Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin

A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a gerany lgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibioti c, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki , K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65 :1627-1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP b iosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, wh ich encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivate d by gene disruptions. The mutants produced no TP, confirming that these cy clase genes are essential for the production of TP. The two cyclase genes w ere also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transforman t produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpe ntetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is t he first report of a eubacterial DC.