Knowledge Resource Center for Ecological Environment in Arid Area
| 特色放线菌天然产物库的构建及阿维菌素产量提高 | |
| 其他题名 | Natural Product Library of Propreitary Actinomycetes and Avermectin Overproduction |
| 刘梅 | |
| 出版年 | 2017 |
| 学位类型 | 博士 |
| 导师 | 凌立成 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 微生物能够产生多样性的次级代谢产物，被称为新药发现的宝库。放线菌是微生物药物的重要来源，构建特色放线菌的天然产物库可以作为微生物药物筛选的基础。本论文应用一株菌多种次级代谢产物（One strain many compounds, OSMAC）的研究方法，选择自新疆不同生态环境中分离获得的10株放线菌，分别属于Nocardia、Micromonospora、Streptomyces和Prauserella 4个放线菌菌属，作为代表菌株进行天然产物库构建方法的优化。选择了营养成分多样化的15种发酵培养基，培养不同周期，制备天然产物粗提物450份。应用HPLC指纹图谱评价粗提物的化学多样性；应用抗真菌、协同抗真菌、抗MRSA（耐甲氧西林金黄色葡萄球菌）、抗BCG（牛型结核分枝杆菌减毒株）等筛选模型评价粗提物的活性多样性。最终确定M9、M12、M21培养基，培养周期10天，作为新疆放线菌天然产物库制备的发酵培养条件。针对不同生境的特色放线菌，采用其最优的发酵条件和流程，构建了特色放线菌菌种库及天然产物库，对一株分离自新疆沙漠生境的放线菌进行系统发育分析及多项分类，鉴定为普劳斯氏菌属的新种Prauserella shujinwangii sp. nov.。对构建的特色放线菌天然产物库进行高通量筛选，发现其中一株链霉菌可产生具有与酮康唑协同抗真菌新活性的化合物N-formyl-staurosporine 、N-acetyl-staurosporine，MIC分别为6.25μg/mL和0.78μg/mL，有望成为抗真菌新药的候选化合物。本实验室从上述天然产物库中筛选出具有与甲氧西林协同抗MRSA活性的阿维菌素。阿维菌素作为生物杀虫剂在农业、畜牧业等领域的应用越来越广泛，近年来又不断发现其抗感染等新活性，对其产量提高的需求也更加强烈。本实验室的阿维菌素产生菌产量较低，未能达到产业化生产的要求。本论文应用传统诱变方法，进行阿维菌素高产菌株的选育。在紫外线、NTG（亚硝基胍）等物理、化学诱变的基础上，首次将强磁重力环境（High Magneto-Gravitational Environment，HMGE）作为新型诱变方法，复合核糖体工程等进行诱变育种，并对HMGE的诱变效应作了初步分析。应用Bootstrap方法对紫外线辐射、NTG、HMGE等五种突变文库以及对照进行分析，结果表明HMGE获得的突变库表型差异更大，说明这种环境中菌株的突变幅度更大，是一种较好的新型诱变方法。为提高诱变育种效率，本论文建立了基于96孔板固体发酵、紫外检测阿维菌素产量的高通量筛选方法，大大提高了突变菌株的筛选量。最终，经过包括HMGE在内的多轮诱变育种，筛选获得了产量提高93%的阿维菌素高产菌株，印证了传统诱变育种方法在菌株优化上的可行性。发酵条件是影响微生物次级代谢产物产量的重要因素之一，本论文利用Plackett-Burman方法，通过12组发酵实验，确立了阿维链霉菌发酵培养基成分中的最主要影响因素为玉米淀粉和酵母粉，又通过最陡坡度实验确定两个因素的最佳范围，再通过响应曲面法优化两个因素的最佳配比，获得最优结果为：玉米淀粉149.57 g/L，酵母粉8.92 g/L。在上述优化的发酵培养基条件下，高产菌株表现出优良的阿维菌素产量水平。在180m3发酵罐的放大试验中，根据该菌株的代谢特点，建立了补料等发酵工艺，使得阿维菌素B1a发酵水平达到7000 mg/L以上。本论文从特色放线菌天然产物库构建、挖掘和阿维菌素产量提高两方面进行研究，尝试突破微生物药物研发的瓶颈，为其它微生物天然产物药物的发现及高效生产提供可借鉴的思路和方法。 |
| 英文摘要 | Microorganism can produce a variety of natural products with diverse bioactivities, thus they are a treasure trove for new drug discovery. Actinomycetes are very talented for microbial drugs. Construction of a high-quality natural product library of actinomycetes is critical for microbial drug screening. In this thesis, the strategy of one strain many compounds (OSMAC) is employed. 10 actinomycetes strains isolated from different ecological environments in Xinjiang were selected as a case study to streamline the process for the natural product library. The 10 strains were identified and they belong to the family of Nocardia, Micromonospora, Streptomycesand Prauserella, respectively. 15 fermentation media with diversified nutrients were selected, and 450 crude extracts were prepared. For the crude extracts, the chemical diversity was evaluated by HPLC fingerprinting, and the bioactivity diversity was evaluated by screening against the antifungal, synergistic antifungal, anti-MRSA, anti-BCG (attenuated strains of M. bovis) models. The final fermentation condition to prepare the Xinjiang derived actinomycetes natural product library was 10 days culture in the M9, M12 and M21 medium. Using the optimized condition, the actinomycetes strain library and natural product library were constructed. The strain library contains multiple new actinomycetes, exemplified by 1 the new Prauserella shujinwangii sp. nov., which was characterized taxonomically. Bioactivity and biochemical diversities analyses revealed more than 150 natural products with novel bioactivities or novel chemical backbones. Further, compounds N-formyl-staurosporine and N-acetyl-staurosporine capable of synergistic antifungal activity with azole drugs (MIC = 6.25 μg/mL and 0.78μg/ mL, respectively) were identified from 1 Streptomyces strain in the library, which are promising candidate compounds as new antifungal agents.One of the hit compounds having synergistic anti-MRSA activity with methicillin, is avermectin, a biological pesticides used widely in agriculture, animal husbandry and other fields. Recently, new activities of avermectin, such as anti-infection, have been reported. The compound has shown potential to be a drug candidate, therefore improvement of its production is in great need. The yield of avermectin-producing strain screened from the microbial strain library in our laboratory is low and cannot meet the requirements for industrial application. In this thesis, traditional mutagenesis methods were used to breed avermectin high-producers. Except the traditional physical and chemical mutagenesis methods, such as UV and NTG mutagenesis, the High Magneto-Gravitational Environment (HMGE) was first used as a mutagenic method together with the method of ribosome engineering, to breed avermectin high-producers. The mutagenic effect of HMGE was analyzed, and compared with UV, NTG and other mutagenesis methods. The results showed that the mutation phenotype of HMGE was more diverse, indicating the HMGE is a better mutagenesis method. In order to improve the screening efficiency, the high throughput screening method based on 96-well plate solid-state fermentation and UV detection for avermectin production has been established. The method has greatly increased the throughput during screening and improved the efficiency of mutation breeding. Finally, after multiple rounds of mutagenesis including HMGE, avermectin production was increased by 93%. The results proved the feasibility of the traditional mutation breeding methods in strain optimization.Medium composition is one of the major factors affecting the yield of microbial secondary metabolites. In this thesis, using Plackett-Burman design, starch and yeast extract have been revealed as the main factors affecting the production of secondary metabolites of Streptomyces avermitilis. The optimal range of the two factors was determined by the steepest ascend method. Finally, the optimal concentration of the two factors was optimized by response surface method. The optimal concentration was: corn starch 149.57 g/L, yeast 8.92 g/L respectively. Under the optimized fermentation medium conditions, the high-yield strain showed excellent avermectin production level. Under the same condition, the fermentation was scaled up in 180 m3 fermenter, using fed-batch fermentation, the yield could reach over 7000 mg/L. Two parts of work has been done in this thesis: construction of microbial natural products library and improvement of avermectin’s production. The work should open the way to discover and produce more microbial natural products, and break through the bottleneck of the microbial drug research and development. |
| 中文关键词 | 放线菌 ; 天然产物 ; 阿维菌素 ; 产量提高 |
| 英文关键词 | Actinomycetes Natural products Avermectin Overproduction |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 生物化学与分子生物学 |
| 来源机构 | 中国科学院微生物研究所 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/287840 |
| 推荐引用方式 GB/T 7714 | 刘梅. 特色放线菌天然产物库的构建及阿维菌素产量提高[D]. 中国科学院大学,2017. |
| 条目包含的文件 | 条目无相关文件。 | |||||
| 个性服务 |
| 推荐该条目 |
| 保存到收藏夹 |
| 导出为Endnote文件 |
| 谷歌学术 |
| 谷歌学术中相似的文章 |
| [刘梅]的文章 |
| 百度学术 |
| 百度学术中相似的文章 |
| [刘梅]的文章 |
| 必应学术 |
| 必应学术中相似的文章 |
| [刘梅]的文章 |
| 相关权益政策 |
| 暂无数据 |
| 收藏/分享 |
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。
