生二素链霉菌orf1~*c基因的功能研究及菌株改造  

Function of orf1~*c gene biosynthesizing spiramycin produced by Streptomyces ambofaciens and strain improvement

作　　者：刘德玉[1] 赵群飞[2] 高淑红[1] 叶蕊芳[1] 陈长华[1] 刘文[2] 

机构地区：[1]华东理工大学生物工程学院,上海200237 [2]中国科学院上海有机化学研究所,上海200032

出　　处：《江苏农业学报》2013年第1期39-45,共7页Jiangsu Journal of Agricultural Sciences

基　　金：生物反应器国家重点实验室项目

摘　　要：研究螺旋霉素生物合成基因中orf1*c的功能,优化菌株,减少出发菌株发酵液m/z为875的杂质组分。试验选取pKC1139和pSET152作为质粒载体,构建重组质粒pKC1139-orf1*c和pSET-orf1*c。然后分别对螺旋霉素工业生产菌株Streptomyce ambofaciens的orf1*c基因进行敲除和倍增,得到相应的突变株L-D-1*c和L-P1*c-7,将突变株进行发酵、生物效价测定和LC-MS检测。发现突变株L-D-1*c完全丧失了生产螺旋霉素组分的能力并产生了相应的中间产物,突变株L-P1*c-7具有有效减少m/z为875杂质组分的生产能力。通过序列比对和中间产物分析,表明基因orf1*c负责编码螺旋霉素合成中的N-二甲基转移酶,得到了初步的优化菌株L-P1*c-7。Abstract： In order to determine the role of orf/＊c from the biosynthetic gene cluster of spiramycin in its production, and to improve the strain which produces spiramycin, the recombinant plasmids pKC1139-orf/＂c and pSET-orf/＊c were constructed. After in-frame deletion of orfl ＊c and duplication of orf/＊c in an spiramycin-producing strain Streptomyces ambofac/ens followed by transformation and screening, mutant strains L-D-1 ＊c and L-P1 ＊c-7 were achieved, respectively. Bio-assay and HPLC/LC-MS analysis of the two constructed mutants L-D-1 ＊c （ for off~/＊c in-frame deletion） and L-PI ＊c-7 （ for orf/＊c duplication） revealed that L-D-1 ＊c had already lost the production ability of spiramcin; in stead, it produced some intermediate metabolites. Orfl ＊c plays a role in coding N-dimethyltransferase which helps to synthesize spiramycin and L-P1 ＊c-7 was the better mutant.

关 键 词：生二素链霉菌 螺旋霉素 属间接合转移 基因倍增 

分 类 号：R978.1[医药卫生—药品]