title: Identification of diterpene biosynthetic gene clusters and functional analysis of labdane-related diterpene cyclases in Phomopsis amygdali.
abstract: Two diterpene biosynthesis gene clusters in the fusicoccin-producing fungus, Phomopsis amygdali, were identified by genome walking from PaGGS1 and PaGGS4 which encode the geranylgeranyl diphosphate (GGDP) synthases. The diterpene cyclase-like genes, PaDC1 and PaDC2, were respectively located proximal to PaGGS1 and PaGGS4. The amino acid sequences of these two enzymes were similar to those of fungal labdane-related diterpene cyclases. Recombinant PaDC1 converted GGDP mainly into phyllocladan-16 alpha-ol via (+)-copalyl diphosphate (CDP) and trace amounts of several labdane-related hydrocarbons which had been identified from the P. amygdali F6 mycelia. Since phyllocladan-16 alpha-ol had not been identified in P. amygdali F6 mycelia, we isolated phyllocladan-16 alpha-ol from the mycelia. Recombinant PaDC2 converted GGDP into (+)-CDP. Furthermore, we isolated the novel diterpenoid, phyllocladan-11 alpha,16 alpha,18-triol, which is a possible metabolite of phyllocladan-16 alpha-ol in the mycelia. We propose that genome walking offers a useful strategy for the discovery of novel natural products in fungi.
authors.affiliation: Department of Bioresource Engineering, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan. toyomasu@tds1.tr.yamagata-u.ac.jp
authors.forename: Tomonobu
authors.initials: T
authors.lastname: Toyomasu
authors.forename: Rie
authors.initials: R
authors.lastname: Niida
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Kenmoku
authors.forename: Yuri
authors.initials: Y
authors.lastname: Kanno
authors.forename: Shigeyoshi
authors.initials: S
authors.lastname: Miura
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nakano
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Shiono
authors.forename: Wataru
authors.initials: W
authors.lastname: Mitsuhashi
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Toshima
authors.forename: Hideaki
authors.initials: H
authors.lastname: Oikawa
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Hoshino
authors.forename: Tohru
authors.initials: T
authors.lastname: Dairi
authors.forename: Nobuo
authors.initials: N
authors.lastname: Kato
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sassa
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary; D004224:Diterpenes / Q000378:metabolism*; D016681:Genome, Fungal; D008190:Lyases / Q000235:genetics* / Q000378:metabolism*; D005810:Multigene Family; D025282:Mycelium / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D004224:Diterpenes; D010940:Plant Proteins; D011106:Polyisoprenyl Phosphates; D019883:Alkyl and Aryl Transferases; C090963:ent-kaurene synthetase A; D008190:Lyases; C002963:geranylgeranyl pyrophosphate