本文選題：鏈霉菌 + PCR篩選��； 參考：《山東大學(xué)》2017年博士論文

【摘要】：土壤放線菌是各種活性化合物尤其是抗生素的重要來(lái)源。其中,安莎霉素類抗生素具有獨(dú)特的結(jié)構(gòu)和生物活性,從土壤放線菌中發(fā)掘具有成藥潛力的新型安莎霉素類化合物是我們的研究目標(biāo)。傳統(tǒng)的天然產(chǎn)物發(fā)掘周期長(zhǎng)、效率低,且具有盲目性�；贏HBA合酶是安莎霉素類化合物合成的關(guān)鍵酶,且AHBA合酶基因在不同菌株中具有高度保守性,我們依次通過(guò)土壤采集、放線菌分離和逐級(jí)PCR篩選的方法獲得33株AHBA(3-amino-5-hydroxybenzoic acid)合酶基因陽(yáng)性菌。本論文通過(guò)2次菌株篩選,結(jié)合菌株產(chǎn)量和基因進(jìn)化樹(shù),進(jìn)一步選擇8株潛在安莎霉素產(chǎn)生菌深入研究,并從其次級(jí)代謝產(chǎn)物中分離得到不同種類的安莎霉素和其他類型化合物,同時(shí)對(duì)發(fā)掘到的新安莎霉素類化合物進(jìn)行活性評(píng)價(jià)。對(duì)菌株Streptomyces sp.S011、S013、S014和S027的固體燕麥培養(yǎng)基發(fā)酵產(chǎn)物進(jìn)行分離和結(jié)構(gòu)鑒定,共得到7個(gè)化合物,包括1個(gè)已知8酮安莎霉素(17-O-demethylgeldanamycin),2 個(gè)已知 9 酮安莎霉素(hygrocin C 和 hygrocin D)和1個(gè)新萘酚類化合物S11B(2)。對(duì)菌株Streptomyces sp.S015、S045和Kitasatospora sp.S023 的固體燕麥培養(yǎng)基發(fā)酵產(chǎn)物進(jìn)行分離和結(jié)構(gòu)鑒定,共得到19個(gè)化合物,其中2個(gè)新化合物,即1個(gè)呋喃酮類化合物(S45E),1個(gè)喹啉類化合物(S45P),未得到安莎霉素類抗生素,推測(cè)安莎基因簇在這3株菌中低表達(dá)或者不表達(dá)。對(duì)菌株Streptomyces sp.S012的固體燕麥培養(yǎng)基發(fā)酵產(chǎn)物進(jìn)行分離和結(jié)構(gòu)鑒定,共得到22個(gè)化合物,其中12個(gè)新化合物,包括9個(gè)結(jié)構(gòu)類型豐富的新安莎霉素和3個(gè)安莎霉素生物合成中的前體。經(jīng)結(jié)構(gòu)鑒定,此9個(gè)新安莎霉素屬于曲張鏈絲菌素類化合物(streptovaricins),命名為 ansavaricins A-I(34-38 和 42-45),與利福霉素同屬于11酮安莎霉素。從結(jié)構(gòu)上分析,化合物34、35、42-45的脂肪側(cè)鏈(ansa chain)與C-5斷裂,形成新的開(kāi)環(huán)類曲張鏈絲菌素,目前除本文外僅有1個(gè)此類化合物(streptovaricin U)被報(bào)道。此外,我們對(duì)分離得到的安莎霉素34-45進(jìn)行了生物活性測(cè)試,發(fā)現(xiàn)新化合物ansavaricinE(38)能夠顯著抑制Ⅲ型分泌系統(tǒng)毒性蛋白的分泌,并且不影響鼠傷寒沙門氏菌的生長(zhǎng),從而降低了耐藥性的產(chǎn)生。通過(guò)拓?fù)洚悩?gòu)酶體外抑制實(shí)驗(yàn),證明ansavaricin H(44)具有較強(qiáng)的抑制拓?fù)洚悩?gòu)酶Ⅰ和Ⅱα的活性。此外我們還發(fā)現(xiàn),在所篩化合物中,開(kāi)環(huán)的曲張鏈絲菌素(42-45)對(duì)拓?fù)洚悩?gòu)酶的抑制活性要強(qiáng)于閉環(huán)的化合物。在細(xì)胞毒活性測(cè)試中,ansavaricin　H(44)對(duì)HeLa和MDA-MB-453細(xì)胞的增殖有明顯的抑制作用(IC_(50)均為50 μM)。通過(guò)Western Blot和免疫熒光實(shí)驗(yàn)推測(cè)ansavaricin H(44)能夠引起Hela細(xì)胞DNA損傷。
[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，

本文編號(hào)：2113025