【作者】 张熠；

【导师】 洪文荣；

【作者基本信息】 福州大学 ， 生物化学与分子生物学， 2014， 硕士

【摘要】 庆大霉素由绛红色小单孢菌产生,已在临床上得到广泛应用。但庆大霉素生物合成途径与功能基因的研究却刚刚开始。本研究在分子水平上探索庆大霉素生物合成与功能基因的关系,完成了GKN27、GKP116、G091和GKB3226等小单孢工程菌的构建。具体内容包括以下三个方面：第一,利用生物信息学技术,预测庆大霉素生物合成基因簇的相关基因功能。选取四个功能基因(genN、genO、genP和genB3)进行研究。经同源比对,分析基因编码蛋白(GenN、 GenO、GenP和GenB3)的结构域和同源建模等,推测基因genN可能是修饰庆大霉素6’C-N的甲基化酶基因；基因genO的结构域含有甲基化序列,与庆大霉素生物合成途径中的甲基化修饰有关；基因genP可能是庆大霉素C3’,C4’双脱氧反应中的磷酸转移酶基因;基因genB3与庆大霉素C6’的氨基转移有关。第二,功能基因的研究。根据同源重组原理,设计功能基因(genN、genO、genP)框内敲除的实验方法。利用基因工程技术,成功构建基因genN缺失小单孢工程菌M. purpwea GKN27。工程菌GKN27不产生庆大霉素C族复合物,而积累四种庆大霉素生物合成中间体,表明genN基因功能与生物信息学的理论推测不符,即genN可能不是编码庆大霉素6’C-N的甲基化酶基因；该研究已整理论文被工业微生物杂志录用。敲除基因genP,获得小单孢工程菌M. purpwea GKP116,研究表明,genP基因与庆大霉素生物合成过程密切相关。利用同样的方法,获得基因genO缺失的小单孢工程菌M purpwea G091,结果表明,基因genO与庆大霉素6’C-N的甲基化反应有关。第三,庆大霉素X2小单孢工程菌的构建。庆大霉素X2是进一步开发抗原虫,抗病毒以及抗肿瘤药物的重要前体。选取工程菌M. purpurea GK1101为出发菌,以其染色体DNA为模板,利用PCR扩增庆大霉素合成基因簇中氨基转移酶基因genB3的上、下游序列作为同源交换臂。通过分子克隆技术构建重组质粒pFU803。重组质粒pFU803经接合转移,导入绛红色小单孢菌GK1011。经筛选得到目标小单孢工程菌M. purpurea GKB3226。根据其代谢产物的TLC和EIS-MS分析,确认其代谢产物主要为庆大霉素X2和少量的庆大霉素JI-20A。结果表明,genB3基因功能与庆大霉素Π-20A双脱羟基反应有关,敲除genB3阻断了庆大霉素Π-20A至西索米星的生物合成,获得小单孢工程菌M. purpurea GKB3226.填补了国内外研究空白,申请了国家发明专利。更多还原

【Abstract】 Gentamicin producted by micromonospora purpurea, had been widely used in clinical. However the study on gentamicin biosynthesis and gene function has just begun. In this study, we explored the relationship of gentamicin biosynthesis and function of genes at the molecular level. And the micromonospora engineering strains (GKB3226, GKN27, GKP116, GO91) were constructed by gene knockout method. There are three aspects involved in this study:Firstly, bioinformatics analysis. The functions of the key genes from gentamicin biological gene cluster were predicted by bioinformatics analysis. This study was focus on four functional genes on the base of industrial application and gentamicin biosynthetic pathway. The functions of genes (genN, genO, genP, genB3) were speculated by homologous comparison, domain analysis and homology modeling research. The gene genN belongs to the methyltransferase superfamily; The gene genB3 was associated with gentamicin C6’aminotransferase, The gene genO enconding tRNA queosine ribosyl transferase, is related with the reaction of methylation in gentamicin biosynthetic pathway; The gene genP may be charge of 3’-phosphotransferase.Secondly, the research of functional genes (genN, genO, genP). According to the principle of homologous recombination, the experimental method of gene (genN, genO, genP) frame knockout was designed. The micromonospora engineering strain GKN27 (deletion of genN gene) was successfully constructed by genetic engineering technology. Instead of gentamicin C complex, the engineering strain M. Purpurea GKN27 generates four intermediates of gentamicin biosynthesis pathway. And it indicated that genN was not associated with methyl transferase. The study parper has been recieved by the journal of industial microbiology. The engineering strain M. Purpurea GKP116, the gene genP of which was knocked out from M. purpurea GK1101, was constructed by the same method. The analysis of metabolites from GKP116 suggested that genP is closely related to gentamicin biosynthesis. The engineering strain In. Purpurea G091, the genO gene of which was knocked out from M. purpurea G1008. The results showed that genO gene is association with the reaction of 6’C-N methylation.Thirdly, construction of gentamicin X2 micromonospora engineering strain. Gentamicin X2 is an important precursor of antiprotozoal, antiviral and antitumor drugs. The homologous fragments were amplified with the chromosome DNA of M. purpurea GK1101. The recombinant plasmid pFU803 was constructed by molecular cloning technology. Then the recombinant plasmid pFU803 was introduced into the M. purpurea GK1101 through the conjugal transfer method. After repeated screening, we successfully obtained double exchange engineering strain M. purpurea GKB3226. According to the results of TLC and EIS-MS analysis, we substantiated that the engineering strain GKB3226 accumulates gentamicin X2 by inactivation of genB3. The micromonospora engineering strains M. purpurea GKB3226, special biosynthesis gentamicin X2, fills the gaps in the domestic and foreign research.更多还原