淮河沉积物来源翅孢壳属真菌D3-12中次级代谢产物的分子多样性和活性研究

The Huaihe River basin is a typical south-north climate transitional zone. Microorganisms isolated from the Huaihe River sediments have been living in this special environment for a long time and have the potential to generate novel chemical skeletons and secondary metabolites with diverse activities, which is an important source for new natural products that have not been fully explored. A large number of studies have shown that most of the genes related to secondary metabolites of microorganisms keep in silence under the traditional culture conditions and how to activate the silent genes by various methods has become a frontier and hot issue in recent years. In order to make full use of microbial resources and obtain secondary metabolites with diverse structures and activities, approaches including One Strain Many Compounds (OSMAC) and the co-culture will be adopted in this project to study the structural diversity and bioactivity of secondary metabolites from fungi isolated from sediments of Huaihe River. In the previous research, we have found that Emericellopsis sp. D3-12 have the ability to generate diverse metabolites with strong antitumor activities and the OSMAC and co-culture approaches will be applied to activate the silence genes. Thus, this project will provide novel structural types for the study of natural products and the discovery of drug lead compounds.

淮河流域是典型的气候过渡带地区，该来源微生物长期处于此特殊环境中，具有生成新颖骨架和活性多样的次级代谢产物的潜力，是未被充分发掘的新天然产物的重要来源。大量研究表明，在传统培养条件下，微生物体内与次级代谢有关的基因大部分处于沉默状态，如何运用多种方法激活沉默基因成为了近年来的研究热点。为了充分利用微生物资源，获得结构类型多样的活性次级代谢产物，本项目将采用单菌多次级代谢产物（OSMAC）策略和共培养等技术手段，对淮河沉积物来源真菌次级代谢产物的分子多样性和活性进行研究。前期通过筛选发现一株翅孢壳属真菌D3-12在常规培养条件下有丰富的代谢产物及明显的抗肿瘤活性，本项目拟将其作为研究对象，对其常规培养条件下的代谢产物进行研究，并采用OSMAC和共培养策略激活目标菌株中未表达的沉默代谢途径，对其代谢潜能进行深度挖掘研究，从而为天然产物研究乃至药物先导化合物的发现提供新颖的结构类型。

淮河淡水真菌来源的微生物是未被充分挖掘的天然产物的巨大宝库，本项目拟以两株淮河淡水真菌为研究对象，探究其结构独特的次级代谢产物，发现新化合物，深入挖掘具有抗肿瘤或抗菌等生物活性的天然产物，从而为药物先导化合物的发现提供新颖的结构类型。主要取得的研究进展及成果如下：.①自建淮河沉积物来源菌株库的完善.本项目对前期分离得到的真菌（编号BMC-1~87，排重后剩余70株）进行了统一编号、三点拍照以及显微拍照入库，并构建了菌株种属的系统发育树，对菌株种属进行了鉴定。为后期继续探究淮河淡水真菌来源天然产物提供了菌株资源。.②两株目标菌株中次级代谢产物的分离鉴定.分别从目标菌株Acremonium tubakii BMC-58和Talaromyces stipitatus BMC-16中分离得到5个多肽类化合物cephaibols A-E和13个多苯环聚酮二聚体，其中新化合物9个，均来自于菌株Talaromyces stipitatus BMC-16。.③化合物的活性筛选及机制研究.生物活性结果显示，多肽类化合物cephaibol A具有明显抗肿瘤作用（IC50：5.58~11.73 μM）和抗菌活性（MIC：4.1~16.2 μg/mL），且其抗肿瘤活性为首次发现。对其抗肿瘤作用机制进行了初步探索，结果显示cephaibol A可以诱导肿瘤细胞MDA-MB-231细胞周期停滞在S期，且能抑制肿瘤细胞的迁移和侵袭。进一步研究发现cephaibol A能够导致线粒体功能障碍，减少细胞内ATP的产生，同时增加活性氧(ROS)的积累。线粒体膜电位(MOMP)测定结果表明，cephaibol A通过影响Bcl/Bax含量，降低MOMP，激活caspase级联反应，最终诱导细胞凋亡。以上结果为抗肿瘤天然肽类化合物的进一步开发提供理论依据。.④两株目标菌株的代谢潜能挖掘.通过OSMAC策略对两株目标菌株的代谢潜能进行了挖掘，结果显示Talaromyces stipitatus BMC-16高产且敏感性较强，改变培养基成分和培养条件，其代谢谱发生了变化，有明显新峰产生，可为后续进一步发现结构新颖的药物先导化合物提供物质基础，有待进一步研究。.⑤发表论文5篇，其中SCI论文4篇，申请专利1项，培养研究2名。