基因组挖掘导向的深海链霉菌NA4中新型环肽类抗生素的发现和活性研究

Secondary metabolites from deep-sea-derived Streptomyces are important sources of lead compounds for drug and pesticides. Cyclic peptides have unique structures with cyclic ring, and they possess special specificities as candidates for drugs. These characteristics lead to the great attention of marine derived cyclic peptides as drug leads for further screening and exploitation. We have isolated a deep sea-derived Streptomyces sp. NA4 strain, and the strain has excellent bioactivities. Genome mining analysis results of NA4 showed that the strain has gene clusters for nonribosomal peptide and ribosomal peptide biosynthesis. However, we have only identified PKS-I type antibiotics, bafilomycins, with antifungal activities from the strain, this phenomenon provides clues that the gene clusters of these cyclic peptide are probably silenced or with low expression during fermentation procedure. Our main goals of the project are the activation of these gene clusters for cyclic peptides biosynthesis and develop these peptides as drug leads or pesticides. We have several steps to fulfill this project: 1) We will use OSMAC (one strain many compounds) strategy to randomly activate the ‘silenced’ biosynthesis pathways for cyclic peptides biosynthesis; 2) We will use genetic engineering techniques-guided approach to construct the mutants that can overproduce certain peptides, by regulation or activation of ‘silenced’ gene clusters of peptides in molecular level; 3) Isolation and identification of the chemical structures of new peptidic products via various spectrometric and spectroscopic methods; 4) Evaluation of the biological activities of these cyclic peptides, and find out the most promising peptides which have potential to be developed as drug leads and pesticides. To sum up, this project will provide essential knowledge for developing the drug leads from marine peptides, and also shed new lights for the activation of diverse ‘silenced’ gene clusters from other microbes.

深海链霉菌产生的次生代谢产物是创新医药和农药先导化合物的重要新来源，尤其环肽类物质结构奇特，具有众多优秀的成药特性，在药物筛选和开发中备受关注。前期研究发现深海来源链霉菌NA4具有多样的生物活性，基因组学分析显示其含有丰富的NRPs和RPs合成基因簇，然而我们仅从其中分离到PKS I型抗真菌物质bafilomycins，提示这些肽类合成基因簇沉默或者低表达。为了激活这些丰富的环肽类代谢产物，并发掘它们在医药和农药中利用的潜力。本项目拟：1）采用培养调控增加其化学多样性，随机激活环肽类化合物；2）利用遗传工程改造和异源表达技术，定向激活目标基因簇编码的新颖环肽类产物；3）运用多种色谱和波谱手段分离鉴定目标环肽；4）对获得的环肽单体进行多模型生物活性筛选和评价，发现结构新颖、活性显著先导化合物。该研究将为开发具有自主知识产权海洋药物提供化学实体，同时为其他类型沉默次生代产物的激活提供参考。

海洋微生物产生的次生代谢产物是创新医药和农药先导化合物的重要新来源，尤其环肽类物质结构新奇，具有优异的成药特性，受到众多化学家和药物学家的关注。使用基因组序列分析发现链霉菌NA4具有丰富和新颖的肽类次级代谢产物合成基因簇，是发掘肽类活性产物的优良菌种资源。本项目以链霉菌NA4等为例，建立和发展了一系列海洋微生物肽类次级代谢产物的高效挖掘技术。通过基于OSMAC策略的培养调控技术一共发掘了5类、共计14个环肽类的次级代谢产物，其中新环肽类物质5个；它们对多种植物病原真菌展示出良好的抑制活性，特别是部分先导在体内对番茄灰霉病菌显示出显著的防控效果，具有进一步开发的潜力。采用主成分bafilomycin合成基因灭活、强化目标肽类基因簇表达和敲除其负调控基因等策略，构建一系列菌株NA4中肽类基因簇激活的工程菌株，直接或间接激活了部分隐性次级代谢产物合成基因簇。进一步从2株工程菌中分离和鉴定了新出现的17个次级代谢产物，包括新结构3个，其中肽类化合物7个，来自3种不同的生物合成途径；部分化合物表现出良好的抗真菌、抗氧化、抗肿瘤细胞等活性，具有深入研究的价值。通过本项目的实施，发现了一些具有杀真菌、抗氧化、抗肿瘤等作用的肽类先导分子，同时为其他微生物次级代谢产物隐性生物合成基因簇的激活提供技术支持和参考。