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非核糖体肽(NRP)是具有化学结构和生物活性多样性的一类重要的天然产物,其肽骨架的生物合成是由非核糖体肽合成酶(NRPS)催化完成。所有的NRPS含有三个最基本的催化功能域:腺苷化功能域(A domain),肽载体蛋白功能域(PCP)和缩合功能域(C domain)。在菌株优选和天然产物的探索发现中,基于天然产物生物合成中重要蛋白质的保守氨基酸序列设计简并引物而进行的PCR筛选已经成为一种十分有效的方法。在本研究中,基于NRPS中腺苷化功能域的保守序列设计简并引物,用PCR的方法从100株土壤放线菌中筛选得到了21株“阳性”菌株。在对21株“阳性”菌株的小规模发酵粗提物的HPLC分析基础上,选择了其中一株链霉菌HMU0027进行大规模发酵培养,通过色谱分离和谱学技术鉴定得到了七个NRPS产生的thiazostatin类嗜铁素(siderophore)类似物(1–7)。其中化合物1是一个新化合物,其含有罕见的苯酚类嗜铁素与糖基团连接的结构特色。本研究为进一步对这些thiazostatin类似物的生物合成研究打下了坚实基础,并展示了基于生物合成知识的基因组挖掘技术在非核糖体肽类天然产物探索发现中的巨大潜力。
Non-ribosomal peptides (NRPs) are a class of important natural products with a variety of chemical structures and biological activities. The biosynthesis of the peptide backbone is catalyzed by non-ribosomal peptide synthase (NRPS). All NRPSs contain three basic catalytic domains: the A domain, the peptide carrier protein domain (PCP) and the C domain. In exploratory discoveries of strain preferences and natural products, PCR screening using degenerate primers based on conserved amino acid sequences of important proteins in natural product biosynthesis has become a very effective method. In this study, degenerate primers were designed based on the conserved sequences in the adenosine functional domain of NRPS, and 21 “positive” strains were screened from 100 soil actinomycetes by PCR. Based on the HPLC analysis of the small-scale fermentation crude extracts of 21 “positive” strains, one of the strains, Streptomyces HMU0027, was selected for large-scale fermentation. Seven NRPSs were identified by chromatographic separation and spectroscopic techniques The resulting thiazostatin class of siderophore analogues (1-7). Among them, compound 1 is a new compound, which contains the structural features of the rare phenolic siderophore linked to sugar groups. The present study laid a solid foundation for further biosynthesis studies of these thiazostatin analogues and demonstrated the potential of genome-mining techniques based on the knowledge of biosynthesis for the discovery of natural products for non-ribosomal peptides.