【作者】 魏杰；

【导师】 孟宪军；

【作者基本信息】 沈阳农业大学 ， 食品科学与工程， 2009， 博士

【摘要】 金褐链霉菌(Streptomyces aureofuscus)是从我国土壤中分离得到的链霉菌新种,其产生一种四烯大环内酯类抗真菌抗生素——金褐霉素(Aureofuscin),金褐霉素化学结构与国外文献报道的纳他霉素(Natamycin)结构相似。金褐霉素对霉菌、多种酵母菌及丝状真菌有很强的抗菌作用,但不抗细菌。临床上用于治疗真菌性角膜炎,其疗效优于两性霉素B,同时对一些真菌引起的皮肤病及霉菌性阴道炎等也有很好疗效。金褐霉素早在1975年发现时只作了早期化学结构和初步药效试验后,一直未作深入研究和进行产业化开发。鉴于金褐霉素与纳他霉素有着相似的化学结构和生物活性,而纳他霉素现在主要依赖进口。因此,我国具有自主知识产权的金褐霉素的研究开发在真菌疾病的防治和食品防腐上显示出了极好的应用前景和市场潜力,这对促进新药研制、食品防腐和发酵工业的发展都具有积极意义。金褐霉素是一种天然的生物防腐剂,目前市场的需求量很大,但限制其生产的主要原因就是发酵产量太低。本文对金褐霉素生物合成基因簇中调控基因进行了初步研究,实验主要通过过度表达线性反应中某一基因来提高最终产物产量,以期获得较为高产的金褐霉素生产菌株,以及发酵条件优化来提高金褐霉素产生菌的发酵产金褐霉素水平。1)确立了高效液相色谱法(HPLC)测定发酵液中的金褐霉素的条件。检测波长:303nm;流动相:甲醇:水体积比为60∶40,流速:1.00mL/min,保留时间为6.957min。线性回归方程为Y=8E+07X-381894,R~2=0.9998。当信噪比为3时的最小检测限为0.25μg/mL。样品的平均回收率(n=3)为100.09%、100.90%、102.41%,RSD为0.18%、1.53%、1.29%。2)本试验发现金褐链霉菌DNA中存在纳他霉素生物合成基因簇中基因pimM的一个同源基因,并将这段基因命名为AURJ3M。两者的基因序列具有95%的相似性,这两种蛋白的氨基酸序列同源性为97%。将获得的新基因序列登录GenBank,登录号为EU697915。3)本试验将纳他霉素生物合成基因簇的正调控基因pimM置于PermE之下进行整合性表达,克隆到高效表达质粒载体pSET152中,成功构建大肠杆菌ET12567与金褐链霉菌SYAU0709属间接合转移重组质粒PBJPIM,将PBJPIM转入金褐链霉菌野生型菌株SYAU0709染色体DNA上,其转化子的金褐霉素产量提高3-4倍,说明pimM基因在金褐链霉菌中能够促进金褐霉素的合成。4)本试验将pimM的一个同源基因AURJ3M置于PermE之下进行整合性表达,克隆到高效表达质粒载体pSET152中,成功构建大肠杆菌ET12567与金褐链霉菌SYAU0709属间接合转移重组质粒PBJJ3M,将PBJJ3M转入金褐链霉菌野生型菌株SYAU0709染色体DNA上,其转化子的金褐霉素产量提高5-6倍,说明AURJ3M基因在金褐链霉菌中促进了金褐霉素的合成,并且遗传性能稳定。5)以金褐霉素摇瓶发酵为基础,进行由摇瓶到5L发酵罐的放大,确定培养条件为:通气量4L/min,搅拌转速220r/min,发酵的溶氧界点不低于20%、发酵周期84h和产素期pH应控制在5.5,最佳的接种量10%,金褐霉素产量为3.85mg/mL。6)X-5大孔吸附树脂对金褐霉素的吸附率和解吸率都较高,可以作为纯化金褐霉素的树脂。纯化条件为吸附8h,解吸5h,上样液pH值5.0,洗脱液pH值7.0,吸附流速为1.0BV/h,洗脱流速为1mL/min。此方法得到了金褐霉素的粗结晶。更多还原

