An efficient plasmid transformation system for S. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S. mycarofaciens 1748, the gene disruption experiment was carried out. For t...An efficient plasmid transformation system for S. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S. mycarofaciens 1748, the gene disruption experiment was carried out. For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S. mycarofaciens 1748. This suggested that homologous recombination between plasmid-borne MKR gene sequence and the chromosome of S. mycarofaciens 1748 had occurred. A Southern hybridization experiment using α- P-labelled MKR gene as probe indicated that the desired integration event had occurred in the re-combinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S. mycarofa-ciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was in-cubated with fermentation medium optimised for midecamycin production. A TLC assay showed that the recombinant strain produced midecamycin in quantities comparable to that of S. mycarofaciens 1748. A pCN8B12 was a clone from genomic library of midecamycin producing strain which contained a 28-kb DNA insert. The 28-kb DNA fragment contained act I -homologous and actⅢ-homologous regions. The PKS (act I -homologous) and MKR (act Ⅲ-homolo-gous) genes that define spore pigment of midecamycin producing strain were localized by restriction endonuclease diges-tion with pCN8B12, indicating that they are separated by about 10 kb DNA. The polyketide synthase gene cluster of similar organization has not been reported yet.展开更多
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。展开更多
基金Project supported by the National Natural Science Foundation of China.
文摘An efficient plasmid transformation system for S. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S. mycarofaciens 1748, the gene disruption experiment was carried out. For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S. mycarofaciens 1748. This suggested that homologous recombination between plasmid-borne MKR gene sequence and the chromosome of S. mycarofaciens 1748 had occurred. A Southern hybridization experiment using α- P-labelled MKR gene as probe indicated that the desired integration event had occurred in the re-combinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S. mycarofa-ciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was in-cubated with fermentation medium optimised for midecamycin production. A TLC assay showed that the recombinant strain produced midecamycin in quantities comparable to that of S. mycarofaciens 1748. A pCN8B12 was a clone from genomic library of midecamycin producing strain which contained a 28-kb DNA insert. The 28-kb DNA fragment contained act I -homologous and actⅢ-homologous regions. The PKS (act I -homologous) and MKR (act Ⅲ-homolo-gous) genes that define spore pigment of midecamycin producing strain were localized by restriction endonuclease diges-tion with pCN8B12, indicating that they are separated by about 10 kb DNA. The polyketide synthase gene cluster of similar organization has not been reported yet.
