表观遗传修饰增加海洋真菌次生代谢产物结构多样性和活性研究

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	表观遗传修饰增加海洋真菌次生代谢产物结构多样性和活性研究
	刘洋
学位类型	博士
导师	王斌贵
	2015-05
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	海洋生物学
关键词	海洋真菌次生代谢生物活性表观遗传修饰
摘要	天然产物是新药开发的重要途径之一，而海洋真菌来源的天然产物大多具有新颖的结构和显著的生物活性，正引起研究者们的极大兴趣。对真菌全基因组测序的研究表明，真菌的大多数基因是沉默的，在常规的实验培养条件下并不发生表达。利用表观遗传修饰的策略激活某些沉默的表达途径、深入挖掘真菌的代谢潜力、发现结构新颖和具有生物活性的化合物成为目前研究的热点。本文采用表观遗传修饰的策略开展了海洋真菌次生代谢产物分子多样性及活性研究。以海绵内生真菌类阿达青霉(Penicillium adametzioides AS-53)、木榄根际土壤来源真菌赭曲霉(Aspergillus ochraceus MA-15)、榄李叶内生真菌桔青霉(Penicillium citrinum MA-197)和红海榄内生真菌柄篮状菌(Talaromyces stipitatus MA-205)为研究对象，采用不同的培养基优化培养，获得最佳培养条件并进行规模化发酵，从上述四株真菌中分离、鉴定了106个次生代谢产物，发现17个新结构化合物和2个新天然产物，结构类型包括含硫二酮哌嗪衍生物、喹唑啉酮类、吲哚二酮哌嗪类、倍半萜及衍生物、混源萜类、甾体类及衍生物、聚酮类和二苯醚类等。对分离获得的部分单体化合物进行了抗细菌、抗真菌、抗肿瘤和卤虫致死等生物活性测试。具体结果如下：对海绵内生真菌类阿达青霉Penicillium adametzioides AS-53进行了表观遗传修饰增加代谢产物结构多样性和活性的初步研究，对20种培养条件下的代谢产物进行HPLC比较分析，发现改变培养条件可以使该菌株的次生代谢产物产生较大变化。选择改良PDB 和大米培养基分别对菌株AS-53进行培养，从不同培养条件的发酵物中分离得到不同的化合物。从该菌株的发酵产物中共分离并鉴定了40个化合物，其中新化合物7个，包括从改良PDB培养基中分离获得4个含硫二酮哌嗪类新化合物（PAP1~PAP4）和1个喹诺唑啉酮类新化合物（PAP6）以及从大米培养基中分离获得2个倍半萜类新化合物（PAR1~PAR2），其中PAP4具有spiro[furan-2,7’-pyrazino[1,2-b][1,2]oxazine]新骨架片段。对分离得到的单体化合物进行了卤虫致死活性、抗菌活性和细胞毒活性测试。结果显示，新化合物PAP1具有较好的卤虫致死活性，LD50值为4.76 µM，优于阳性对照秋水仙碱(LD50值为8.09 µM)。新化合物PAP1对致病菌金黄色葡萄球菌、嗜水气单胞菌、哈氏弧菌、副溶血性弧菌和小麦全蚀病菌具有不同程度的抑制活性，MIC值分别为8.0, 8.0, 32.0, 8.0, 16.0 μg/mL，其中对嗜水气单胞菌抑制活性优于阳性对照氯霉素(MIC值为16 μg/mL)。新化合物PAR2对肿瘤细胞NCI-H446具有选择性抑制活性，其IC50 = 5.0±0.9 μM，阳性对照阿霉素(doxorubicin)IC50 = 4.0±0.4 μM。从菌株赭曲霉MA-15中分离鉴定了28个化合物，其中6个新化合物，包括5个小分子聚酮类新化合物（AO1~AO3、AO8、AO15）和1个单苯环类新化合物（AO19）。从菌株桔青霉MA-197中分离鉴定了23个化合物，其中首次通过单晶衍射实验确定了倍半萜衍生物PC17的绝对构型。从菌株柄篮状菌MA-205中分离鉴定了18个化合物，其中4个二苯醚类新化合物（TS1~TS3、TS6）和2个新天然产物（TS7、TS17）。对获得的部分单体化合物进行了抗菌、抗肿瘤、卤虫致死活性测试。新化合物AO1和已知化合物AO13对嗜水气单胞菌、鳗弧菌和哈氏弧菌均具有一定的抑制活性，其MIC值分别8.0, 16.0, 8.0和1.0, 32.0, 0.5 μg/mL，阳性对照氯霉素MIC值为4.0, 1.0, 8.0 μg/mL。化合物PC5对金黄色葡萄球菌具有一定的抑制活性，MIC为4.0 μg/mL，是阳性对照氯霉素的2倍，化合物PC18对藤黄微球菌具有很强的抑制活性，其MIC为0.25 μg/mL，是阳性对照氯霉素的8倍，化合物PC13和PC15分别对柑橘炭疽病菌和小麦全蚀病菌具有与阳性对照博莱霉素相当的抑制活性，MIC分别为4.0和2.0 μg/mL。本论文通过改变培养条件探索海洋真菌的次生代谢潜力，诱导其产生了分子结构多样性的代谢产物，为深入开发海洋真菌次生代谢能力、获得结构新颖和具有生物活性的海洋天然产物提供了一定的依据。
其他摘要	Natural products are important source of developing new drugs, especially those from marine-derived fungi which possess novel structures and significant biological activities have attracted more and more attentions. The whole genome sequencing studies of fungi have indicated that most genes of fungi were silent and not expressed in the conventional experimental training conditions. It has been a hot pot nowadays to search for new and bioactive compounds using the strategy of epigenetic modification. In the dissertation, secondary metabolites and their bioactivities for four marined-derived fungi were researched using the strategy of epigenetic modification. One hundred and six compounds were isolated and elucidated from four marine-derived fungi, Penicillium adametzioides AS-53 from an unidentified marine sponge, Aspergillus ochraceus MA-15 from the rhizosphere soil of marine mangrove plant Bruguiera gymnorrhiza, Penicillium citrinum MA-197 from the leaves of mangrove plant Lumnitzera racemosa, and Talaromyces stipitatus MA-205 from the mangrove plant Rhizophora stylosa Griff. Seventeen of the compounds were new structures and two of the others were new natural compounds. Antimicrobial, cytotoxic, as