[1]孟晓露,高凯,冯治洋.基于序列宏基因组学技术的芳香聚酮抗生素的发现[J].南京农业大学学报,2019,42(1):168-176.[doi:10.7685/jnau.201803013]
 MENG Xiaolu,GAO Kai,FENG Zhiyang.Discovery of aromatic polyketide antibiotics using sequence-based metagenomic technology[J].Journal of Nanjing Agricultural University,2019,42(1):168-176.[doi:10.7685/jnau.201803013]
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基于序列宏基因组学技术的芳香聚酮抗生素的发现
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
42卷
期数:
2019年1期
页码:
168-176
栏目:
食品与工程
出版日期:
2019-01-09

文章信息/Info

Title:
Discovery of aromatic polyketide antibiotics using sequence-based metagenomic technology
作者:
孟晓露 高凯 冯治洋
南京农业大学食品科技学院, 江苏 南京 210095
Author(s):
MENG Xiaolu GAO Kai FENG Zhiyang
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
关键词:
宏基因组学序列筛选异源表达芳香聚酮抗生素
Keywords:
metagenomicssequence-based screeningheterologous expressionaromatic polyketide antibiotics
分类号:
Q786
DOI:
10.7685/jnau.201803013
摘要:
[目的]本文旨在筛选土壤微生物来源的芳香聚酮抗生素。[方法]通过宏基因组学技术,利用基于酮基合成酶基因(KSα)保守序列设计的简并引物筛选土壤宏基因组文库,获得含有芳香聚酮生物合成基因簇的阳性克隆。对阳性克隆质粒测序并通过BLASTx同源比对分析其所包含的芳香聚酮生物合成基因簇。通过接合转移方法将基因簇整合进异源表达宿主白色链霉菌基因组中,培养发酵接合子,利用高效液相色谱技术确定克隆特异性产物并对发酵产物进行抑菌活性检测。[结果]从珠穆朗玛峰土壤宏基因组文库第493号混合文库中扩增得到了1个KSα片段ZF493,蛋白序列分析显示其与酮基合成酶同源,文库筛选得到了含有其对应芳香聚酮生物合成基因簇的阳性克隆cos493。对cos493的测序分析显示其插入片段大小为34 kb,包括酮基合成酶基因(KSαorf 21)、链长因子基因(KSβorf 20)、酰基载体蛋白基因(ACP,orf 19)、环化酶基因(CYC,orf 17orf 18)、糖基转移酶基因(orf 12)、单加氧酶基因(orf 16)等芳香聚酮合成相关基因。ORF 21与化合物calixanthomycin A聚酮合酶的KSα有67%的相似性,ORF 20与化合物griseorhodin A的KSβ有49%的相似性。通过接合转移使含芳香聚酮合酶基因簇的cos493整合进白色链霉菌宿主基因组中。高效液相色谱分析发酵粗提物发现了2个克隆特异化合物峰,紫外光谱分析显示化合物1在223、296和420 nm处有特征吸收峰,化合物2在214、261和447 nm处有特征吸收峰,特异峰具备芳香聚酮化合物的紫外吸收特征。对发酵粗提物的抑菌活性检测发现,其对金黄色葡萄球菌有抑制作用。[结论]本研究运用基于序列的宏基因组学技术,在链霉菌宿主中表达了来源于土壤微生物的芳香聚酮合酶基因簇并产生了具有抗菌活性的化合物。
Abstract:
[Objectives]This paper aimed to study aromatic polyketide antibiotics from soil microbe. [Methods]Sequence-based metagenomic technology was used for aromatic polyketides discovery. Using the conserved ketosynthase alpha(KSα) gene as the probe,the soil metagenomic libraries were screened and the positive clone harboring aromatic polyketide biosynthetic(PKS) gene cluster was identified. The PKS gene cluster of the positive cosmid was analyzed by BLASTx. The cosmid was transferred into Streptomyces albus J1074 by conjugation,and then the transformants were cultured in fermentation media for heterologous expression of the PKS gene cluster. Extracts of the fermentation broths were analyzed by high performance liquid chromatography(HPLC) and antibacterial assay. [Results]A soil-derived KSα gene fragment named ZF493 was amplified from Mount Qomolangma library using KSα primers. BLASTx analysis showed that the deduced amino acids of ZF493 showed homology with the KSα in aromatic polyketide biosynthesis. The cosmid cos493 which contained PKS gene cluster was isolated from the Mount Qomolangma library and was sequenced. The insert of cos493 was 34 kb in size and contained aromatic polyketide biosynthetic genes:ketosynthase alpha gene(KSα,orf 21),ketosynthase beta gene(KSβ,orf 20),acyl carrier protein gene(ACP,orf 19),cyclase gene(CYC,orf 17 and orf 18),glycosyltransferase gene(orf 12),monooxygenase gene(orf 16). ORF 21 shared 67% sequence identity with the KSα in calixanthomycin A polyketone synthase,while ORF 20 shared 49% sequence identity with the KSβ in griseorhodin A biosynthesis. The cos493 was integrated into the heterologous expression host S.albus J1074. Two clone-specific peaks were identified by HPLC analysis of extracts of the fermentation broths. UV absorption spectra of compound 1 and compound 2 were observed the strongest at 223,296,420 nm and 214,261,447 nm,respectively,which showed typical features of aromatic compounds. The extracts of the fermentation broths exhibited activity against Staphylococcus aureus. [Conclusions]Soil eDNA-derived aromatic polyketide biosynthetic gene cluster was expressed in Streptomyces host and compounds with antibacterial activity were discovered using sequence-based metagenomic technology.

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备注/Memo

备注/Memo:
收稿日期:2018-03-23。
基金项目:国家自然科学基金项目(31370088,31770049)
作者简介:孟晓露,硕士研究生。
通信作者:冯治洋,教授,主要从事环境微生物资源挖掘研究,E-mail:zfeng@njau.edu.cn。
更新日期/Last Update: 1900-01-01