. While total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin from its immediate precursor, artemisinic acid, can be used for artemisinin production. Artemisinic acid is synthesized by three consecutive oxidations at the C12 isopropenyl group of sesquiterpene hydrocarbon, amorpha-4,11-diene. A cytochrome P450 monooxygenase (P450) was proposed to catalyze the first oxygenation. Since sesquiterpene lactone is common in Asteraceae, we undertook a comparative genomics analysis of plants in the Asteraceae family (cgpdb.ucdavis.edu) to decipher the sesquiterpene lactone biosynthetic pathway in A. annua. Among ESTs of lettuce, sunflower, and A. annua, a single highly conserved P450 was identified and a new subfamily name CYP71AV1 was assigned to this novel P450. This enzyme catalyzes three consecutive oxidations at the C12 position of amorpha-4,11-diene to yield artemisinic acid in in vitro assays. We demonstrate de novo synthesis of up to 100 mg of artemisinic acid per liter culture in an engineered yeast strain expressing CYP71AV1.