【摘要】 以碳霉素４″异戊酰基转移酶基因（ｃａｒＥ）为探针，从麦迪霉素产生菌基因文库中，经菌落杂交获得阳性菌落ｐｃＮ１０Ｆ５。经分子杂交证明ｐｃＮ１０Ｆ５ＤＮＡ的ＢａｍＨ１－ＢａｍＨＩ８．０ｋｂ片段与ＣａｒＥ基因同源。将ｐＣＮ１０Ｆ５ＢａｍＨＩ－ＢａｍＨ１８．０ｋｂ片段分别克隆到ｐＩＪ６８０、ｐＷＨＭ３、ｐＷＨＭ６０１质粒载体上，获得重组质粒ｐ６Ｆｓ、ｐｗＦ５和ＰＧＦ５。含上述重组质粒的螺旋霉素产生菌克隆菌株均产生与丙酰螺旋霉素相类似的产物，且以ｐＩＪ６８０为载体的克隆菌株产生丙酰螺旋霉素的比例最高，最稳定。含ｐ６Ｆ５克隆菌株的发酵产物经提取、纯化后，进行理化性质及各种光谱数据的分析，证明其主要产物为丙酰螺旋霉素Ⅲ及Ⅱ。分子杂交实验证明螺旋霉素产生菌克隆菌株中，确实含有ｐ６Ｆ５ＢａｍＨＩ－ＢａｍＨＩ８．０ｋｂ片段。经分子杂交、亚克隆及基因表达实验，将麦迪霉素４″羟基丙酰基转移酶基因，定位于ＥｃｏＲＩ－ＥｃｏＲＩ－ｐｓｔＩ的２．６ｓｋｂ片段上。核昔酸序列分析结果显示该基因由１１６４个核昔酸组成，Ｇ＋Ｃ％为７０．ｏ％，ＳＤ序列为ＧＡＧＧＴ，起始密码为ＡＴＧ，终止密码为ＴＧＡ，共编码３８８个氨基酸残基，蛋白的分子量为４２．?更多还原

【Abstract】 A genetically engineered hybrid antibioticPropionylspiram-ycin producing strain was constructed by expression of Midecamycin 4″- propionyltransferase(MPT)gene in the Spiramycin producing strain (S. sp- iramyceticus),The MPT gene was identified from the genomic library ofMidecamycin producing S,mycarofaciens 1748 strain using S. ihermotole-rans 4″-isovaleryltransferase(CarE)gene as a probe,Southern blot hybri-dization of the pCN10F5 cosmid clone localized the CarE homology in an8.0kb BamHI DNA fragment.This fragment was subcloned into plasmidspIJ680,pWHM3 and pWNM601 to give respective recombinant plasmidsp6F5,pWF5 and pGF5.Transfomation of S. spiramyceticus and S. livid- ans with these plasmids resulted in production or conversion of exogenou-sly added Spiramycin to a hybrid antibiotic,Based on their physicochem-ical properties and spectral evidences the main components were 4″propio-nylspiramycin Ⅱ&Ⅲ. Southern hybridization confirmed that the BamHI-BamHI 8.0kb fragment was cloned in the the Spiramycin producing stra-in. Restriction map of BamHI8.0kb fragment was constructed.The MPTgene was localized on a EcoRI-EcoRI-pstI 2.65kb fragment through Sou-thern hybridization,subcloning and its expression in S. lividans as wellas in S. spiramyceficus.The nucleotide sequeiice of the MPT gene hasbeen determined.The codon utilization program predicts that the codingregion for MPT contains 1164 nucleotide,which encodes a protein of 388amino acids,with ATG as a start codon and TGA as a terminator cod-on,The deduced amino acids sequence of MPT shows 67.9% identity wi-th CarE amino acids sequence,The genetically engineered Propionylspira-mycin producing strain was very stable when put into successive passageson slant medium or in batchwise fermentation.更多还原