【摘要】 从含麦迪霉素生物合成基因的初级克隆pCN6C5中,发现并分离了麦迪霉素4″酰化酶基因,与质粒载体p1J680相连,获得重组质粒p66B,在螺旋霉素产生菌中得到表达,其主要产物为4″异戊酰螺旋霉素。以p66B DNA BamHI-BamHI 2.3kb插入片段为探针,从麦迪霉素产生菌基因文库中获得了另一阳性克隆pCN10F5,Southern分子杂交确定pCN10F5 BamHI-BamHI 8.0kb为同源片段。以pWHM3及pIJ680为载体,获得重组质粒pWF5及p6F5,分子大小分别为15.2kb及13.3kb。通过DNA转化,并经分子杂交实验证明,获得含重组质粒的螺旋霉素产生菌克隆菌株。其主要产物经分离、纯化后,分析其理化性质和光谱数据,鉴定为丙酰螺旋霉素Ⅲ和Ⅱ。研究还表明,麦迪霉素基因文库中只有pCN10F5 DNA与碳霉素产生菌的4″异戊酰化酶基因同源,提示pCN6C5克隆携带的麦迪霉素4″酰化酶基因与pCN10F5的4″丙酰化酶基因及碳霉素4″异戊酰化酶基因有一定的区别。更多还原

【Abstract】 A midecamycin 4"-aeyltransferase gene was discovered from primary clone pCN6C5 (48.5 kb), containing mideeamyein biosynthetic gene. Subcloning of pCN6C5 DNA into plasmid vector pIJ680 BamHI site resulted in obtaining of recombinant plasmid p66B. Transformation of p66B DNA into spiramycin producing strains resulted in direct production of 4"-isovalerylspiramyein. In order to identify the completeness of cloned midecamycin 4"-isovaleryltransferase gene on plasmid p66B from pCN6C5 DNA and the possible existence of other 4"-acyltransferase gene in the midecamycin producing strain S. mycarofaciens 1748 genome. Colony hybridization of genomic library of S. mycarofaciens 1748 was performed by using the BamHI-BamHI 2.3kb insert on p66B DNA as probe. Another positive clone pCN10F5 was obtained. A BamHI-BamHI 8.0kb homologous region on pCN10F5 was determined by Southern hybridization and was subcloned into plasmid pWHM3 as well as plJ680. Recombinant plasmids pWF5 and p6F5 were obtained. Transformation of these plasmids into spiramycin producing strains resulted in production of two major acylspiramycin components. Based on their physicochemical properties and spectral evidences, component I has been identified as 4"-propionylspiramycin Ⅲ, component Ⅱ as 4"-propionylspiramycinll. Southern hybridization confirmed that the BamHI-BamHI 8.0kb fragment was cloned in the spiramycin producing strain. Only pCN10F5 clone was identified from genomic library of S. mycarofaciens 1748 when the 4"-isovaleryltransferase gene of carbomycin producing strain S. thermotolerans was used as a probe in colony hybridization, that suggested there is a difference between the 4"-acyltransferase genes in the pCN6C5 and in pCN10F5 clones.更多还原