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  

Molecular cloning and functional analysis of a gene encoding echinocandin B deacylase from Streptomyces sp. NCPC-1020

作　　者：徐冬梅[1,2] 可爱兵[2] 路新华[2] 陈文青[1,3] 邓子新[1,3] 

机构地区：[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 [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

出　　处：《微生物学通报》2014年第8期1564-1573,共10页Microbiology China

基　　金：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。[Objective] loning 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 ofECB 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.

关 键 词：链霉菌 棘白霉素B Degenerate PCR TAIL PCR  脱酰基酶 

分 类 号：Q78[生物学—分子生物学]