产macrolactins的海洋细菌x-2中ⅰ型pks基因簇的筛选鉴定与功能分析.docVIP

产Macrolactins的海洋细菌X-2中型PKS基因簇的筛选鉴定与功能分析Macrolactins是近年来从Actinomadura sp.Bacillus sp.等海洋来源的微生物中分离的一群24元大环内酯类化合物, 具有抗菌、抗病毒和抗肿瘤活性。本室从东海海泥中分离到一株海洋细菌X-2, 可产生多种Macrolactin。本研究自行设计了一套针对型PKS)基因中酮缩合酶(ketosynthase, KS)片段的简并引物, 使用PCR方法直接克隆到了X-2基因组中的一段长约645 bp的型KS基因片段EF486351, 并以之为探针筛选X-2的fosmid基因组文库, 得到了3个阳性克隆, 测序获得的序列经同源性分析和功能预测, 初步该菌中负责Macrolactin的部分型PKS基因簇。acrolactin, 聚酮合酶枯草芽孢杆菌Screening, Identifying and Function Analysis of Polyketide Synthase I Cluster from the Environmental Strain X-2 Which Produce Macrolactins DONG Xiao-Yi WANG Liang-Hua SUN Ming-Juan ZONG Ying JIAO Yu-Liang JIAO Bing-Hua* (Department of Biochemistry and Molecular Biology, Faculty of Basic Medical Science, Second Military Medical University, Shanghai 200433) Abstract: Macrolactins are 24-membered macrolides produced by unidentified marine bacterium, Actinomadura sp. and Bacillus sp., which exhibit both antibacterial and antitumor activities in vitro. The environmental strain X-2 which was isolated from the sediment of the East China Sea produce Macrolatin A, B and O. In this study, a set of degenerate oligonucleotide primers, designed for amplification ketosynthase(KS) domains, had been employed to identify KS gene fragments of the X-2 DNA samples. One 645 bp KS fragment(GenBank accession no. EF486351)had been cloned and used as a probe to screen the genome DNA fosmid library of X-2. Three positive clones were selected and sequenced, Homologous analysis and the function prediction of the obtained PKS gene fragments suggested that macrolactin is the Polyketide Biosynthesis Product of the gene cluster obtained in the environmental strain X-2. Keywords: Macrolactin, Polyketide synthase, Bacillus sp.利用组合生物学手段对不同来源的次级代谢产物的生物合成基因进行重新设计和组合, 产生大量具有新的药效的“非天然”药物是新药开发的新方向。但这些工作的前提条件是次级代谢产物生物合成基因(簇)的克隆与功能分析[1]。 Macrolactins是一类24员大环内酯类化合物, 近年来又发现其可有效抑制鲨烯合酶的活性, 提示其可作为胆固醇合成的抑制剂[2]。目前Macrolactins已经有1个家族成员(Macrolactin A－)[3,4,6?8], 主要来自海洋来源的Actinomadura sp.和Bacillus sp., 在陆地土壤中的Streptomyces sp.中也有发现[2]。海洋细菌X-2