【作者】 徐培培；

【导师】 沈月毛； 李越中；

【作者基本信息】 山东大学 ， 微生物学， 2013， 博士

【摘要】 放线菌是一类具有复杂生活史的革兰氏阳性细菌,产生了目前已知的50%以上的抗生素。放线菌主要分布在陆地和海洋,极端环境和植物组织内部也成为新的放线菌资源的重要来源。安莎霉素是一类结构独特的抗生素,这一类化合物通常具有很强的抗细菌、抗真菌、抗癌和抗病毒活性,主要分布在放线菌中,其中一个子类maytansinoids也存在高等植物和苔藓中。安莎霉素属于I性聚酮类化合物,具有共同的生物合成起始单元AHBA(3-氨基-5-羟基苯甲酸),特异的AHBA合酶基因探针可以用于安莎霉素生物合成基因簇的筛选。安莎霉素的生物合成除了受细胞中次级代谢的全局调控因子的调控外,很多安莎基因簇中存在LuxR家族的途径特异性正调控因子,因此可以通过对LuxR调控因子进行过表达来激活沉默的基因簇或提高抗生素产量。天然产物是最重要的药物来源,也是寻找新的安莎类抗生素的巨大宝库。宏基因组技术的兴起为寻找蕴藏在天然产物中的药物资源提供了有力支持。本论文尝试从两个方面发掘新的安莎类抗生素。第一种是构建BAC文库,希望能够一次性获得较大的完整的安莎类生物合成基因簇。首先构建了一系列各具特色的E. coli-Strptomycets穿梭载体,其中pCCESAC82载体具有以下优点：诱导型复制子,红霉素强启动子,支持BstⅨ的接头连接,蔗糖致死基因sacB可降低空载率,CIS元件支持向链霉菌中的接合转移和染色体整合。探索了构建链霉菌BAC文库的方法,从如何提取高质量的HMW DNA(high molecular weight DNA),提高连接效率,制备超高效电转感受态细胞和提高转化效率等方面进行了方法学探索。构建了Actinosynnema pretiosum31565的BAC文库,获得了超过100kb插入片段的克隆。第二种是利用Red重组方法拼接重叠的不完整基因簇,在大肠杆菌中进行基因调控后转入合适宿主中异源表达。利用构建的pCCESAC2载体和fosmid载体pCC1FOS的两段同源区以及两段基因簇插入片段之间的一段同源区,进行两轮Red重组,将完整的生物合成基因簇成功捕获到穿梭载体pCCESAC2上。创立了将插入在fosmid载体上的不完整基因簇进行拼接的简便方法。该方法较已经报道的方法步骤简化,不需要对载体进行额外操作,不需要体外的基因操作创建同源臂和筛选标记,获得的完整基因簇不需要从载体上切下和重新连接,可以直接通过接合转移转入宿主中进行异源表达。利用该方法,成功拼接了滑桃树宏基因组来源的新的五酮安莎类生物合成基因簇tam,还实现了三个重叠基因簇片段的拼接,获得了Streptomyces sp. LZ35中的未知安莎类抗生素nas的完整基因簇。将获得tam基因簇和nas基因簇分别转入Streptomyces coelicolor ZM12, Amycolatopsis mediterranei s699△rifA和Thermophilic Streptomyces sp.4F中进行异源表达,并进行了基因表达调控和产物检测。更多还原

【Abstract】 Actinomycetes is a kind of Gram-positive bacterial with complex life style, which produces more than half of the currently known antibiotics. It mainly distributed in terrestrial and marine while extreme environment and plant tissue became the new source of actinomycetes.Ansamycins is a family of antibiotics, which often possess strong antimicrobial, antifungal, anticancer and antiviral activity. Most members of this family were isolated from actinomycetes. A subfamily named maytansinoids was found in higher plants and mosses. Ansamycins is Type Ⅰ PKS compound. It has a common biosynthetic starting unit AHBA. By using the DNA probe of AHB A, we can screen for ansamycin biosynthetic gene cluster. The biosynthesis of ansamycin is not only under global regulation of the cell but the pathway-specific regulation. Many of the ansamycin biosynthetic regulators belong to the LuxR family, by overexpression of which we can activate the silent ansamycin biosynthetic gene cluster or improve the production.Natural products was the most important source of drugs, but also a great treasure of new ansamycins. Megagenomic technology provides a strong support for the extraction of drugs from natural products.This thesis discussed two main ways of extracting new ansamycins.Approach Ⅰ:construct BAC library and expect to capture large integrated ansamycin biosynthetic gene cluster. First, a serials of E.coli-Streptomycets shuttle vector were constructed. pCCESAC82has advantages such as inducible replicon, PermE promoter, BstⅪ adaptor supported ligation, capability of conjugation and integration based on CIS element. Methods of constructing BAC library including extraction of high quality HMW DNA, preparation of high-efficient competent cells and improvement of ligation and transformation efficiency was investigated. Then a BAC library of Actinosynnema pretiosum31565was constructed and clones with an insertion of up to100kb was obtained. Approach Ⅱ:reassemble of overlapped DNA fragments by using Red recombination and heterologous expression of the biosynthetic gene cluster in suitable host. Utilizing the homologous arms between pCCESAC2and pCC1FOS, and the overlap fragment between the two fosmid insertion, two rounds of Red recombination was carried out resulting in the capture of the whole biosynthetic gene cluster in pCCESAC2. So, we developed a simple method for reassembling large DNA fragments into one vector. This method was much simple than the reported ones. It doesn’t need any additional modifications of donor fosmids and in vitro preparation of the homologous arms and selected marker. The plasmid can be directly conjugated to host for expression.We successfully constructed tam gene cluster obtained from metagenomic library of Trewia nudiflora and the unknown nas gene cluster of Streptomyces sp. LZ35. The obtained clusters were transformed into Streptomyces coelicolor ZM12, Amycolatopsis mediterranei s699△rifA and Thermophilic Streptomyces sp.4F for heterologous expression and product detection.更多还原