八株土壤放线菌次级代谢产物的研究

发布时间：2018-07-10 10:01

  本文选题：链霉菌 + PCR筛选　； 参考：《山东大学》2017年博士论文

【摘要】：土壤放线菌是各种活性化合物尤其是抗生素的重要来源。其中,安莎霉素类抗生素具有独特的结构和生物活性,从土壤放线菌中发掘具有成药潜力的新型安莎霉素类化合物是我们的研究目标。传统的天然产物发掘周期长、效率低,且具有盲目性。基于AHBA合酶是安莎霉素类化合物合成的关键酶,且AHBA合酶基因在不同菌株中具有高度保守性,我们依次通过土壤采集、放线菌分离和逐级PCR筛选的方法获得33株AHBA(3-amino-5-hydroxybenzoic acid)合酶基因阳性菌。本论文通过2次菌株筛选,结合菌株产量和基因进化树,进一步选择8株潜在安莎霉素产生菌深入研究,并从其次级代谢产物中分离得到不同种类的安莎霉素和其他类型化合物,同时对发掘到的新安莎霉素类化合物进行活性评价。对菌株Streptomyces sp.S011、S013、S014和S027的固体燕麦培养基发酵产物进行分离和结构鉴定,共得到7个化合物,包括1个已知8酮安莎霉素(17-O-demethylgeldanamycin),2 个已知 9 酮安莎霉素(hygrocin C 和 hygrocin D)和1个新萘酚类化合物S11B(2)。对菌株Streptomyces sp.S015、S045和Kitasatospora sp.S023 的固体燕麦培养基发酵产物进行分离和结构鉴定,共得到19个化合物,其中2个新化合物,即1个呋喃酮类化合物(S45E),1个喹啉类化合物(S45P),未得到安莎霉素类抗生素,推测安莎基因簇在这3株菌中低表达或者不表达。对菌株Streptomyces sp.S012的固体燕麦培养基发酵产物进行分离和结构鉴定,共得到22个化合物,其中12个新化合物,包括9个结构类型丰富的新安莎霉素和3个安莎霉素生物合成中的前体。经结构鉴定,此9个新安莎霉素属于曲张链丝菌素类化合物(streptovaricins),命名为 ansavaricins A-I(34-38 和 42-45),与利福霉素同属于11酮安莎霉素。从结构上分析,化合物34、35、42-45的脂肪侧链(ansa chain)与C-5断裂,形成新的开环类曲张链丝菌素,目前除本文外仅有1个此类化合物(streptovaricin U)被报道。此外,我们对分离得到的安莎霉素34-45进行了生物活性测试,发现新化合物ansavaricinE(38)能够显著抑制Ⅲ型分泌系统毒性蛋白的分泌,并且不影响鼠伤寒沙门氏菌的生长,从而降低了耐药性的产生。通过拓扑异构酶体外抑制实验,证明ansavaricin H(44)具有较强的抑制拓扑异构酶Ⅰ和Ⅱα的活性。此外我们还发现,在所筛化合物中,开环的曲张链丝菌素(42-45)对拓扑异构酶的抑制活性要强于闭环的化合物。在细胞毒活性测试中,ansavaricin　H(44)对HeLa和MDA-MB-453细胞的增殖有明显的抑制作用(IC_(50)均为50 μM)。通过Western Blot和免疫荧光实验推测ansavaricin H(44)能够引起Hela细胞DNA损伤。
[Abstract]:Soil actinomycetes are important sources of various active compounds, especially antibiotics. Among them, Ansanamycin antibiotics have unique structure and biological activity. It is our research goal to explore new Ansanamycin compounds with the potential of patent medicine from soil actinomycetes. The traditional natural product discovery cycle is long, the efficiency is low, and has the blindness. Based on the fact that AHBA synthase is the key enzyme in the synthesis of Ansanamycin compounds, and the AHBA synthase gene is highly conserved in different strains, we collected it from soil. 33 strains of AHBA (3-amino-5-hydroxybenzoic acid) synthase gene positive strains were isolated from actinomycetes and screened by stepwise PCR. In this paper, 8 strains of potential Ansanamycin producing bacteria were selected for further study through two strains screening, combined with strain yield and gene evolution tree. Different kinds of Ansanamycin and other compounds were isolated from their secondary metabolites, and the activity of these compounds was evaluated. The fermentation products of Streptomyces sp.011S013S014 and S027 in solid oat culture medium were isolated and identified, and 7 compounds were obtained. It consists of one known 8-keto-demethylgeldanamycin and two known 9-ketoanamycin (hygrocin C and hygrocin D) and one neonaphthalene phenolic compound S11B (2). The fermentation products from solid oat medium of Streptomyces sp.015S045 and Kitasatospora sp.023 were isolated and identified. A total of 19 compounds were obtained, 2 of which were new compounds. One furanone compound (S45E) and one quinoline compound (S45P) were not obtained Ansanamycin antibiotics. The fermentation products from solid oat medium of Streptomyces sp.012 were isolated and identified. A total of 22 compounds were obtained, 12 of which were new compounds, including 9 precursors of nisamycin and 3 precursors of Ansanamycin biosynthesis. The nine nisamycin belong to varicose streptomycin compound (streptovaricins), named ansavaricins A-I (34-38 and 42-45) and belong to 11 ketoanamycin with rifamycin. Structurally, the adipose side chain (ansa chain) and C-5 of compound 34N 35N 42-45 were broken off to form a new ring-opening streptavidin. Only one such compound (streptovaricin U) has been reported so far except in this paper. In addition, we tested the bioactivity of Ansanamycin 34-45, and found that Anansavaricin E (38) could significantly inhibit the secretion of toxic protein of type 鈪，

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