新型聚酮合酶基因簇的分离与部分鉴定  

Isolation and partial characterization of new PKS gene clusters

作　　者：焦豫良[1] 王梁华[1] 董晓毅[1] 宗英[1] 高云[1] 任娜[1] 郭爱芸[1] 张兴群[1] 焦炳华[1] 

机构地区：[1]第二军医大学基础医学部,上海200433

出　　处：《中国抗生素杂志》2008年第3期134-139,共6页Chinese Journal of Antibiotics

基　　金：国家自然科学基金项目(30670043);863计划海洋技术领域专题项目(2006AA09Z434)资助

摘　　要：目的从海洋沉积物中分离新型聚酮合酶(PKS)基因簇,并解析其基因结构,为研究PKS的组合生物合成提供新组件。方法采集中国东海沉积物样本,并提取宏基因组DNA,以其为模板,通过简并PCR扩增新型PKS片段,用地高辛标记正确的扩增片段作为探针,然后从本研究中构建的海洋沉积物样本Fosmid宏基因组文库中筛选完整基因簇,测序、结构分析。结果(1)所分离得到的海洋沉积物宏基因组DNA可直接作为PCR模板,简并PCR扩增得到26个新型KS编码片段,并成功提交GenBank;(2)所得DNA构建成滴度约5×108cfu/ml的Fosmid文库,插入片段平均长度约为40kb;(3)将其中的一个KS片段(GenBank登录号DQ942531)用地高辛标记并筛选部分文库,得到4个克隆,测序得到7981bp的序列(GenBank登录号EF568935),结构分析发现含典型的I型PKS组件,如酮缩酶(KS)、酰基转移酶(AT)、酰基载体蛋白(ACP)等。结论简并引物扩增、宏基因组文库筛选、测序、结构分析,在本研究中被证明是有效的分离新PKS基因簇的途径;通过系统进化树分析得出26条新KS片段属于Ⅰ型KS片断和杂合PKS/NRPS基因簇,主要来自放线菌门、δ变形菌门、蓝藻门和β变形菌门的微生物。Objectives To isolate new poiyketide synthase （PKS）gene clusters from marine sediment sampie and characterize the gene structure of them for contributing new elements to PKS combinatorial biosynthesis. Methods Metagenome DNA was extracted from marine sediment samples gathered from the East China Sea; using Consensus-Degenerate Hybrid Oligonucleotide Primers（CODEHOP） PCR method to amplify new PKS gene fragments from the metagenome DNA. Using a Dig-labeled KS gene fragment（GenBank accession no. DQ942531）as probe to screen out holo-PKS gene clusters from marine sediment sample metagenome fosmid library constructed for this study. Results （1）The metagenome DNA extracted from marine sediment can be used directly as template for PCR. Using CODEHOP PCR, 26 new ketoacyl synthase （KS） fragments were isolated, identified and submitted to GenBank. （2）A marine sediment sample metagenome DNA fosmid library composed of 40kb inserts was constructed. The titer of it is 5 × 10^8cfu/ml approximately; （3）one KS fragment（GenBank accession no. DQ924531） was used as probe to screen the library and 4 positive clones were selected; the clones were sequenced and a 7981bp partial type Ⅰ gene cluster （GenBank accession no. EF568935）was identified; typical PKS gene structures, such as KS, AT and ACP were found after analyzing EF568935. Conclusions CODEHOP PCR amplification of conserved PKS domain and metagenome library screening are useful ways to isolate new PKS gene clusters; the 26 new KS fragments belong to type Ⅰ ketosyntase, as well as hybrid or mixed PKS/NRPS enzymes complexes primarily originating from marine actinobacteria,deha-proteobacteria, cyanobateria, and beta-proteobacteria by phylogenetic analysis.

关 键 词：海洋沉积物 聚酮合酶 酮缩酶 基因簇 组合生物合成 

分 类 号：Q78[生物学—分子生物学]