Presence of copalyl diphosphate synthase gene in an actinomycete possessing the mevalonate pathway
Presence of copalyl diphosphate synthase gene in an actinomycete possessing the mevalonate pathway
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DOI：
10.7164/antibiotics.57.739
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发表时间：
2004-11-01
影响因子：
3.3
通讯作者：
Dairi, T
中科院分区：
文献类型：
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作者：
Kawasaki, T;Kuzuyama, T;Dairi, T
我们先前已经证明，生物合成松油菌素和倍半萜抗生素BE-40644的基因簇位于相邻的甲氧戊酸途径基因簇中。在本研究中，从链霉菌中克隆了甲氧戊酸途径基因簇。以已从菌株KO-3988克隆的3-羟基-3-甲基戊二酰辅酶A(HMG-CoA)还原酶基因为探针，对已知的产生呋喃西林A的菌株KO-3988进行了杂交实验。通过测序，我们在甲氧戊酸途径基因簇的上游区域发现了四个开放阅读框，编码一个可能的细胞色素P450(ORF1)、一个类异戊二烯环酶(ORF2)、一个未知蛋白(ORF3)和一个聚戊烯基二磷酸合成酶基因(ORF4)，但我们没有发现任何与呋喃喹诺酮A生物合成相关的基因。这两个ORF(ORF2和ORF4)在大肠杆菌中以重组酶的形式表达，并用于研究这些产物的功能。ORF4产物经鉴定为香叶基香叶二磷酸(GGDP，C-20)合成酶。ORF2产物被证明催化GGDP转化为二磷酸钴，这是具有这种功能的原核酶的第一个例子。这些结果再次表明，具有甲羟戊酸途径的放线菌通常会产生一种异戊二烯，其生物合成基因簇存在于邻近甲羟戊酸途径的基因簇中。
We have previously shown that gene clusters for biosyntheses of terpentecin and BE-40644, a diterpene antibiotic and a sesquiterpene antibiotic, respectively, were located in the adjacent mevalonate pathway gene clusters. In this study, a mevalonate pathway gene cluster was cloned from Streptomyces sp. strain KO-3988, which was known to produce furaquinocin A, employing a hybridization experiment using a 3-hydroxy-3-methyl glutaryl CoA (HMG-CoA) reductase gene, which had been previously cloned from the strain KO-3988, as a probe. By sequencing flanking regions, we found four open reading frames that could encode a putative cytochrome P450 (ORF1), an isoprenoid cyclase (ORF2), an unknown protein (ORF3), and a polyprenyl diphosphate synthase gene (ORF4) in the upstream region of the mevalonate pathway gene cluster, though we did not find any genes related to furaquinocin A biosynthesis. The two ORFs (ORF2 and 4) were expressed as recombinant enzymes in E. coli and used for studies to investigate functions of these products. The ORF4 product was confirmed to be a geranylgeranyl diphosphate (GGDP, C-20) synthase. The ORF2 product proved to catalyze a conversion of GGDP into copalyl diphosphate, the first example of an enzyme with this function of prokaryotic origin. These results again showed that actinomycetes possessing the mevalonate pathway usually produce an isoprenoid and that its biosynthetic gene cluster exists in adjacent the mevalonate pathway gene cluster.