诺卡氏菌来源活性天然产物的发现及其生物合成机制的研究

Bacteria of the genus Nocardia are aerobic Gram-positive, non-motile filamentous actinomycetes that are widely spread in soil, water, human and animal tissues. Nocardia speicies have been classified as one of the rare actinomycetes since they were less extensively exploited in relative to streptomycetes. One third of Nocardia species are pathogenic or opportunistically pathogenic to human, causing serious infectious disease or fatal death, referred to as nocardiosis. Many natural products with bioactivities of antibacterial, antifungal, antitumor and immunosuppressive agents have been reported in the past from various pathogenic Nocardia. However, the opportunistic pathogenic nature of Nocardia may hamper the discovery of new potential drug candidates from the other two thirds of non-pathogenic Nocardia...All sequenced Nocardia strains from NCBI accessible genome data have been intensively analyzed using antiSMASH. Bioinformatic analysis revealed that each strain contains 30-40 secondary metabolite biosynthetic gene clusters from 77 Nocardia strains investigated, indicating that Nocardia is another rich source of bioactive natural products. Genome mining has become a promising and efficient strategy of tapping the new bioactive metabolites that are encoded by orphan gene clusters or overlooked under standard lab conditions. ..N. coubleae isolated from oil-contaminated soil was chosen for this proposal since no study on isolation of natural products from this strain has been reported before. Bioinformatic analysis of entire genome sequence of N. coubleae revealed the presence of 33 biosynthetic gene clusters encoding 16 nonribosomal peptides, 5 terpenes, 7 polyketides, etc. In this proposal, we will focus on two completely annotated NRPS gene clusters, one is about 45 kb, which might be responsible for the biosynthesis of a new cyclopeptide containing 12 amino acids; the other one is about 17 kb, sharing 87% sequence identity to nocobactin NA gene cluster from N. farcinica. Investigation of biosynthesis of these new peptides will be performed by TAR (Tranformation-associated recombination) cloning in yeast or Gibson assembly and then individually heterologous expression in Streptomyces lividans K4-114, followed by isolation and structural elucidation of peptides, bioactivity tests and functional analysis of essential genes via gene knock-out and in vitro assay. This project will pave the way for basic drug discovery from Nocardia strains and provide new natural products and useful gene resources for drug discovery.

近些年，许多研究发现稀有放线菌诺卡式菌能产生多种具有抗癌、抗菌、免疫抑制剂以及降解复杂有机化合物和工业污染物等生物活性的天然产物，可以作为未来新药开发的重要来源之一。Nocardia coubleae 分离自被石油污染过的土壤中，目前尚未有研究报道过它的次级代谢产物。申请人对该菌的基因组DNA进行了测序，通过生物信息学分析发现这个菌株染色体上含有33个未知次级代谢产物合成基因簇，所以可以作为新药开发的潜在研究对象。本项目拟通过基因组挖掘的技术，综合利用生物信息学、分子生物学、分析化学以及生物活性测定等方法有针对性地从N. coubleae中获得具有新结构和生物活性的两个非核糖体肽及其衍生物并揭示它们的生物合成机制，最终为从诺卡氏菌中挖掘新颖活性天然产物提供研究基础，从而为新药研发提供先导化合物和重要的基因资源。

前期研究表明稀有放线菌诺卡氏菌可以产生多种具有生物活性和结构多样性的天然产物，是尚未被广泛研究的一类放线菌，有望成为未来新药发现的重要来源之一。本项目选择了一株分离自石油污染过土壤来源的诺卡氏菌Nocardia coubleae, 通过对其进行全基因组测序、生物信息学分析，进而采用基因组挖掘技术以及天然产物分离技术从中发现具有潜在新颖活性与结构的非核糖体肽类并揭示它们的生物合成机制，最终为从诺卡氏菌中挖掘新颖活性天然产物提供研究基础，从而为新药研发提供先导化合物和重要的基因资源。. 根据项目研究计划，本项目选用RecET同源重组大片段克隆技术对N. coubleae基因组中包括项目重点关注的Cluster 25和Cluster22，和其他7个非核糖体肽类基因簇同时展开了研究，最终成功构建重组异源表达质粒 6个，获得含有目标基因簇的异源表达菌株共6株，经发酵培养和液质分析后发现异源表达菌中并没有产生潜在的目标产物。与此同时，我们也对野生菌N. coubleae开展了基于OSMAC策略的发酵分析，确定了适宜的培养基，并对其进行大量发酵，最终从中分离并鉴定出25个化合物，其中有8个是新的nocobactin NA类铁载体化合物（命名为coublebactins），具有极强的铁结合能力。初步活性检测，发现这些这类铁载体对酵母具有抑菌活性，有望作为一种抗真菌药物筛选的先导化合物。进一步通过对NCBI数据库中已报道的105种诺卡氏菌基因组进行生物信息学分析发现，其中有90株诺卡式菌基因组都含有一个nocobactin NA类似的铁载体基因簇，证明这类铁载体在诺卡氏菌生长和发育过程中发挥着非常重要的作用，结合这类化合物的结构以及预测的基因簇信息，我们推断了这类铁载体的潜在生物合成途径。目前正在对铁载体分离制备这部分研究进行论文和专利撰写。