Hamano Y, Dairi T, Yamamoto M, Kawasaki T, Kaneda K, Kuzuyama T, Itoh N, Seto H
Biotechnology Research Center, Toyama Prefectural University, Japan.
Biosci Biotechnol Biochem. 2001 Jul;65(7):1627-35. doi: 10.1271/bbb.65.1627.
A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (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 E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.
通过搜索先前已通过互补分离出的3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶基因的侧翼区域，从类萜抗生素特戊菌素产生菌灰色链霉菌菌株MF730-N6中分离出了一个编码甲羟戊酸途径相关酶的基因簇。通过对一个8.9 kb的BamHI片段进行DNA测序，推测7个基因按此顺序存在，它们分别编码香叶基香叶基二磷酸合酶（GGDPS）、甲羟戊酸激酶（MK）、甲羟戊酸二磷酸脱羧酶（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。这些基因在仅具有非甲羟戊酸途径的大肠杆菌和变铅青链霉菌中进行异源表达，表明甲羟戊酸途径的基因包含在克隆的DNA片段中。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异构酶被证实具有预期活性。据我们所知，这是关于真细菌MK有直接证据的首次报道。
