化学表观遗传学修饰法激活植物内生真菌沉默基因挖掘抗MRSA感染新天然产物探究

Searching brand new active small molecule from microbial secondary metabolites for anti-MRSA is one of the effective way for research and exploitation of antibiotic drugs. However, the traditional high throughput screening for new anti-infective agents occupies a great deal of resource and economic costs, and the genome oriented mining emerging in recent years is inefficiency and heavy investment. In our pilot experiment, several strains from Paris polyphylla var. yunnanensis had been induced with chemical epigenetic modification. The anti-MRSA activity of 12 samples were significantly enhanced and the HPLC analysis of the secondary metabolites showed significant different compared to normal fermentation. So we put forward the hypothesis that the microbial secondary metabolites for anti-MRSA could be found quickly and efficiently by activating their silent genes/ gene cluster with chemical epigenetic modification. To test this hypothesis, kinds of DNA methyltransferase inhibitors and DNA deacetylase inhibitors were used to active the silent genes/ gene cluster of the strains from Paris polyphylla var. yunnanensis. The brand new active small molecules for anti-MRSA could be obtained by screening and bioassay-guided investigation with the method of filter paper diffusion. The research provides a new perspective view for mining brand new active small molecule from microbial secondary metabolites for anti-MRSA by activating their silent genes/ gene cluster with chemical epigenetic modification and a new train of thought for resolving the increasingly serious dilemma of anti-MRSA.

从微生物次级代谢产物出发寻找全新抗耐甲氧西林金黄色葡萄球菌(MRSA)感染活性小分子化合物，是研究和开发抗细菌感染药物的有效途径。然而，传统的高通量筛选需要耗费大量的资源成本和经济成本。我们的预实验针对多株滇重楼内生真菌以化学表观遗传修饰法进行诱导，其中12个样品抗MRSA活性显著增强，HPLC分析次生代谢产物与常规发酵相比有显著差异。为此我们提出假说，化学表观遗传修饰法激活植物内生真菌沉默基因/基因簇，或可快速高效地发现抗MRSA感染活性新天然产物。为验证这一假说，我们将采用多种DNA甲基化酶抑制剂和DNA去乙酰化酶抑制剂诱导处理多株滇重楼内生真菌，激活其沉默基因和基因簇，采用滤纸片法进行导向分离，快速发现具有抗MRSA感染的新颖化合物。本研究将利用化学表观遗传修饰法激活植物内生真菌沉默基因/基因簇，挖掘抗MRSA感染全新天然产物这个新视点，为解决日渐严峻的MRSA感染困境提供新的思路。

项目组在前期抗MRSA活性筛选的基础上，对10株植物内生真菌进行化学表观遗传学修饰后的次生代谢产物研究，其中的链格孢属菌株YNCA0322、拟茎点霉属菌株YNCA1227、高卢蜜环菌YNCA2012进行了放大发酵。同时对6株未经化学表观遗传学修饰的菌株进行了次生代谢产物研究。16株植物内生真菌发酵产物中共分离到169个单体化合物，其中新化合物21个，抗MRSA活性化合物52个，活性较强的11个，同时对部分化合物进行了抗肿瘤和抗病毒活性筛选。研究表明化学表观遗传修饰法可有效激活部分植物内生真菌沉默基因/基因簇，提高化合物的多样性，快速高效地发现抗MRSA活性天然产物。.项目执行期内发表研究论文9篇，其中7篇为SCI收录期刊，申请发明专利4件，完成了计划任务书规定的任务。