title: Identification of a biosynthetic gene cluster in rice for momilactones.
abstract: Rice diterpenoid phytoalexins such as momilactones and phytocassanes are produced in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor and in rice leaves irradiated with UV light. The common substrate geranylgeranyl diphosphate is converted into diterpene hydrocarbon precursors via a two-step sequential cyclization and then into the bioactive phytoalexins via several oxidation steps. It has been suggested that microsomal cytochrome P-450 monooxygenases (P-450s) are involved in the downstream oxidation of the diterpene hydrocarbons leading to the phytoalexins and that a dehydrogenase is involved in momilactone biosynthesis. However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. In this study, we found that a putative dehydrogenase gene (AK103462) and two functionally unknown P-450 genes (CYP99A2 and CYP99A3) form a chitin oligosaccharide elicitor- and UV-inducible gene cluster, together with OsKS4 and OsCyc1, the diterpene cyclase genes involved in momilactone biosynthesis. Functional analysis by heterologous expression in Escherichia coli followed by enzyme assays demonstrated that the AK103462 protein catalyzes the conversion of 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A. The double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor-inducible production of momilactones, strongly suggesting that CYP99A2, CYP99A3, or both are involved in momilactone biosynthesis. These results provide strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis.
authors.affiliation: Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Shimura
authors.forename: Atsushi
authors.initials: A
authors.lastname: Okada
authors.forename: Kazunori
authors.initials: K
authors.lastname: Okada
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Jikumaru
authors.forename: Kwang-Wook
authors.initials: KW
authors.lastname: Ko
authors.forename: Tomonobu
authors.initials: T
authors.lastname: Toyomasu
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sassa
authors.forename: Morifumi
authors.initials: M
authors.lastname: Hasegawa
authors.forename: Osamu
authors.initials: O
authors.lastname: Kodama
authors.forename: Naoto
authors.initials: N
authors.lastname: Shibuya
authors.forename: Jinichiro
authors.initials: J
authors.lastname: Koga
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nojiri
authors.forename: Hisakazu
authors.initials: H
authors.lastname: Yamane
mesh_terms: D002686:Chitin / Q000494:pharmacology; D032461:Chromosomes, Plant / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004224:Diterpenes / Q000378:metabolism*; D005810:Multigene Family / Q000502:physiology* / Q000528:radiation effects; D009844:Oligosaccharides / Q000494:pharmacology; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction / Q000187:drug effects / Q000528:radiation effects; D018515:Plant Leaves / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D004224:Diterpenes; D009844:Oligosaccharides; D010940:Plant Proteins; D011106:Polyisoprenyl Phosphates; D002686:Chitin; C116023:momilactone A; D003577:Cytochrome P-450 Enzyme System; C002963:geranylgeranyl pyrophosphate