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