well as brine shrimp (Artemia salina) lethality were measured for the compounds. The preliminary research of the fermentation of P. adametzioides AS-53 using the strategy of epigenetic modification was reported in this dissertation. The results showed that different fermentation factors of this fungus could produce diverse secondary metabolites. Two fermentation conditions were performed and different compounds from each fermentation extracts were isolated. Thirty-one compounds were isolated and elucidated from the cultivation on a liquid potato-dextrose broth (PDB) culture medium of the fungus, five of which were new structures, including four bisthiodiketopiperazines (PAP1~PAP4) and one spiroquinazoline (PAP6). Nine distinct compounds were isolated from a rice solid culture medium of this fungus, including two new acorane sesquiterpenes (PAR1~PAR2). All these compounds were examined for antimicrobial and cytotoxic activity as well as brine shrimp (Artemia salina) lethality. New compound PAP1 showed brine shrimp lethality, with LD50 value of 4.76 µM, and the positive control colchicin was 8.09 µM. Furthermore, PAP1 showed potential activity against Staphyloccocus aureus, Aeromonas hydrophilia, Vibrio harveyi, V. parahaemolyticus, and Gaeumannomyces graminis with MIC values of 8.0, 8.0, 32.0, 8.0, and 16.0 μg/mL, respectively. New compound PAR2 showed selective activity against the NCI-H446 cell line with IC50 value of 5.0 μM, while the positive control doxorubicin was 4.0±0.4 μM. Twenty-eight compounds were isolated and elucidated from the extracts of A. ochraceus MA-15, six of which were new compounds, including five polyketides (AO1~AO3, AO8, AO15) and one phenolic derivative (AO19). Twenty-three compounds were isolated and elucidated from the extracts of P. citrinum MA-197. Eighteen compounds were isolated and elucidated from the extracts of T. stipitatus MA-205, including four new diphenyl ester compounds (TS1~TS3, TS6) and two new natural products (TS7, TS17). These compounds were tested for their antimicrobial, cytotoxic, and brine shrimp (Artemia salina) lethality. Compounds AO1 and AO13 exhibited inhibitory activities against A. hydrophilia, V. anguillarum, V. harveyi, with MIC values of 8.0, 16.0, 8.0 and 1.0, 32.0, 0.5 μg/mL, respectively, and the positive control chloramphenicol was 4.0, 1.0, 8.0 μg/mL. Compound PC5 showed inhibition activity against S. aureus with MIC value of 4.0 μg/mL, which was two times as much as positive control (chloramphenicol), while compound PC18 showed strong antibacterial activity against Micrococcus luteus, with MIC value of 0.25 μg/mL, and the positive control chloramphenicol was 2.0 μg/mL. Compounds PC13 and PC15 showed considerable inhibitory activities against Colletotrichum gloeosprioides and G. graminis with MIC values of 4.0 and 2.0 μg/mL, respectively. The research for fermentation explored the potential of the fungus to produce secondary metabolites, and became successful application for epigenetic modification in fungal secondary metabolites.
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/22771
专题	实验海洋生物学重点实验室
作者单位	1.中国科学院大学 2.中国科学院海洋研究所
推荐引用方式 GB/T 7714	刘洋. 表观遗传修饰增加海洋真菌次生代谢产物结构多样性和活性研究[D]. 北京. 中国科学院研究生院,2015.

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