新骨架chaetomugilide类嗜氮酮生物碱及其生物合成研究

An increasing number of studies have shown that plant endophytic fungi are an important and novel resource of natural bioactive compounds. More than twenty chlorine-containing azaphilone analogues have been discovered from Chaetomium globosum inhabited in Ginkgo biloba during the course of our ongoing search for biologically active metabolites from endophytic fungi. Four of these analogues are chaetomugilide type alkaloids with new skeleton, these alkaloids showed significant antitumor activity. The traditional single culture method restricts the expression of genes encoding cryptic metabolic pathways in fungi. Considering this situation, the strategy of one strain-many compounds (OSMC) will be conducted to produce more novel alkaloids by the application of as many different cultivation conditions as possible or the inhibition of histone acetycation and DNA methylation to activate the cryptic biosynthetic genes, and more lead compounds may be found from these newly derived metabolites. In order to reveal their biosynthetic pathway, especially the meachanism of the formation of the novel nitrogen ring skeleton in chaetomugilide, we are going to sequence the whole genome of chaetomugilide producing strain, cloning, and characterization of the chaetomugilide biosynthetic gene cluster. The implementation of the proposed project can provide molecular template for the further chaetomugilide-derived antitumor drug development, and lay the foundation for combinatorial biosynthesis of chaetomugilide metabolites in the future.

越来越多的研究证明植物内生真菌已经成为发现新颖活性天然产物的新资源。在长期从内生真菌中寻找活性化合物过程中，我们从银杏内生球毛壳菌（Chaetomium globosum）中发现了20多个结构新颖的5位氯代嗜氮酮类化合物，其中4个为具有新骨架的chaetomugilide生物碱，这些生物碱显示出优异的抗肿瘤活性。传统单一培养方法限制了真菌中大量次生代谢途径的表达，所以项目拟采用单菌多次级产物（OSMAC）策略，通过改变菌株培养条件及抑制组蛋白乙酰化和DNA甲基化等多种方法来诱导相关沉默基因的表达，产生更多新颖生物碱，从中发现高活性先导化合物；同时，为了揭示这类成分的生物合成途径，尤其新颖的氮环骨架的形成机理，项目拟对菌株进行测序，并对其生物合成基因簇进行克隆和鉴定。项目的顺利实施可为进一步的chaetomugilide抗肿瘤药物开发提供分子模板，也为将来组合生物合成这类化合物打下基础。

研究证明植物内生真菌已经成为发现新颖活性天然产物的新资源。在国家自然科学基金资助下我们采用单菌多次级产物（OSMAC）策略，通过改变菌株培养条件及抑制组蛋白乙酰化和 DNA 甲基化等多种方法来诱导相关沉默基因的表达，产生多种新颖嗜氮酮类化合物，从中发现高活性先导化合物；项目对天才菌株进行测序，并对其生物合成基因簇进行克隆和鉴定。研究结果如下：从该天然菌株的不同培养条件的提取物中共跟踪分离到52种这类化学成分，包括13种新结构，通过活性测试发现都具有细胞毒性，其中三个化合物对三种肿瘤细胞表现出了强细胞毒性，有望作为先导化合物进一步深入研究。对该天才菌株进行了全基因组测序，并对可能的代谢通路进行了分析，发现了嗜氮酮类的合成基因簇，来源于聚酮类合成途径，对结构中5位上的负责氯原子取代的氯化酶正在验证。项目研究筛选出了强抗肿瘤先导化合物三个，为后续的抗肿瘤药物研究打下基础。另外，研究结果发表了7篇SCI论文，培养了多名研究生。