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

摘要:

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。

Abstract:

Abstract:［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.