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-16α-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-16α-ol had not been identified in P. amygdali F6 mycelia, we isolated phyllocladan-16α-ol from the mycelia. Recombinant PaDC2 converted GGDP into (+)-CDP. Furthermore, we isolated the novel diterpenoid, phyllocladan-11α,16α,18-triol, which is a possible metabolite of phyllocladan-16α-ol in the mycelia. We propose that genome walking offers a useful strategy for the discovery of novel natural products in fungi.