西沙珊瑚共附生真菌表观遗传修饰及新颖结构活性次级代谢产物发现

The symbiotic fungi of the corals have proven to be a rich source for searching for secondary metabolites with novel structures and potential bioactivities. In recent year, the coral-derived fungi have been considered as a new hot resource for the discovery of bioactive marine natural products and lead compounds. Epigenetic modification could activate the silent genes of fungi and regulate the fungal metabolism, resulting in the improvement of structure diversity and novelty of their secondary metabolites. Therefore, epigenetic modification has been used as a feasible approach to find novel secondary metabolites. In our previous work, a series of bioactive new compounds have been isolated from the fingi derived from the corals collected from the coral reef in Xisha Islands, including alkaloids, macrolides, and terpenoids with such as acetylcholinesterase (AchE) inhibitory activity. In this research proposal, epigenetic modification will be applied to three coral-derived fungal strains to activate their unique metabolic pathways. The chemical diversity and high bioactivities should be prospected for the culture of the treated fungal strains with chemical genetic modification agents. The large scale culture of the treated strains should be conducted under the optimal fermentation conditions with the selected genetic modification agents. The bioactive secondary metabolites will be isolated and identified from the fermentation broth under bioassay guidance, especially the novel compounds with AchE inhibitory activity. The structure-activity relationship will be discussed for the acitive compounds to find the lead structures of AchE inhibitors.

珊瑚共附生微生物具有丰富的物种多样性，能够产生结构新颖、活性显著的次级代谢产物，已成为活性海洋天然产物和药物先导化合物发现的新的热点资源。表观遗传修饰能激活真菌沉默基因，调控真菌代谢，提高次级代谢产物结构多样性和新颖性，是新颖结构活性次级代谢产物发现的有效手段。在前期工作中，我们从西沙珊瑚共附生真菌中发现了一系列结构新颖的生物碱、大环内酯、萜类等化合物，具有抑制乙酰胆碱酯酶(Acetylcholinesterase, AchE)等活性。本项目拟在已有工作基础上，对3株西沙珊瑚共附生真菌进行表观遗传修饰，以激活真菌独特的代谢途径，增加真菌次级代谢产物的化学多样性和生物活性；在集成筛选和发酵条件优化基础上，以添加最佳表观遗传修饰剂的最优发酵条件进行发酵，重点从发酵提取物中追踪分离鉴定结构新颖的具有AchE抑制活性的化合物，系统分析构效关系，为新颖AchE抑制剂先导结构的发现提供基础资料。

针对海洋真菌天然产物重复分离、代谢产物隐秘基因沉默表达的问题，开展了海洋真菌表观遗传修饰及新颖结构活性次级代谢产物发现研究。对中国南海西沙珊瑚共生真菌曲霉Aspergillus sp. XS-20090066、月状旋孢腔菌Cochliobolus lunatus TA26-46、哈茨木霉Trichoderma harzianum XS-20090075及同属真菌进行表观遗传修，结合OSMAC方法，筛选使得真菌代谢发生显著变化的表观遗传修剂及培养条件。运用组蛋白去乙酰化酶抑制剂和DNA甲基转移酶抑制剂对真菌进行表观遗传修饰，真菌代谢谱发生了显著变化。综合运用TLC，HPLC-UV，UPLC-PDA-MS，分子网络，1H NMR等技术，考察了真菌的代谢差异表达，对比分析了表观遗传修饰剂调控组和对照组的次级代谢产物的异同，追踪分离获得了差异表达的化合物，进而在结构表征的基础上，分析了真菌次级代谢产物的化学多样性与结构类型的变化特征。通过表观遗传修饰，结合OSMAC策略和分子网络技术，从真菌修饰产物中分离鉴定获得了新产生的次级代谢产物170余个，发掘获得新颖结构核苷类、生物碱、倍半萜、二萜、大环内酯、聚酮等化合物50余个。运用乙酰胆碱酯酶AChE抑制、拓扑异构酶Top I抑制、蛋白酪氨酸磷酸酶PTP1B抑制、细胞毒、抗菌等活性模型评价化合物的生物活性，系统分析构效关系，发现了10余个高活性化合物，显示了进一步药物研究价值。对真菌进行了基因组及其功能分析，探讨了表观遗传修饰剂对海洋真菌的代谢调控作用，推测了海洋真菌被激活的沉默基因及其独特的代谢途径，初步揭示了表观遗传修饰代谢调控机制。我们的探索研究表明，表观遗传修饰剂有效激活了海洋真菌的沉默基因，显著增加了真菌次级代谢产物的化学多样性，发掘获得了真菌隐秘次级代谢产物。表观遗传修饰法是发掘海洋真菌新颖结构活性次级代谢产物的有效途径。