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.

从二萜类抗生素、TP 生产者灰孢链霉菌 MF730-N6 中克隆了两个含有甲羟戊酸途径基因和萜烯 (TP) 生物合成基因的基因簇。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)，甲羟戊酸二磷酸脱碳基酶(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合酶被认为以该顺序存在于大肠杆菌和链霉菌中。仅具有非甲羟戊酸途径，表明克隆的 DNA 中涉及甲羟戊酸途径的基因GGDPS、MK、MDPD、PMK、IPP异构酶和HMG-CoA合酶在大肠杆菌中表达，其中重组GGDPS、MK和IPP异构酶在后一簇中被证实具有预期的活性。，两个 ORF，ORF11 和 ORF12，编码与真核二萜环化酶 (DC) 相似的蛋白质，以及一个分别发现真细菌五烯合酶基因在Streptomyces Iividans中表达，转化体产生了一种新型环状二萜，ent-clerod-3,13(16),14-三烯(terpentetriene)，其具有共同的碱性。与 TP 一样，这两种酶在大肠杆菌中过量产生并纯化至同质。 ORF11产物将GGDP转化为二磷酸中间体，然后通过重组ORF12产物将其转化为萜烯，据我们所知，这是关于真细菌DC的第一篇报道。