Tang L, Wang Y G, Zhu X W
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing.
Chin J Biotechnol. 1991;7(1):33-42.
The plasmid containing the spiramycin polyketide synthase genes, pCN3H8, was obtained from the genomic library of spiramycin producing strain, S. spiramyceticus U-1941, using homologous DNA to actI and actIII genes as hybridization probes. Restriction analysis of the plasmid pCN3H8 showed that the molecular weight was 44kb. The regions homologous to the actI & actIII genes were localized by Southern hybridization, and corresponding DNA fragments were subcloned onto E. coli-Streptomyces shuttle vector pWHM3. A recombinant plasmid pCG4 was obtained. Transformation of the pCN3H8 DNA into the polyketide synthase deficient mutant of midecamycin producing strain, S. mycarofaciens subsp. No. 68, resulted in the production of midecamycin A by UV analysis. Transformation of the pCN3H8 DNA into the polyketide synthase deficient mutant of actinorhodin producing strain, S. coelicolor TK17, resulted in the production of an antibacterial compound which was neither similar to actinorhodin in color nor to spiramycin by paper chromatographic analysis. Transformant of S. lividans with pCN3H8 DNA produced an antibacterial compound as well. Resistance to spiramycin was expressed in transformants of spiramycin sensitive strain, S. griseofuscus, with pCN3H8 DNA. A plasmid pSG3 DNA with molecular weight of 7.0kb, isolated from the transformant, might be a result of in vivo deletion of pCN3H8 in S. griseofuscus. Retransformation of pSG3 DNA into S. griseofuscus confirmed that the gene of resistance to spiramycin was on the plasmid pSG3 DNA. Transformation of spiramycin producing strain, S. ambofaciens, with pCG4 or pSG3 DNA increased spiramycin production in fermentation broth.
含有螺旋霉素聚酮合酶基因的质粒pCN3H8，是从螺旋霉素产生菌弗氏链霉菌U-1941的基因组文库中获得的，使用与actI和actIII基因同源的DNA作为杂交探针。对质粒pCN3H8的限制性分析表明其分子量为44kb。通过Southern杂交确定了与actI和actIII基因同源的区域，并将相应的DNA片段亚克隆到大肠杆菌-链霉菌穿梭载体pWHM3上。获得了重组质粒pCG4。将pCN3H8 DNA转化到麦迪霉素产生菌弗氏链霉菌亚种68的聚酮合酶缺陷型突变体中，通过紫外分析检测到产生了麦迪霉素A。将pCN3H8 DNA转化到放线紫红素产生菌天蓝色链霉菌TK17的聚酮合酶缺陷型突变体中，通过纸色谱分析检测到产生了一种抗菌化合物，其颜色既不同于放线紫红素，也不同于螺旋霉素。用pCN3H8 DNA转化变铅青链霉菌也产生了一种抗菌化合物。用pCN3H8 DNA转化螺旋霉素敏感菌灰褐链霉菌，其转化子表现出对螺旋霉素的抗性。从该转化子中分离出的分子量为7.0kb的质粒pSG3 DNA，可能是pCN3H8在灰褐链霉菌体内缺失的结果。将pSG3 DNA重新转化到灰褐链霉菌中，证实螺旋霉素抗性基因位于质粒pSG3 DNA上。用pCG4或pSG3 DNA转化螺旋霉素产生菌浅青紫链霉菌，可提高发酵液中螺旋霉素的产量。
