北极海洋链霉菌604F的卤化酶基因克隆及特征  

Cloning and analysis of a halogenase gene of Streptomyces sp. 604F from the Arctic Ocean

作　　者：陈瑞勤[1,2] 廖丽[2] 张晓华[3] 陈波[1,2] 

机构地区：[1]华东理工大学生物工程学院,上海200237 [2]中国极地研究中心国家海洋局极地科学重点实验室,上海200136 [3]中国海洋大学海洋生命学院,山东青岛266003

出　　处：《微生物学报》2014年第6期703-712,共10页Acta Microbiologica Sinica

基　　金：国家"863计划"(2012AA092105);南北极环境综合考察与评估专项(CHINARE01-06);上海市自然科学基金项目(13ZR1462700)~~

摘　　要：【目的】本研究旨在克隆来自北极海洋、具有合成卤化物潜力的链霉菌(Streptomyces sp.)604F中的一个卤化酶基因,为后续克隆卤化物合成基因簇、分离鉴定卤化物提供指导。【方法】利用琼脂块法初步测试抗菌活性,借助液相色谱-飞行时间串联质谱技术(LC-Tof MS)初步寻找Streptomyces sp.604F发酵粗提液中的卤化物,并以简并PCR扩增合成次级代谢产物的指示基因(I型、II型聚酮合酶及非核糖体多肽合成酶编码基因);根据依赖黄素腺嘌呤二核苷酸的卤化酶基因保守区设计的简并引物,扩增基因片段并测序分析;采用高效热不对称交错PCR(hiTAIL-PCR)技术扩增卤化酶基因全长。【结果】Streptomyces sp.604F具有较强的抗白色念珠菌活性,其基因组同时含有编码I型聚酮合酶、II型聚酮合酶和非核糖体多肽合成酶基因,以及卤化酶基因;通过染色体步移克隆该卤化酶基因全长,共1443 bp,编码一个新的非色氨酸卤化酶,在合成已知卤化物的卤化酶数据库中,与其同源关系最近的为一类参与合成糖肽类次级代谢产物的卤化酶。【结论】Streptomyces sp.604F具有新的非色氨酸卤化酶,且推测参与糖肽类化合物的卤化修饰,为后续寻找目的卤化物提供了指导,也为研究该合成基因簇奠定基础。[ Objective] To clone a halogenase gene from halometabolite-producing Streptomyces sp. 604F to facilitate identification of potential halometabolites and its biosynthetic gene cluster. [ Methods] We used agar block method to detect the antimicrobial activity of Streptomyces sp. 604F. We further amplified the conserved regions of type Ⅰ polyketide synthase （PKS Ⅰ） , type Ⅱ polyketide synthase （PKS Ⅱ） and nonribosomal peptide synthetase （NRPS） encoding genes by degenerative PCR. We detected halometabolites in fermentation extracts of Streptomyces sp. 604F analyzed by liquid chromatography-time of flight mass spectrometry （LC-Tof MS）. Next, we amplified halogenase gene fragment from Streptomyces sp. 604F by using degenerative primers targeting reduced flavin adenine dinucleotide （FADH2 ） -dependent halogenase genes. Finally, we cloned the full halogenase gene and its flanking sequences through high-efficiency thermal asymmetric interlaced PCR （hiTAIL-PCR）. [ Results] Streptomyces sp. 604F showed promising antifungal activity against Candida albicans ATCC 10231, and its genome contained genes encoding PKS Ⅰ, PKS Ⅱ, NRPS and a halogenase with 1443 bp. The halogenase is a new non-tryptophan halogenase, and most closely related to halogenases catalyzing the chlorination of glycopeptides. [ Conclusion] Streptomyces sp. 604F possessed a new non-tryptophan halogenase, which may be involved in .halogenation of glycopeptide-like metabolites. The cloning and analysis of this halogenase have provided guidance for searching target halometabolites, and laid the foundation for obtaining the biosynthetic gene cluster.

关 键 词：卤化酶 放线菌 北极 

分 类 号：Q78[生物学—分子生物学]