麦迪霉素生物合成基因克隆研究  

CLONING OF MIDECAMYCIN BIOSYNTHETIC GENE FROM STREPTOMYCES MYCAROFACIENS 1748

作　　者：王以光[1] 

机构地区：[1]中国医学科学院医药生物技术研究所

出　　处：《生物工程学报》1989年第4期261-269,共9页Chinese Journal of Biotechnology

摘　　要：利用穿梭粘粒载体pNJ_1组建了麦迪霉素产生菌Streptomyces mycarofaciens 1748的基因文库,用与麦迪霉素生物合成有类似途径的放线紫红素聚酮合成酶基因Act Ⅰ,Act Ⅲ为探针,从麦迪霉素产生菌的基因文库中,获得了与Act Ⅰ,Act Ⅲ基因有同源性的阳性克隆,对其中PCNSB12、6C5及11E11克隆DNA进行了酶切分析,其分子量分别为36kb,48.5kb及41.6kb。PCN6C5 DNA中包含有PCN 8812的全部DNA片段,PCN JIEII与PCN 8B12及PCN 6C5有6.75kb的重叠区。通过分子杂交实验,初步将麦迪霉素聚酮合成酶基因定位在PCN 8812 DNA的EcoR Ⅰ-BamH Ⅰ 4.02kb片段上(与Act Ⅲ基因有同源性)及PCN 8812(6C5),PCN11E11DNA Bg Ⅲ-Bg Ⅲ 2.42kb与PCN11E11 Bgl Ⅱ-Bgl Ⅱ 10kb片段上(与Act Ⅰ基因有同源性)。PCN 8812及PCN 6C5克隆DNA在麦迪霉素聚酮合成酶基因缺陷型变株Streptomyces mycarofaciens var.68及不产抗生素的变铅青链霉菌Streptomyces lividans TK24受体菌的表达产物,经TLC及HPLC等分析表明与麦迪霉素标准品相似。Genomic library from midecamycin producing strain Streptomyces mycarofaciens 1748 has been constructed by using bifunctional cosmid vector PNJ1. According to the DNA homology between the different polyketide synthase gene, actinorhodin polyketide synthase gene Act Ⅰ , Act Ⅲ have been used as probes to identify biosynthetic gone from midecamycin producing strain Streptomyces mycarofaciens 1748. Several clones were obtaincd. Restriction analysis of cloned DNA PCN 8B12, PCN6CS, PCN11E11 has shown that the molocular weight of them were 36kb, 48.6kb and 41.6kb respectively. The PCN6C5 DNA contains whole PCN8B12 DNA fragment, the PCN11E11 DNA has 6.75kb overlay region with PCN8B12 and PCN6C5 DNA. Southern hybridization indicated the preliminary location of the polyketide synthase genes of rnidecamycin in the cloned DNA. Cloncd DNA were introduced into blocked mutant of midecamycin producing strain (Str. mycarofaciens var. 68) and Str. lividans. The transformants produced an antibiotic similar to midecamycin according to TLC and HPLC analysis.

关 键 词：麦迪霉素 生物合成基因 克隆 

分 类 号：Q785[生物学—分子生物学]