| 硫藤黄链霉菌基因组表达文库的构建及筛选 |
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| 引用本文： | 杨丽鸳,朱梦奕,何璟.硫藤黄链霉菌基因组表达文库的构建及筛选[J].华中农业大学学报,2016,35(5):39-44. |
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| 作者姓名： | 杨丽鸳 朱梦奕 何璟 |
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| 作者单位： | 华中农业大学农业微生物学国家重点实验室,武汉,430070 |
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| 基金项目： | 国家自然科学基金项目（30970059，31270136）；教育部留学回国人员科研启动基金项目(2009-1590)；教育部新世纪人才支持计划项目（NCET-08-0779） |
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| 摘 要： | 为了探讨硫藤黄链霉菌中可能存在的次级代谢产物合成及调控机制,以链霉菌整合型质粒pJTU2554为载体,构建了硫藤黄链霉菌的基因组表达文库。以模式菌株变铅青链霉菌为异源表达宿主,通过生物活性筛选获得9株具有抑菌活性的异源表达突变菌株,同时通过PCR筛选获得多个含有聚酮合酶和非核糖体多肽合成酶基因的克隆子。TLC及HPLC-MS分析发现6个携带有aureothin生物合成基因簇的cosmid,通过异源表达在变铅青链霉菌中成功合成聚酮类抗生素aureothin,证实文库异源表达及筛选的有效性。
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| 关 键 词： | 硫藤黄链霉菌 天然产物 基因组文库 异源表达 文库筛选 |
| 收稿时间： | 2015/6/13 0:00:00 |
Constructing and screening a genomic expression library of Streptomyces thioluteus |
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| Abstract: | Genome sequencing and bioinformatics analyses indicated that Streptomyces thioluteus has great potential to produce a variety of bioactive natural products and possesses complex regulatory mechanisms of secondary metabolites. In order to investigate the biosynthetic pathways of putative secondary metabolites and regulation mechanisms in this strain, a genomic expression library of S. thioluteus was constructed by using an integrative Streptomyces plasmid pJTU2554 as the vector. Nine heterologous expression mutant strains with antibacterial activities were obtained by bioassay screening of the expression library in the heterologous host of Streptomyces lividans ZX1. Positive clones carrying the putative polyketide synthase (PKS) and noribosomal peptide synthetase (NRPS) genes were screened from the library by PCR. Results of TLC and HPLC-MS analyses showed that heterolgous expression of 6 positive cosmids containing the aureothin biosynthetic gene cluster in S. lividans ZX1 resulted in the production of the polyketide antibiotic, aureothin. It is indicated that heterologous expression and screening of the library was successful in S. thioluteus. |
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| Keywords: | Streptomyces thioluteus natural products genomic library heterolgous expression library screening |
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