产新颖次生代谢产物海洋微生物及其农用抗生素的快速发掘

Marine microorganisms are an important source for the discovery of new antibiotics in medicine and agriculture due to their vast majority and rich biodiversity as well as abundant and unique metabolites. Through the implementation of the National Natural Science Funds of China (Grant No. 41006088), we found that strains with the genetic potential to produce new secondary metabolites were key for the efficient discovery of new antibiotics from marine microorganisms. In this proposal, in order to efficiently discover new agricultural antibiotics, strains with the genetic potential to produce new secondary metabolites will be selective isolated based on nucleotide sequences of PKS and NRPS biosynthesis genes with bioactive substances from marine environments or biological materials. And then OSMAC (one strain many compounds) approach will be employed to enhance chemical diversity of secondary metabolites of target strains and fermentation conditions to produce new secondary metabolites will be determined by transcriptional analysis, a variety of biological screening for antibacterial, antifungal and insecticidal activity as well as chemical screening. Finally, novel active metabolites for control agricultural diseases will be isolated using extensive chromatographic techniques including solid phase extraction, silica gel flash chromatography, ODS flash chromatography, gel filtration chromatography over Sephadex LH20, and semi-preparative HPLC, and then identified by interpretation of the NMR and MS spectroscopic data. To our best knowledge, this is the first attempt about selective isolation strains with the genetic potential to produce new secondary metabolite from marine environments or biological samples. The implementation of our project will improve the ability of our country's development of marine microbial resources, also provide valuable references for the efficient discovery of other new lead compounds. Our project is significant to China’s R&D of novel marine microbial pesticides and fungicides with self-owned intellectual property rights.

海洋微生物是新型医药和农用抗生素的重要来源。申请者青年科学基金的研究表明，获得具有潜在产生新型活性次生代谢产物的菌株（简称，潜力菌株）是发现新型活性化合物的关键。为了定向筛选和高效发现新型农用抗生素，本项目拟通过对海洋环境和生物样品完成PKS和NRPS基因的多样性研究和生物信息学分析，揭示其蕴含新型次生代谢产物合成基因簇及其相应微生物的潜能；然后依据优选的目标基因序列设计特异性引物，使用序列指导的方式选择性分离获得目标潜力菌株；再次优选其中5株以上潜力菌株采用OSMAC策略发酵，提高其产生次生代谢产物的化学多样性，并结合转录水平的RT-PCR筛选和多种植物病害生物模型筛选，确定目标化合物的最佳发酵条件；最终使用固相萃取、HPLC、MS 和NMR等技术实现新颖活性化合物的分离和鉴定。该项目的实施有利于提高我国海洋微生物资源开发能力，并为研发我国自主知识产权的新型海洋微生物农药奠定基础。

海洋微生物是新型医药和农用抗生素的重要来源。为了实现产新颖次生代谢产物海洋微生物及其药用先导化合物的高效发掘，本项目成功构建了一条利用基因组挖掘和化学识别技术快速发现新颖先导化合物的高效途径。在本项目中，对17株潜力海洋微生物进行了基因组测序，包括6个基因组完成图和11个基因组扫描图，并对它们的次级代谢潜能进行预测，发现了一批潜在合成新型次级代谢产物的生物合成基因簇；通过多相分类学研究鉴定了3株海洋微生物新种；优选5株潜力菌株，通过放大发酵，活性追踪和化学结构识别，分离和鉴定具有农药、医药用生物活性物质27个，其中新化合物7个，新天然产物2个；对化合物brefeldin A进行了结构修饰获得了18个新的衍生物，其中衍生物13b对肿瘤细胞有较高选择性，并阐明了其诱导肿瘤细胞凋亡的作用机制，具有开发前景；发现了1条合成ditryptophenaline类环二肽二聚体化合物的非NRPS的新生物合成途径。以上部分结果已发表科技论文7篇，其中项目负责人以第一或通讯作者在Applied Microbiology and Biotechnology、European Journal of Medicinal Chemistry等权威期刊发表SCI论文4篇；授权专利4件，审查阶段专利2件；做大会报告或分组报告7次，会议论文3篇；上述成果将为海洋微生物资源的开发利用提供菌种资源和技术贮备，也为研制具有自主知识产权的新型微生物药物奠定物质基础。