麦迪霉素4"-O-丙酰基转移酶(mpt)基因结构的研究  

STUDIES ON MIDECAMYCIN 4" -O-PROPIONYLTRANSFERASE GENE STRUCTURE

作　　者：张叙伦[1] 王以光[1] 

机构地区：[1]中国医学科学院中国协和医科大学医药生物技术研究所,北京100050

出　　处：《微生物学报》1996年第6期417-422,共6页Acta Microbiologica Sinica

基　　金：国家"863"高技术支持项目

摘　　要：对含有麦迪霉素4"-O-丙酰基转移酶(mpt)基因的BamHI-BamHI 8.0kb的DNA片段进行限制性酶切分析,绘制出了含有21个酶切位点的限制性酶切图谱。以含有碳霉素异戊酰基转移酶基因(CarE)的2.4kb DNA片段为探针,经Southern blot分子杂交,将mpt定位于一个EcoRI-EcoRI-Pstl 3.0kb的DNA片段上,将该片段克隆至大肠杆菌/链霉菌穿梭质粒载体pWHM3上,获得重组质粒pWFPE。含有pWFPE的螺旋霉素产生菌产二素链霉菌(S.ambofaciens)及变铅青链霉菌(J.lividans)TK24均可将内源产生的或外源加入的螺旋霉素酰化为4"-O-丙酰螺旋霉素。对EcoRI-EcoRI-PstI 3.0kbDNA片段上mpt基因进行序列分析,在该片段上有一个开放阅读框架,它以ATG为起始密码子,以TGA为终止密码子,与其序列对应的编码产物含有388个氨基酸。mpt基因的G+C mol％为68.0,密码子第三位上G+C mol％为91.5。mpt基因编码的氨基酸序列与CarE基因编码的氨基酸序列的相同性为67.6％,相似性为86.4％。在起始密码子上游6bp处存在一保守的SD序列GAGGT,同时还发现存在较保守的启动子,在终止密码子下游有一反向重复顺序,充当转录终止子。A BamHI-BamHI 8.0kb DNA fragment which contains midecamycin propionyltransferase (mpt) gene was digested with different restriction enzymes and the restriction map was made. The mpt gene was localized in a EcoRI-EcoRI-PstI3.0 kb DNA fragment by Southern blot analysis using a 2.4 kb DNA fragment of the CarE gene as a probe. The 3.0 kb DNA fragment of mpt gene was cloned into E. coli/ Streptomyces shyttle vector pWHM3 and a recombinant plasmid pWFPE was obtained. S. ambofaciens(pWFPE) and S. lividans(pWFPE) can convert endogenously synthesized or exogenously added spiramycin into 4 ' -O-propionylspiramycin, respectively. Sequence analysis of mpt gene demonstrated an open reading frame in the EcoRI-EcoRI-PstI3.0 kb DNA fragment, which starts with ATG and ends with TGA. Mpt gene encodes a product of 388 aa. G+C mol% of mpt is 68.0 and G+C mol% of 3rd codon position is 91.5. The putative product of mpt has a identity of 67.6% and a similarity of 86.4% with CarE product. A consensus RBS GAGGT in the 6bp upstream from ATG and a promoter region were found. An inverted repeat sequence in the downstream from TGA acts as transcriptional terminator.

关 键 词：药物化学 麦迪霉素 4″-O-丙酰基 转移酶基因 

分 类 号：R978.19[医药卫生—药品] R914[医药卫生—药学]