摘要
在万古霉素产生菌东方拟无枝酸菌(Amycolatopsis orientalis)中异源表达Balhimycin产生菌来源的糖基转移酶基因bgtfA,从而在工程菌发酵液中获得杂合万古霉素,以探索糖基转移酶BgtfA能否作为一个独立的元件在异源宿主中发挥催化作用。首先将bgtfA基因克隆至载体质粒pULVK2AE,构建重组质粒pLYEBA2AE,然后采用电转化方法将质粒导入东方拟无枝酸菌,用HPLC和LC-MS检测、验证工程菌发酵液中是否存在BgtfA的催化产物。成功地在东方拟无枝酸菌(Amycolatopsis orientalis)异源表达bgtfA基因,经HPLC和LC-MS检测分析,在工程菌的发酵液中获得预期的BgtfA催化产物,杂合万古霉素LYV07ww01。这表明BgtfA有一定的底物宽泛性,不仅能够催化表万古糖胺与balhimycin苷元的连接,还能催化万古糖胺连接至万古苷元。
The objective of this study was heterologous expression of the glycosyltranferase gene bgtfA from balhimycin producingstrain in Amycolatopsis orientalis, a vancomycin producing strain, to obtain hybrlde vancomycin from the fermentation broth of the con- structed engineered strain. To obtain an engineered strain, the gene of bgtfA was coloned into vector pULVK2AE, and then the recombinant vector pLYEBA2AE was transformed into Amycolatopsis orientalis by electroporation. The broth of engineered strain was detec- ted and analyzed by HPLC and LC-MS. The results showed that heterologous bgtfA gene was successfully expressed in Amycolatopsis orientalis with the replicative plasmid pLYEBA2AE by eleetroporation and an expected hybride vancomycin named LYV07ww01 was detected and identified by HPLC and LC-MS in the broth of engineered mutant. This indicated that BgtfA has its broad substrate specificity not only to transfer epivancosamine to balhimycin aglycone but also to transfer vancosamine to vancomycin aglycone.
出处
《药物生物技术》
CAS
2013年第3期207-209,共3页
Pharmaceutical Biotechnology
基金
国家重大新药创制课题(No.2012ZX09301002-003)
国家自然科学基金(No.30672558)
关键词
糖基转移酶
异源表达
底物宽泛性
东方拟无枝酸菌
糖肽类抗生素
Glycosyltransferase, Heterologous expression, Broad substrate specificity,Amycolatopsis orientalis, Glyeopeptide antibiotics
