浒苔共附生微生物及其含氮、卤代等克生活性产物研究

Algae are one of the major sources of allelopathic natural products with antimicrobial, anti-red tide, and anti-tumor activities. These active products mainly belong to nitrogen containing and halogenated compounds. Previous study revealed that the seaweed Enteromorpha prolifera blooming into green tide near the seacoast of Qingdao showed anti-red tide against Heterosigma akashiwo, Amphidinium hoefleri, Skeletonema costatum, Karenia mikimitoi and Alexandrium tamarense, indicating that there existed anti-red tide compounds in E. prolifera. And the real source of these allelochemicals might be algicolous microbes that need to be research. Our study also showed that the algicolous fungal metabolites of E. prolifera exhibited cytotoxicity against HL-60 and MCF-7 cell lines with angiogenesis inhibition and antimicrobial effects on Canidia albican, Staphylococcus aureus, Bacillus subtilis, Aerobacter aerogenes, Escherichia coli and Pseudomonas aeruginosa. Based on these findings, the project is planned to obtain antimicrobial, anti-red tide, and anti-tumor products with angiogenesis inhibition from the algicolous actinomyces and fungi associated with E. prolifera. By integrated use of chemical detections (TLC, LC-UV/MS) and bioassay against H. akashiwo, C. albican and MCF-7, as long as the angiogenesis inhibition in vascular fluorescent transgenic zebrafish embryos, the algicolous microbial talented strains will be isolated and identified. And the rest common strains will be converted into the talented strains in this project by means of epigenetic modifications that could be used to activate the silent genes to code fungal secondary metabolites. In addition, biomimetic culture and high salt stress will be applied to induce algicolous actinomyces and fungi to produce structurally new and diverse allelopathic natural products containing nitrogen and halogen atoms. The project, after the implementation, will be predicted to not only provide new candidate compounds for discovery of anti-bacterial and anti-cancer drugs with independent intellectual property rights, but also provide basic data for the prevention of the marine disasters, red tide and green tide, as well as the exploration of the symbiotic relationship between marine algae and their microbes.

海藻是抗菌、抗赤潮、抗肿瘤等克生物质的主要生产者，含氮和卤代化合物是其克生物质的主要类型。近年来在近海海域爆发成"绿潮"的海藻浒苔具有抗赤潮活性，而共附生微生物可能是其克生物质的真正来源，申请者发现其共附生真菌的代谢产物具有抗菌和抗肿瘤活性。本项目拟在此基础上，以赤潮异湾藻、白色念珠菌、血管荧光标记斑马鱼及肿瘤细胞为活性模型，以TLC、LC-UV/MS为化学模型，分离筛选浒苔放线菌和真菌Talented菌株；利用表观遗传修饰，"唤醒"浒苔真菌中的"沉睡"基因，将一般浒苔真菌株转化为Talented菌株；通过拟生态培养和高盐胁迫，诱导浒苔微生物Talented菌株产生具有抗赤潮、抗菌和血管生成抑制作用的含氮、卤代等抗癌活性的克生物质。研究不仅为抗菌和抗癌自主创新药物的研究提供候选化合物及其产生菌，还将为"赤潮"和"绿潮"海洋灾害的防治与利用以及海洋藻类与其微生物之间的共生关系提供基础资料。

以人乳腺癌MCF-7细胞为生物活性模型，对149株浒苔微生物的发酵产物进行了生物活性筛选，发现了14株具有人乳腺癌MCF-7细胞抑制活性的浒苔“Talented菌株”。应用表观遗传修饰技术，将5株无活性的浒苔真菌“非Talented菌株”转化为有肿瘤细胞毒活性或抗菌活性的“Talented菌株”。.采用培养基优化、高盐胁迫、基因突变和化学表观遗传修饰，结合色谱分离、波谱与化学鉴定等方法，系统研究了12株浒苔等来源的海洋微生物“Talented菌株”的活性次级代谢产物，获得115个化合物，其中新化合物40个、含氮化合物49个、具有抗菌、抗病毒、抗肿瘤或α-葡萄糖苷酶抑制作用的活性化合物47个；发现了6个新化合物具有抗菌或抗H1N1病毒活性、10个新化合物具有肿瘤细胞毒活性、7个新化合物具有α-葡萄糖苷酶抑制活性。为治疗糖尿病的α-糖苷酶抑制剂、抗流感病毒和抗肿瘤等新药研发，提供了新的候选化合物或先导结构。.采用化学衍生技术，对毒性大、活性强的微生物产物——十字孢碱进行结构修饰，合成了12个PKC412（米哚妥林，一种治疗急性白血病的新药）的新衍生物，获得了活性相当、但对肺癌细胞A549选择性提高的化合物。为抗肿瘤新药的研发提供了新的先导化合物。.本研究采用真菌表观遗传修饰、混合培养和高盐胁迫等方法，获得了新的活性化合物，其中高盐胁迫获得新的活性次级代谢产物为本课题组首创，为海洋微生物天然产物的研究提供了参考。.发表了标注基金号41376148的SCI论文21篇（第一和第二标注）、9篇CSCD学术论文（其中7篇为第一和第二标注）。主持人5次在全国性学术会议上作学术报告、1次国际学术会议上作学术报告。