螺旋霉素聚酮合成酶基因和抗性基因的克隆与表达的研究

Cloning of Polyketide Synthase Gene and Resistance Gene from Spiramycin Producing Strain Streptomyces spiramyceticus U-1941

根据不同聚酮合成酶基因DNA的同源性,利用放线紫红素聚酮合成酶基因actⅠ,actⅢ作探针,从螺旋霉素产生菌Str.spiramyceticus U-1941 基因文库中检测并分离了螺旋霉素聚酮合成酶基因pCN3H8。限制酶酶切分析表明,其分子量为44kb。通过分子杂交实验,将螺旋霉素聚酮缩合酶基因(与actⅠ有同源性)及聚酮氧化还原酶基因(与actⅢ有同源性)进行了定位。pCN3H8 DNA在麦迪霉素产生菌变株Str.mycarofaciens subsp.68中的表达产物,经紫外光谱分析与麦迪霉素相似。pCN3H8在放线紫红素聚酮缩合酶基因缺陷型变株Str.coelicolor TK17中的表达产物,不具有放线紫红素的色素,其纸层析谱型与螺旋霉素有显著差别。pCN3H8在变青链霉菌Str.lividans TK24中的表达产物,也具有抗菌活性。将pCN3H8 DNA转化对螺旋霉素敏感的Str.griseofuscus原生质体,获得了螺旋霉素抗性的表达。从转化子中分离得到了质粒DNA pSG3,其分子量为7.0kb,可能是pCN3H8DNA转化Str.griseofuscus时在体内缺失而形成。再转化实验证明,宿主菌对螺旋霉素的抗性,确实是由于pSG3 DNA作用的结果。含质粒pCG4,pSG3的螺旋霉素产生菌Str.ambofaciens转化子螺旋霉素的产率明显提高。

According to the DNA homology between the different polyketidesynthase gene, actinorhodin polyketide synthase genes act Ⅰand act Ⅲhave been used as probes to identify biosynthetic genes from the geno-mic library from spiramycin producing strain Str. spiramyceticus U-1941.A clone pCN3H8 was obtained. Restriction analysis of cloned DNApCN3H8 has shown that the molecular weight was 44kb. Southern hyb- ridization indicated the preliminary location of the polyketide synthasegenes in the pCN3H8 DNA.Introduction of pCN3H8 DNA into mutantof midecamycin producing strain Str. mycarofaciens No.68 resulted pro-duction of a midecamycin A. like substance according to UV analysis.The transformants of polyketide synthase gene deficient mutant of acti-norhodin producer Str. coelicolor TK 17 from pCN3H8 produced antiba-cterial substance, that was different from actinorhodin in color andspiramycin in paper chromatographic pattern. Subcloning of polyketidecondensing gene (aet I homologous region) of spiramycin was accompli-shed in vector pWHM3. A recombinant plasmid pCG4 was obtained. A spiramycin resistance gene pSG3 was obtained by transformationof pCN3H8 DNA into spiramycin sensitive strain Str. griseofuscus. Re-transformation of pSC3 into Str. griseofuscus has confirmed that theresistance of spiramycin was associated with this plasmid. The restric-tion enzyme analysis showed that pSG3 has molecular weight of 7kb,implying the pSG3 was formed by in vivo deletion of pCN3H8 in Str.griseofuscus. The plasmids pCG4, pSG3 were introduced to spiramycinproducer Str.ambofaciens. The transformants containing plasmids pCG4.pSG3 showed substantially increased production of spiramycin.