Zhang X, Wang Y
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science, Beijing.
Wei Sheng Wu Xue Bao. 1996 Dec;36(6):417-22.
A BamHI-BamHI 8.0 kb 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 shuttle 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.
一个包含麦迪霉素丙酰基转移酶（mpt）基因的BamHI - BamHI 8.0 kb DNA片段用不同的限制酶进行消化，并绘制了限制酶图谱。通过使用CarE基因的2.4 kb DNA片段作为探针进行Southern印迹分析，将mpt基因定位在一个EcoRI - EcoRI - PstI 3.0 kb DNA片段中。将mpt基因的3.0 kb DNA片段克隆到大肠杆菌/链霉菌穿梭载体pWHM3中，获得了重组质粒pWFPE。产二素链霉菌（pWFPE）和变铅青链霉菌（pWFPE）可分别将内源性合成或外源性添加的螺旋霉素转化为4”-O-丙酰螺旋霉素。mpt基因的序列分析表明，在EcoRI - EcoRI - PstI 3.0 kb DNA片段中有一个开放阅读框，起始于ATG，终止于TGA。mpt基因编码一个388个氨基酸的产物。mpt的G + C摩尔百分比为68.0，第三个密码子位置的G + C摩尔百分比为91.5。mpt的推定产物与CarE产物的同一性为67.6%，相似性为86.4%。在ATG上游6bp处发现了一个共有核糖体结合位点GAGGT和一个启动子区域。TGA下游的一个反向重复序列作为转录终止子。
