权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Studies on the biosynthesis of terpenoids produced by streptomycetes.

链霉菌萜类化合物生物合成的研究。

基本信息

批准号：
12660083
负责人：
DAIRI Tohru
金额：
$ 2.3万
依托单位：
Toyama Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Two gene clusters containing the mevalonate pathway genes and the terpentecin (TP) biosynthetic genes were cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, TP producer. In the former cluster, seven genes enecoding geranylgerairyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarbosylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in Ε. coli and Streptomyces Iividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate paftway were involved in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in Ε. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. In the latter cluster, two ORFs, ORF11 and ORF12 that encode proteins showing similarities to eucaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were found. The two cyclase genes were expressed in Streptomyces Iividans. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has a common basic skeleton as does TP. The two enzymes were overproduced in Ε. coli and purified to homogeneity. The recombinant ORF11 product converted GGDP into an intermediate with diphosphate, and then it was transformed into terpenteteiene by the recombinant ORF12 product. To the best of our knowledge, this is the first report about a eubacterial DC.

从灰孢链霉菌（Streptomyces griseolosporeus）MF 730-N6菌株中克隆了甲羟戊酸代谢途径基因和萜类化合物（tendertecin，TP）生物合成基因。在前一个簇中，有7个基因编码香叶基香叶基二磷酸合酶（GGDPS）、甲羟戊酸激酶（MK）、甲羟戊酸二磷酸脱羧酶（MDPD）、磷酸甲羟戊酸激酶（PMK）、异戊烯基二磷酸（IPP）异构酶、HMG-CoA还原酶和HMG-CoA合酶。这些基因在E. coli和StreptomycesIividans，这两个菌株都只有非甲羟戊酸途径，表明甲羟戊酸途径的基因参与了克隆的DNA片段。GGDPS、MK、MDPD、PMK、IPP异构酶和HMG-CoA合酶在E.杆菌其中，重组GGDPS、MK和IPP异构酶被证实具有预期的活性。在后一个簇中，发现了两个ORF，ORF 11和ORF 12，它们分别编码与真核二萜环化酶（DCs）和真细菌并环戊二烯合酶（pentalenene synthase）相似的蛋白质，这两个环化酶基因在链霉菌中表达。该菌产生了一种新的环状二萜类化合物ent-clerod-3，13（16），14-triene（tenertetriene），它与TP具有共同的基本骨架。这两种酶在E. coli中，并纯化至均一。重组ORF 11产物将GGDP转化为含二磷酸的中间体，然后被重组ORF 12产物转化为萜四烯。据我们所知，这是关于真细菌DC的首次报道。