本文選題：Kigamicin + 生物合成基因簇��； 參考：《福建師范大學》2016年碩士論文

【摘要】：Kigamicin是由Amycolatopsis sp. ML630-mF1發(fā)酵產生的一類多環(huán)氧雜蒽酮類抗生素,具有抗細菌、抗真菌和特異性抗腫瘤的活性。其特異性抗腫瘤活性表現(xiàn)為可特異性抑制營養(yǎng)饑餓狀態(tài)下腫瘤細胞,而對正常狀態(tài)下的細胞無抑制作用,因此可作為特異性抗癌藥物的潛在開發(fā)來源。鑒于Kigamicin獨特的化學結構和生物活性,人們對其結構與功能的關系產生了濃厚的研究興趣。本研究在Kigamicin生物合成基因簇成功克隆的基礎上,通過分子生物學手段對其相關基因功能進行研究,可使我們進一步了解多氧環(huán)雜蒽酮類抗生素生物合成機制,為解釋其獨特的生物活性提供理論依據(jù)。首先,通過生物信息學分析推測Kigamicin生物合成基因簇中orf48和orf49的基因功能,orf48與Xantholipin和Lysolipin生物合成基因xanK和llpK同源性較高,初步推測orf48可能編碼3Fe-S Ferredoxin,參與Kigamicin生物合成途徑中的電子傳遞和氧化還原反應；orf49與Xantholipin生物合成基因xan02功能類似,有可能負責Kigamicin生物合成中亞甲基雙氧橋的形成；但是也可能與Lysolipin生物合成基因llpOIV相似,參與催化Kigamicin中氧雜葸酮環(huán)形成前的環(huán)氧化反應。然后,通過PCR-targeting技術分別構建缺失orf48質粒和缺失orf49質粒,即分別以壯觀霉素抗性基因替換質粒pLL59上的orf48和orf49,并通過抗性篩選轉化子,經(jīng)PCR驗證后,得到突變質粒pLL214和pLL215。而后通過接合轉移將突變質粒分別轉入天藍色鏈霉菌ML154,抗性篩選得到突變株Kiga21468和突變株Kiga21568.通過HPLC-MS檢測突變株Kiga21468、Kiga21568以及野生型ML154發(fā)酵產物,結果比對,發(fā)現(xiàn)突變株Kiga21468和突變株Kiga21568產生了相同的物質組分,并且與Kigamicin具有相同UV特征吸收峰。由此推測,突變株Kiga21468和Kiga21568可能產生Kigamicin結構類似物。對突變株發(fā)酵積累代謝產物[M+H]/Z=497, [M+H]/Z=538, [M+H]/Z=527進行分離純化和結構鑒定,化合物[M+H]/Z=538的分子式為C28H27NO10,其化學結構與Kigamicin結構相比缺少亞甲基雙氧橋結構,故而推測Orf48和Orf49參與了Kigamicin亞甲基雙氧橋結構的生物合成。
[Abstract]:Kigamicin is made by Amycolatopsis sp. A class of polycyclic oxyanthrone antibiotics produced by ML630-mF1 fermentation, with antibacterial, antifungal and specific antitumor activities. Its specific antitumor activity is that it can specifically inhibit tumor cells in the condition of nutritional starvation, but has no inhibitory effect on the cells in normal state, so it can be used as a potential source for the development of specific anticancer drugs. In view of the unique chemical structure and biological activity of Kigamicin, the relationship between its structure and function has attracted much attention. On the basis of the successful cloning of Kigamicin biosynthesis gene cluster, the function of its related genes was studied by molecular biological methods, so that we can further understand the biosynthesis mechanism of polyoxocyclic anthrone antibiotics. It provides theoretical basis for explaining its unique biological activity. Firstly, the gene function of orf48 and orf49 in Kigamicin biosynthesis gene cluster is conjectured by bioinformatics analysis. The gene function of orf48 and orf49 is highly homologous to that of Xantholipin and Lysolipin biosynthesis gene xanK and llpK. It is inferred that orf48 may encode 3Fe-S Ferredoxin, which is involved in the electron transport and redox reaction in the Kigamicin biosynthesis pathway, and may be responsible for the formation of methylene dioxide-bridge in Kigamicin biosynthesis, which is similar to that of Xantholipin biosynthesis gene xan02. But it may also be similar to the Lysolipin biosynthesis gene llpOIV, which is involved in the epoxidation reaction before the formation of oxa-anthracanone ring in Kigamicin. Then, the deletion orf48 plasmid and the deletion orf49 plasmid were constructed by PCR-targeting technique, that is, the orf48 and orf49 on the plasmid pLL59 were replaced with the spectinomycin resistance gene, and the mutant plasmids pLL214 and pLL215were obtained after PCR verification by screening the transformants. Then the mutant plasmids were transferred into Streptomyces cerevisiae ML154 by conjugation transfer. The mutant Kiga21468 and the mutant Kiga21568 were obtained by resistance screening. The fermentation products of the mutant Kiga21468 and wild-type ML154 were detected by HPLC-MS. The results showed that the mutant Kiga21468 and the mutant Kiga21568 produced the same substance components and had the same UV characteristic absorption peak as Kigamicin. It is inferred that Kiga21468 and Kiga21568 may produce Kigamicin structural analogues. The metabolites of [M H] / r ZN 497, [M H] / R ZN 538, [M H] / R ZN 527 were isolated, purified and identified. The molecular formula of compound [M H] / R ZN 538 is C28H27NO10, and its chemical structure lacks methylene dioxide-bridge structure compared with the structure of Kigamicin. Therefore, it is speculated that Orf48 and Orf49 are involved in the biosynthesis of Kigamicin methylene dioxide-bridge structure.
【學位授予單位】：福建師范大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：TQ927;Q78

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本文編號：1904754