Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin
Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin
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DOI：
10.1128/jb.183.20.6085-6094.2001
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发表时间：
2001-10-01
影响因子：
3.2
通讯作者：
Seto, H
中科院分区：
文献类型：
--
作者：
Dairi, T;Hamano, Y;Seto, H
含有用于形成异戊烯基二磷酸的甲羟戊酸途径基因（开放阅读框2 [ORF 2]至ORF 7）和香叶基香叶基二磷酸（GGDP）合酶基因（ORF 1）的基因簇先前已从灰色孢子链霉菌菌株MF 730-N6（一种二萜类抗生素、萜类菌素（TP）产生菌（Y. Hamano，T. Dairi，M.山本，T.川崎，K Kaneda，T. Kuzuyama，N. Itoh和H.濑户，生物科学。Biotech. 65：1627-1635，2001）。对该基因组上游区域的序列分析发现了7个新的ORF，即ORF 8 ~ ORF 14，推测它们可能编码TP生物合成基因。我们构建了两个突变体，其中ORF 11和ORF 12，这编码的蛋白质显示相似的真核二萜环化酶（DC）和真细菌并环戊二烯合酶，分别被基因破坏失活。突变体不产生TP，证实这些环化酶基因是TP产生所必需的。这两个环化酶基因也在变铅青链霉菌中与GGDP合酶基因一起在ermE* 组成型启动子的控制下表达。该化合物是一种新的环状二萜类化合物，即ent-clerod-3，13（16），14-triene（tenertetriene），其基本骨架与TP相同。这两种酶，其中每一个是过量生产的大肠杆菌和纯化的同质化，GGDP转化为萜烯tetriene。据我们所知，这是真细菌DC的首次报道。
A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627-1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.