| 一株链霉菌菌株NCPC-1020中棘白霉素B脱酰基酶基因的克隆与功能分析 |
| |
| 引用本文： | 徐冬梅,可爱兵,路新华,陈文青,邓子新.一株链霉菌菌株NCPC-1020中棘白霉素B脱酰基酶基因的克隆与功能分析[J].微生物学通报,2014,41(8):1564-1573. |
| |
| 作者姓名： | 徐冬梅 可爱兵 路新华 陈文青 邓子新 |
| |
| 作者单位： | 1. 上海交通大学 生命科学技术学院 微生物代谢国家重点实验室 上海 200030；2. 华北制药集团新药研究开发有限责任公司 河北 石家庄 050015;2. 华北制药集团新药研究开发有限责任公司 河北 石家庄 050015;2. 华北制药集团新药研究开发有限责任公司 河北 石家庄 050015;1. 上海交通大学 生命科学技术学院 微生物代谢国家重点实验室 上海 200030；3. 武汉大学 药学院 组合生物合成与药物发现教育部重点实验室 湖北 武汉 430071;1. 上海交通大学 生命科学技术学院 微生物代谢国家重点实验室 上海 200030；3. 武汉大学 药学院 组合生物合成与药物发现教育部重点实验室 湖北 武汉 430071 |
| |
| 基金项目： | 国家973计划项目(No. 2012CB721004) |
| |
| 摘 要： | 【目的】从一株土壤放线菌来源的野生型链霉菌菌株NCPC-1020中克隆一个具有棘白霉素B脱酰基酶活性的新基因。【方法】采用Degenerate和TAIL PCR两种方法,从链霉菌菌株NCPC-1020基因组中快速克隆获得了该基因序列,然后将基因在变铅青链霉菌TK24中进行异源表达,并进行全细胞催化底物脱酰基反应,采用LC-MS检测反应产物。【结果】LC-MS检测证实,棘白霉素B结构中脂肪链被酶促水解,从而证实该基因具有脱酰基酶活性。【结论】采用Degenerate以及TAIL PCR的方法能够快速获得未知功能的新基因。此基因的克隆,奠定了进行半合成棘白霉素类药物的研发基础。
|
| 关 键 词： | 链霉菌，棘白霉素B，Degenerate PCR，TAIL PCR，脱酰基酶 |
Molecular cloning and functional analysis of a gene encoding echinocandin B deacylase from Streptomyces sp. NCPC-1020 |
| |
| XU Dong-Mei,KE Ai-Bing,LU Xin-Hu,CHEN Wen-Qing and DENG Zi-Xin.Molecular cloning and functional analysis of a gene encoding echinocandin B deacylase from Streptomyces sp. NCPC-1020[J].Microbiology,2014,41(8):1564-1573. |
| |
| Authors: | XU Dong-Mei KE Ai-Bing LU Xin-Hu CHEN Wen-Qing and DENG Zi-Xin |
| |
| Institution: | 1. State Key Laboratory of Microbial Metabolism, School of life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China; 2. NCPC New Drug Research and Development Co., Ltd, North China Pharmaceutical Group Corporation, Shijiazhuang, Hebei 050015, China;2. NCPC New Drug Research and Development Co., Ltd, North China Pharmaceutical Group Corporation, Shijiazhuang, Hebei 050015, China;2. NCPC New Drug Research and Development Co., Ltd, North China Pharmaceutical Group Corporation, Shijiazhuang, Hebei 050015, China;1. State Key Laboratory of Microbial Metabolism, School of life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China; 3. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan, Hubei 430071, China;1. State Key Laboratory of Microbial Metabolism, School of life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China; 3. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan, Hubei 430071, China |
| |
| Abstract: | Objective] Cloning of a new gene possessing a function of Echinocandin B (ECB) side-chain deacylation from a wild type Streptomyces strain NCPC-1020 of soil source. Methods] Using degenerate PCR and TAIL PCR strategy, we successfully cloned the target gene, which was heterologously expressed in S. lividans TK24, then a whole-cell catalysis method was developed to dectect the deacylation activity of ECB deacylase by LC-MS. Results] The ECB deacylase gene was cloned and its function was confirmed. Conclusion] Combined the two PCR techniques, degenerate and TAIL PCR, a new gene could be cloned quickly. The obtain of the new ECB deacylase gene lays a good foundation for the researches on semibiosynthetic of ECB related drugs. |
| |
| Keywords: | Streptomyces Echinocandin B Degenerate PCR Thermal asymmetric interlaced PCR Deacylase |
| 本文献已被 CNKI 等数据库收录！ |
| 点击此处可从《微生物学通报》浏览原始摘要信息 |
| 点击此处可从《微生物学通报》下载免费的PDF全文 |
|
