42(P-1) 原核生物起源ジテルペンサイクラーゼ遺伝子に関する研究(ポスター発表の部)

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We have studied a gene cluster responsible for the biosynthesis of the diterpene compound, terpentecin, produced by a Streptomyces strain. We have prepared a mutant lacking the 3-hydroxy-3methylglutaryl CoA (HMG-CoA) reductase activity from this strain, which uses both the mevalonate and nonmevalonate pathways for the formation of isopentenyl diphosphate, by screening terpentecin non-producing mutants. Terpentecin is known to be synthesized via the mevalonate pathway. The gene encoding HMG-CoA reductase (hmgr) was cloned and identified by complementation of the mutant, using a self-cloning system developed in this study for strain MF730-N6. By searching a flanking regions of the HMG-CoA reductase gene, we were able to,identify two gene clusters, which encode the mevalonate pathway enzymes and the terpentecin biosynthetic enzymes. In the former cluster, seven genes encoding geranylgeranyl diphosphate synthase (GGPPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), an unknown protein, HMG-CoA reductase, and HMG-CoA synthase were identified. In the latter cluster, additional seven genes, which were suggested to encode the terpentecin biosynthetic genes, were found. Two genes found in the terpentecin biosynthetic gene cluster, which have similarities to eucaryotic diterpene cyclase genes and a eubacterial pentalenolactone synthase gene, respectively, were expressed in Streptomyces lividans together with the GGPPS gene under the control of the ermE^* promoter. The transformant produced a novel cyclic diterpenoid with the same basic structure as that of terpentecin. To the best of our knowledge, this is the first report about a eubacterial diterpene cyclase. A dxs gene, which encodes a 1-deoxy-D-xylulose 5-phosphate (DXP) synthase participating in the initial step of the nonmevalonate pathway was also cloned from strain MF730-N6. The dxs, hmgr and GGPPS cyclase genes were used as probes for Northern blot analysis to examine temporal expression of the nonmevalonate and mevalonate pathways. A transcript of the dxs gene was detected throughout the cultivation. On the other hand, messages of the hmgr gene and the GGPPS cyclase gene were not detected at early growth phase but appeared when terpentecin production started, suggesting that the nonmevalonate pathway was essential for growth and the mevalonate pathway was mainly used for the secondary metabolism.
天然有機化合物討論会の論文
2000-10-01

42(P-1) 原核生物起源ジテルペンサイクラーゼ遺伝子に関する研究(ポスター発表の部)

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