Cloning, sequencing, and functional analysis of an iterative type I polyketide synthase gene cluster for biosynthesis of the antitumor chlorinated polyenone neocarzilin in "Streptomyces carzinostaticus"
Cloning, sequencing, and functional analysis of an iterative type I polyketide synthase gene cluster for biosynthesis of the antitumor chlorinated polyenone neocarzilin in "Streptomyces carzinostaticus"
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DOI：
10.1128/aac.48.9.3468-3476.2004
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发表时间：
2004-09-01
影响因子：
4.9
通讯作者：
Ebizuka, Y
中科院分区：
文献类型：
--
作者：
Otsuka, M;Ichinose, K;Ebizuka, Y
新卡兹菌素(NCZ)是由卡津链霉菌(Streptomyces Carzinostaticus)产生的抗肿瘤氯化多烯类化合物。F-41克隆并鉴定了与NCZ生物合成有关的基因簇。对33kb的DNA序列进行分析，发现有14个开放阅读框(ORF)，其中3个(ORF4、ORF5和ORF6)编码I型聚酮合成酶(PKS)，由4个模块组成。模块化组织的不寻常特征是在模块2和4上缺乏明显的酰基转移酶结构域，每个模块上存在长度超过200个氨基酸的较长的结构域间区域。通过在天蓝色链霉菌CH999中异源表达PKS基因，证实了PKS基因参与了NCZ的生物合成，该链在天蓝色链霉菌CH999中产生了明显的NCZ生物合成中间产物去氯卡兹林A和去氯酮卡兹林B。ORF5的中断导致了NCZ合成的失败，进一步证明了该簇是NCZ生物合成所必需的。对NCZ形成的机械考虑表明重复使用至少一个PKS模块，该模块随后通过脱羧化释放其产物来生成NCZ骨架，可能是由ORF7编码的II型硫代酯酶催化的。这是一种新型的细菌来源的I型PKS系统，用于生物合成还原的聚酮链。此外，ORF3编码的蛋白位于PKS基因的上游，与依赖FADH(2)的卤代谢酶非常相似，参与了卤代谢物的形成。ORF3蛋白可能负责NCZ的卤化，这是一个独特的例子，展示了一个参与脂肪族卤代谢物生物合成的卤化酶。
Neocarzilins (NCZs) are antitumor chlorinated polyenones produced by "Streptomyces carzinostaticus" var. F-41. The gene cluster responsible for the biosynthesis of NCZs was cloned and characterized. DNA sequence analysis of a 33-kb region revealed a cluster of 14 open reading frames (ORFs), three of which (ORF4, ORF5, and ORF6) encode type I polyketide synthase (PKS), which consists of four modules. Unusual features of the modular organization is the lack of an obvious acyltransferase domain on modules 2 and 4 and the presence of longer interdomain regions more than 200 amino acids in length on each module. Involvement of the PKS genes in NCZ biosynthesis was demonstrated by heterologous expression of the cluster in Streptomyces coelicolor CH999, which produced the apparent NCZ biosynthetic intermediates dechloroneocarzillin A and dechloroneocarzilin B. Disruption of ORF5 resulted in a failure of NCZ production, providing further evidence that the cluster is essential for NCZ biosynthesis. Mechanistic consideration of NCZ formation indicates the iterative use of at least one module of the PKS, which subsequently releases its product by decarboxylation to generate an NCZ skeleton, possibly catalyzed by a type II thioesterase encoded by ORF7. This is a novel type I PKS system of bacterial origin for the biosynthesis of a reduced polyketide chain. Additionally, the protein encoded by ORF3, located upstream of the PKS genes, closely resembles the FADH(2)-dependent halogenases involved in the formation of halometabolites. The ORF3 protein could be responsible for the halogenation of NCZs, presenting a unique example of a halogenase involved in the biosynthesis of an aliphatic halometabolite.