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.

将两个含有甲戊酸途径基因和perpentecin（TP）生物合成基因的基因簇从链霉菌菌株MF730-N6（一种数字抗生素，TP生产者）中克隆。 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 reduction, and建议以该顺序存在HMG-COA合酶。这些基因在ε中的异源表达。大肠杆菌和链霉菌均只有非层状途径，这表明甲戊酸途径的基因参与克隆的DNA片段。 GGDP，MK，MDPD，PMK，IPP异构酶和HMG-COA合酶在ε中表达。大肠杆菌。其中，重组GGDP，MK和IPP异构酶被证实具有预期的活动。在后一个群集中，分别发现了与Eucaryotic二萜循环（DCS）（DCS）和一个Eubacterial Pentalene合酶的两个ORFS，ORF11和ORF12。两个环化酶基因在链霉菌群体中表达。转化剂产生了一种新型的循环双萜类化合物，Ent-Clerod-3,13（16），14-三烯（Terpenterenene），该元素与TP一样具有常见的基本骨骼。两种酶在ε中过量生产。大肠杆菌并纯化为同质性。重组ORF11产物将GGDP转换为与二磷酸盐的中间体，然后通过重组ORF12产物将其转化为TerpenteTeiene。据我们所知，这是关于Eubacterial DC的第一份报告。