Streptomyces diastaticus W2中吩嗪生物合成基因簇的研究

发布时间：2018-05-21 09:18

本文选题：链霉菌W2 + diastaphenazine　；参考：《华中农业大学》2017年硕士论文

【摘要】：链霉菌W2和W4是从药用植物黄花蒿中分离得到的两株内生链霉菌,能够产生两种二聚化吩嗪类化合物——izumiphenazine C和diastaphenazine,其中diastaphenazine是一种结构新颖、具有抗金黄色葡萄球菌和细胞毒活性的新型二聚化吩嗪化合物,目前还没有其生物合成机制的相关报道。本研究首先对二聚化吩嗪产生菌W2和W4进行了背景研究,确定了它们的最佳产孢培养基,分析了它们对不同抗生素的背景抗性以及抑菌谱,推测两种菌之间具有极其相近的亲缘关系。采用PacBio RSII测序平台对链霉菌W2的基因组进行测序分析,获得一个8,074,405 bp的染色体和三个线性大质粒的序列。生物信息学分析发现染色体DNA中含有7993个基因,其中有102个基因与萜类和聚酮化合物代谢相关,65个基因与其他次级代谢产物的代谢相关。整个基因组中共有24个次级代谢产物生物合成基因簇,其中细菌素合成基因簇cluster9、铁载体合成基因簇cluster12和萜类化合物合成基因簇cluster16与已知的生物合成基因簇没有相似性,有可能产生新的代谢产物。在链霉菌W2的基因组中只发现了一个与吩嗪化合物合成相关的基因簇cluster17。以pMSB152B为载体构建了链霉菌W2的cosmid基因组文库,然后根据15个小于40 kb的次级代谢产物合成基因簇的序列设计特异性引物,通过PCR筛选基因组文库,共获得了67个阳性克隆子。将含有细菌素合成基因簇cluster9的阳性克隆导入Streptomyces albus中进行异源表达,发现其中两个异源表达菌株S.albus::21C7和S.albus::22A11表现出抑制枯草芽孢杆菌的活性,并且在异源表达菌株的发酵产物中找到了可能的代谢产物特征吸收峰。将含有铁载体合成基因簇cluster12的阳性克隆导入S.albus中进行异源表达,通过HPLC分析也寻找到了可能的代谢产物特征吸收峰。将5个含有吩嗪合成基因簇cluster17的阳性克隆导入S.albus中进行异源表达,TLC分析发现其中两个异源表达菌株S.albus::23C5和S.albus::20G10可以产生特异性的代谢产物。HPLC及LC-MS检测证实这两个菌株的异源表达产物中含有单体吩嗪-1-羧酸、二聚化吩嗪diastaphenazine和izumiphenazine C,表明cluster17即为二聚化吩嗪diastaphenazine的生物合成基因簇。
[Abstract]:Streptomyces W2 and W4 are two strains of endophytic Streptomyces isolated from the medicinal plant Artemisia Artemisia, and can produce two kinds of dimerization phenazine compounds - izumiphenazine C and diastaphenazine. Diastaphenazine is a novel structure, a new type of dimerization phenazine compounds with anti Staphylococcus aureus and cytotoxic activity. The biosynthesis mechanism was not reported before. This study first studied the background of the dimerization phenazine producing bacteria W2 and W4, determined their best sporulation medium, analyzed their background resistance to different antibiotics and bacteriostasis spectrum, and speculated that there was a close affinity between the two bacteria. The PacBio RSII test was used. Sequencing analysis of the genome of Streptomyces W2 to obtain a sequence of 8074405 BP chromosomes and three linear large plasmids. Bioinformatics analysis found that chromosome DNA contains 7993 genes, of which 102 genes are associated with terpenoids and polyketones metabolism, and 65 genes are associated with other secondary metabolites. There are 24 biosynthetic gene clusters of secondary metabolites in the whole genome, including the bacteriocin synthesis gene cluster cluster9, the ferric carrier synthesis gene cluster cluster12 and the terpenoid compound gene cluster cluster16 and the known biosynthetic gene cluster, which may produce new metabolites. In the genome of Streptomyces W2 Only one gene cluster related to the synthesis of phenazine compound cluster17. was found to construct the cosmid genomic library of Streptomyces W2 with pMSB152B as the carrier. Then, specific primers were designed based on the sequence of 15 sub metabolites less than 40 KB, and 67 positive clones were obtained by PCR screening the gene pool. The positive clones containing the bacteriocin synthetic gene cluster cluster9 were introduced into Streptomyces albus for heterologous expression, and two heterologous expression strains, S.albus:: 21C7 and S.albus:: 22A11, showed the activity of inhibiting Bacillus subtilis and found the potential absorption peak of the metabolites in the fermented products of the heterologous expression strain. The positive clones containing the iron carrier synthesis gene cluster cluster12 were introduced into S.albus for heterologous expression, and the potential absorption peaks of the possible metabolites were found through HPLC analysis. 5 positive clones containing the phenazine synthesis gene cluster cluster17 were introduced into S.albus for heterologous expression, and two heterologous expression strains, S, were found by TLC analysis. .albus:: 23C5 and S.albus:: 20G10 can produce specific metabolites.HPLC and LC-MS detection confirmed that the heterologous expression products of these two strains contain phenazine -1- carboxylic acid, dimerization phenazine diastaphenazine and izumiphenazine C, indicating that cluster17 is the biosynthetic gene cluster of dimerization phenazine diastaphenazine.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q936

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