Identification of diterpene biosynthetic gene clusters and functional analysis of labdane-related diterpene cyclases in Phomopsis amygdali.
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.
Toyomasu
Tomonobu
T
Department of Bioresource Engineering, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan. toyomasu@tds1.tr.yamagata-u.ac.jp
Niida
Rie
R
Kenmoku
Hiromichi
H
Kanno
Yuri
Y
Miura
Shigeyoshi
S
Nakano
Chiaki
C
Shiono
Yoshihito
Y
Mitsuhashi
Wataru
W
Toshima
Hiroaki
H
Oikawa
Hideaki
H
Hoshino
Tsutomu
T
Dairi
Tohru
T
Kato
Nobuo
N
Sassa
Takeshi
T
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
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