Studies on the biosynthesis of terpenoids produced by streptomycetes.

从绿孢链霉菌(Streptomyces Griseolosporreus)二萜类抗生素产生菌MF730-N6中克隆了两个包含甲氧丙酮酸途径基因和松油菌素(TP)生物合成基因的基因簇。在前者中，编码香叶酰二磷酸合成酶(GGDPS)、甲氧戊酸激酶(MK)、甲氧戊酸二磷酸脱羧酶(MDPD)、磷酸戊酸酶(PMK)、异戊烯基二磷酸(IPP)异构酶、HMG-CoA还原酶和HMG-CoA合成酶的7个基因依次存在。这些基因在Ε中的异源表达。Coli和Iividans都只有非甲氧戊酸途径，这表明甲氧戊酸途径的基因参与了克隆的DNA片段。Ε表达GGDPS、MK、MDPD、PMK、IPP异构酶和HMG-CoA合成酶。Coli.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ORF11和ORF12，它们编码的蛋白质分别与真核二萜环酶(DC)和真细菌五烯合酶相似。这两个环化酶基因在艾维链霉菌中得到了表达。转化子产生了一种新的环状二萜-3，13(16)，14-三烯(三戊三烯)，与TP一样具有共同的基本骨架。在Ε中，这两种酶的产量过高。Coli，并纯化至均一。重组ORF11产物将GGDP转化为带有二磷酸的中间体，再由重组ORF12产物转化为萜烯。据我们所知，这是第一次关于真细菌DC的报道。

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.