本文選題：鏈霉菌W2 + diastaphenazine��； 參考：《華中農(nóng)業(yè)大學》2017年碩士論文

【摘要】：鏈霉菌W2和W4是從藥用植物黃花蒿中分離得到的兩株內(nèi)生鏈霉菌,能夠產(chǎn)生兩種二聚化吩嗪類化合物——izumiphenazine C和diastaphenazine,其中diastaphenazine是一種結構新穎、具有抗金黃色葡萄球菌和細胞毒活性的新型二聚化吩嗪化合物,目前還沒有其生物合成機制的相關報道。本研究首先對二聚化吩嗪產(chǎn)生菌W2和W4進行了背景研究,確定了它們的最佳產(chǎn)孢培養(yǎng)基,分析了它們對不同抗生素的背景抗性以及抑菌譜,推測兩種菌之間具有極其相近的親緣關系。采用PacBio RSII測序平臺對鏈霉菌W2的基因組進行測序分析,獲得一個8,074,405 bp的染色體和三個線性大質粒的序列。生物信息學分析發(fā)現(xiàn)染色體DNA中含有7993個基因,其中有102個基因與萜類和聚酮化合物代謝相關,65個基因與其他次級代謝產(chǎn)物的代謝相關。整個基因組中共有24個次級代謝產(chǎn)物生物合成基因簇,其中細菌素合成基因簇cluster9、鐵載體合成基因簇cluster12和萜類化合物合成基因簇cluster16與已知的生物合成基因簇沒有相似性,有可能產(chǎn)生新的代謝產(chǎn)物。在鏈霉菌W2的基因組中只發(fā)現(xiàn)了一個與吩嗪化合物合成相關的基因簇cluster17。以pMSB152B為載體構建了鏈霉菌W2的cosmid基因組文庫,然后根據(jù)15個小于40 kb的次級代謝產(chǎn)物合成基因簇的序列設計特異性引物,通過PCR篩選基因組文庫,共獲得了67個陽性克隆子。將含有細菌素合成基因簇cluster9的陽性克隆導入Streptomyces albus中進行異源表達,發(fā)現(xiàn)其中兩個異源表達菌株S.albus::21C7和S.albus::22A11表現(xiàn)出抑制枯草芽孢桿菌的活性,并且在異源表達菌株的發(fā)酵產(chǎn)物中找到了可能的代謝產(chǎn)物特征吸收峰。將含有鐵載體合成基因簇cluster12的陽性克隆導入S.albus中進行異源表達,通過HPLC分析也尋找到了可能的代謝產(chǎn)物特征吸收峰。將5個含有吩嗪合成基因簇cluster17的陽性克隆導入S.albus中進行異源表達,TLC分析發(fā)現(xiàn)其中兩個異源表達菌株S.albus::23C5和S.albus::20G10可以產(chǎn)生特異性的代謝產(chǎn)物。HPLC及LC-MS檢測證實這兩個菌株的異源表達產(chǎn)物中含有單體吩嗪-1-羧酸、二聚化吩嗪diastaphenazine和izumiphenazine C,表明cluster17即為二聚化吩嗪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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78;Q936

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