利用靶向非核糖体肽合成酶的PCR技术从链霉菌HMU0027中发现thiazostatin类嗜铁素

doi:10.5246/jcps.2017.10.083

中国药学(英文版) ›› 2017, Vol. 26 ›› Issue (10): 737-746.DOI: 10.5246/jcps.2017.10.083

利用靶向非核糖体肽合成酶的PCR技术从链霉菌HMU0027中发现thiazostatin类嗜铁素

刘发旺, 周梦洁, 金晶, 杨小燕, 张英涛, 马明, 杨东辉^*

北京大学医学部药学院天然药物及仿生药物国家重点实验室, 北京 100191

收稿日期:2017-07-07 修回日期:2017-09-04 出版日期:2017-10-31 发布日期:2017-09-15
通讯作者: Tel.: +86-010-82801559, E-mail: ydhui@bjmu.edu.cn
基金资助:
The National Natural Science Foundation of China (Grant No. 81573326, Grant No. 81673332).

Discovery of thiazostatin siderphores from Streptomyces sp. HMU0027 with a genomic DNA-based PCR assay targeting nonribosomal peptide synthetase

Fawang Liu, Mengjie Zhou, Jing Jin, Xiaoyan Yang, Yingtao Zhang, Ming Ma, Donghui Yang^*

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

Received:2017-07-07 Revised:2017-09-04 Online:2017-10-31 Published:2017-09-15
Contact: Tel.: +86-010-82801559, E-mail: ydhui@bjmu.edu.cn
Supported by:
The National Natural Science Foundation of China (Grant No. 81573326, Grant No. 81673332).

摘要/Abstract

摘要：

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。

关键词: 基因组挖掘, 非核糖体肽合成酶, 嗜铁素, 菌株优选, Thiazostatin

Abstract:

Nonribosomal peptides (NRPs) represent a large family of natural products with great diversities of chemical structures and biological activities. The peptide backbones of NRPs are synthesized by nonribosomal peptide synthetases (NRPSs) that minimally consist of one adenylation (A) domain, one peptidyl carrier protein (PCP) and one condensation (C) domain. In this study, we carried out a PCR screening and identified 21 “positive” strains from 100 actinomycete strains with the degenerate primers designed from the conserved sequences of A domains of NRPSs. One of the 21 “positive” strains, Streptomyces sp. HMU0027, was selected for large-scale fermentation (9 L) based on HPLC analysis, and subsequent isolation and structural elucidation afforded seven NRPS-synthesized thiazostatin siderophore analogues (1–7).Compound 1 was a new compound containing an unusual linkage between a phenolate siderophore and a sugar moiety. These results laid the foundation for further biosynthetic research of these thiazostatin analogues and highlighted the power of genome mining technologies based on biosynthetic knowledge in NRP discovery.

Key words: Genome mining, Nonribosomal peptide synthetase, Siderophore, Strain prioritization, Thiazostation

中图分类号:

R284

Supporting:

刘发旺, 周梦洁, 金晶, 杨小燕, 张英涛, 马明, 杨东辉. 利用靶向非核糖体肽合成酶的PCR技术从链霉菌HMU0027中发现thiazostatin类嗜铁素[J]. 中国药学(英文版), 2017, 26(10): 737-746.

Fawang Liu, Mengjie Zhou, Jing Jin, Xiaoyan Yang, Yingtao Zhang, Ming Ma, Donghui Yang. Discovery of thiazostatin siderphores from Streptomyces sp. HMU0027 with a genomic DNA-based PCR assay targeting nonribosomal peptide synthetase[J]. Journal of Chinese Pharmaceutical Sciences, 2017, 26(10): 737-746.

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利用靶向非核糖体肽合成酶的PCR技术从链霉菌HMU0027中发现thiazostatin类嗜铁素

Discovery of thiazostatin siderphores from Streptomyces sp. HMU0027 with a genomic DNA-based PCR assay targeting nonribosomal peptide synthetase

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