Functional expression of a bark beetle cytochrome P450 that hydroxylates myrcene to ipsdienol

The final steps in the pheromone-biosynthetic pathway of the pine engraver beetle, Ips pini (Say) (Coleoptera: Scolytidae) are unknown, but likely involve myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation to produce the aggregation pheromone component, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol). We have isolated a full-length L pini cDNA encoding a cytochrome P450, CYP9T2. The recovered cDNA is 1.83 kb and the open reading frame encodes a 532 amino acid protein. CYP9T2 is regulated by the same physiological factors that induce pheromone production. Quantitative real-time PCR experiments showed that feeding on host phloem induced CYP9T2 expression in males, but not females, and that basal expression levels are highest in male midguts, similar to other I pini pheromone-biosynthetic genes. Microsomes prepared from Sf9 cells co-expressing baculoviral-mediated recombinant CYP9T2 and housefly (Musca domestica) NADPH-cytochrome P450 reductase converted myrcene to ipsdienol. The product identified by coupled GC-MS was mostly (4R)-(-)-ipsdienol, an important aggregation pheromone component for western North American I. pini. These results are consistent with CYP9T2 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway. (c) 2006 Elsevier Ltd. All rights reserved.