Terpentecin

Eubacterial diterpene cyclase genes had previously been cloned from a diterpenoid antibiotic terpentecin producer (Dairi, T., Hamano, Y., Kuzuyama, T., Itoh, N., Furihata, K., and Seto, H. (2001) J. Bacteriol. 183, 6085-6094). Their products, open reading frame 11 (ORF11) and ORF12, were essential for the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene (TTE) that had the same basic skeleton as terpentecin. In this study, functional analyses of these two enzymes were performed by using purified recombinant enzymes. The ORF11 product converted GGDP into a cyclized intermediate, terpentedienol diphosphate (TDP), which was then transformed into TTE by the ORF12 product. Interestingly, the ORF12 product directly catalyzed the conversion of GGDP into three olefinic compounds. Moreover, the ORF12 product utilized farnesyl diphosphate as a substrate to give three olefinic compounds, which had the same structures as those formed from GGDP with the exception of the chain lengths. These results suggested that the ORF11 product with a DXDD motif converted GGDP into TDP by a protonation-initiated cyclization and that the ORF12 product with a DDXXD motif completed the transformation of TDP to the olefin, terpentetriene by an ionization-initiated reaction followed by deprotonation. The kinetics of the ORF12 product indicated that the affinity for TDP and GGDP were higher than that of farnesyl diphosphate and that the relative activity of the reaction converting TDP into TTE was highest among the reactions using TDP, GGDP, or farnesyl diphosphate as the substrate. These results suggested that an actual reaction catalyzed by the ORF12 was the conversion of TDP into TTE in vivo.

Most Streptomyces strains are equipped with only the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the formation of isopentenyl diphosphate, a common precursor of isoprenoids. In addition to this pathway, some Streptomyces strains possess the mevalonate (MV) pathway via which isoprenoid antibiotics are produced. We have recently cloned and analyzed the MV pathway gene clusters and their flanking regions from terpentecin, BE-40644, and furaquinocin A producers. All these clusters contained genes coding for mevalonate kinase, mevalonate diphosphate decarboxylase, phosphomevalonate kinase, type 2 IPP isomerase, HMG-CoA reductase, and HMG-CoA synthase. The order of each of the open reading frames (ORFs) is also the same, and the respective homologous ORFs show more than 70% amino acid identity with each other. In contrast to these conservative gene organizations, the biosynthetic genes of terpentecin, BE-40644, and furaquinocin A were located just upstream and/or downstream of the MV pathway gene cluster. These facts suggested that all the actinomycete strains possessing both the MV and MEP pathways produce isoprenoid compounds and the biosynthetic genes of one of these isoprenoids usually exist adjacent to the MV pathway gene cluster. Therefore, when the presence of the MV cluster is detected by molecular genetic techniques, isoprenoids may be produced by the cultivation of these actinomycete strains. During the course of these studies, we identified diterpene cyclases possessing unique primary structures that differ from those of eukaryotes and catalyze unique reactions.

We report here the draft genome sequence of Kitasatospora griseola strain MF730-N6, a known producer of bafilomycin, terpentecin, and satosporins. The current assembly comprises 8 contigs covering 7.97 Mb. Genome annotation revealed 7,225 protein coding sequences, 100 tRNAs, 40 rRNA genes, and 23 secondary metabolite biosynthetic gene clusters.