【Abstract】 Aureofuscin is a tetraene macrolides antifungal antibiotic produced by Streptomyces aureofuscus which was isolatated from the soil in China. The chemical constitution of Aureofuscin is similar to Natamycin. They exhibit high activity against human pathogens including mycetes, yeast fungus and mycelial fungus, but they do not exhibit high activity against bacterium. The clinical efficacy of Aureofuscin surpass amphotericin B in treating keratomycosis, fungus-caused dermatoses and colpitis mycotica. Aureofuscin was discovered on chemical constitution and initial pharmacodynamic tests in1975, but not lucubrating constantly and industrialization development. In view of the chemical constitution and bioactivity of Aureofuscin similar to Natamycin which is mainly depended on the import, the research and development of Aureofuscin possessing the China independent intellectual property right shows the splendiferous applying perspective and market potential on the antifungal agents and food preservative, also possesses active meaning on promoting development of new drugs trituration, food antiseptic and fermentation industry.Aureofuscin is a natural antiseptic with market demands. But the primary cause limited its production is the low fermentation output. In this article a systematic investigation was carried out to enhance the Aureofuscin -producing by over-expression of the certain gene in Aureofuscin biosynthesis gene cluster in the linear response and fermentation condition optimization were adopted for high Aureofuscin-producing.1) The HPLC method of Aureofuscin detection in fermentation broth was developed. The optimum determination conditions were detection wavelength: 303nm, mobile phase: methanol: water: 60:40, flow speed: 1.00mL/min, keeping time: 6.957min, log-linear regression equation Y=8E+07X -381894, R~2=0.9998. The minimal detectability was 0.25μg/mL when the signal noise ratio was 3. Averaged recovery was 100.09%、100.90%、102.41% and averaged RSD was 0.18%、1.53%、1.29%.2) A pimM-homologene in Aureofuscin biosynthesis gene cluster was discovered, named AURJ3M.Sequence analysis indicated that the sequence of the cloned AURJ3M gene is the 95% and the amino acid sequence is the 97% similarity as the published PimM gene in Natamycin biosynthesis gene cluster. The AURJ3M was registed in GenBank, and the Accession number was EU697915.3) The high-copy-number plasmid pBJPIM, containing the S.natalensis pimM gene under the promoter PermE , was constructed as follows. This fragment was ligated with pSET152, resulting in plasmid pBJPIM. It was introduced into wild-type S.aureofuscus SYAU0709 via E.coli-Streptomyces conjugation. The Aureofuscin yield improved 3-4 times in the transformants. Results showed that an extra copy of the pimM gene was existed in the recombinant strains after the integration of a single copy of recombinant plasmid pBJPIM into the chromosome of S.aureofuscus SYAU0709. Overexpression of the pimM gene causes overproduction of Aureofuscin in wild-type strain.4) The high-copy-number plasmid pBJJ3M, containing the S.aureofuscus AURJ3M gene under the promoter PermE , was constructed as follows. This fragment was ligated with pSET152, resulting in plasmid pBJJ3M. It was introduced into wild-type S.aureofuscus SYAU0709 via E.coli-Streptomyces conjugation. The Aureofuscin yield improved 5-6 times in the transformants. Results showed that an extra copy of the AURJ3M gene was existed in the recombinant strains after the integration of a single copy of recombinant plasmid pBJJ3M into the chromosome of S.aureofuscus SYAU0709. Overexpression of the AURJ3M gene causes overproduction of Aureofuscin in wild-type strain. The recombinant strains have the stable heritage.5) Base on the above optimum condition, the batch fermentation was performed in 5-liter fermentor. And the optimal fermentation conditions were that 4L/min ventilatory capacity, 220r/min rotational speed, dissolves oxygen not be lower than 20%, fermentation period 84h and pH should control nearby 5.5, and the yield of Aureofuscin is 3.85mg/mL.6) The Aureofuscin purification using X-5 macroporous resin was studied. X-5 resin possessed higher absorption and desorption rate. The time of adsorption and desorption is respectively 8h and 5h. Sample pH is 6.0 and flowed pH is 7.0. Speed of adsorption and desorption is 1.0 BV/h and 1ml/min. The mecrocrystalline of Aureofuscin was obtained.更多还原