Studies on the biosynthesis of terpenoids produced by streptomycetes.

从二萜类抗生素、TP产生菌灰孢链霉菌MF730-N6中克隆出甲戊酸途径基因和terpentecin （TP）合成基因两个基因簇。在前一簇中，7个基因依次编码香叶酰二磷酸合成酶（GGDPS）、甲戊酸激酶（MK）、甲戊酸二磷酸脱羧酶（MDPD）、磷酸戊酸激酶（PMK）、二磷酸异戊烯基异构酶（IPP）、HMG-CoA还原酶和HMG-CoA合成酶。这些基因在Ε中的异源表达。大肠杆菌和绿链霉菌都只有非甲羟戊酸途径，这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、HMG-CoA合成酶在Ε中表达。杆菌。其中，重组GGDPS、MK和IPP异构酶被证实具有预期的活性。在后一簇中，发现了两个orf， ORF11和ORF12，分别编码与真核生物二萜环化酶（DCs）和真细菌五烯合成酶相似的蛋白质。这两个环化酶基因均在猪链霉菌中表达。这种转化产生了一种新型的环二萜，即邻戊二烯-3,13(16)，14-三烯（terpentetriene），它与TP具有共同的基本骨架。这两种酶在Ε中过量产生。大肠杆菌纯化至均质。重组ORF11产物将GGDP转化为带二磷酸的中间体，再由重组ORF12产物转化为萜烯。据我们所知，这是关于真菌性DC的第一篇报道。

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.