【作者】 韩锋；

【导师】 夏焕章； 王以光；

【作者基本信息】 沈阳药科大学 ， 微生物与生化药学， 2004， 硕士

【摘要】 必特螺旋霉素(4″酰化螺旋霉素)是含4″-异戊酰基转移酶基因(4″-isovaleryltransferase gene，ist)的螺旋霉素产生菌的产物，即以4″-OH螺旋霉素为底物在异戊酰基转移酶催化下，在4″位加入异戊酰基侧链形成以4″位异戊酰基螺旋霉素为主成份的新抗生素。目前菌种的发酵产物是一个多组分的混合物，含有酰化的和未酰化的螺旋霉素，生产中希望尽可能提高4″酰化螺旋霉素在发酵产物中所占的比例，因此我们决定用DNA改组(DNA shuffling)的方法提高4″-异戊酰基转移酶的酰化能力，以期提高必特螺旋霉素在发酵产物中的比例。 DNA改组是一种分子水平上的定向进化(directed evolution)，它基于有性PCR原理，通过DNA碎片反复交换模板的重聚反应，能够迅速得到理想变异。 我们从6株必特螺旋霉素高产菌株中克隆得到它们的ist基因，进行改组，将最终获得的全长基因构建到链霉菌一大肠杆菌穿梭、链霉菌整合型载体pSET152上，转化到Streptomyces spiramyceticusB183中，传代后发酵，TLC检测其发酵产物，得到5株必特螺旋霉素产量比例明显增加的菌株，进一步做HPLC检测，确定了其中的4株确实产生了比例较大的4″酰化螺旋霉素，与初始基因的作用相对比，4″酰化螺旋霉素所占的比例提高了61.5％～169.7％。将这4株的ist基因送交测序，结果有2株的基因在它们的终止密码子之后发生了一些突变。更多还原

【Abstract】 Biotechmycin(4″acylspiramycin),a mixture of spiramycin derivatives, is a new antibiotic produced by a genetic engineering strain constructed by introducing ist gene from Streptomyces thermotolerans into the chromosome of Streptomyces spiramyceticus through homologous double-crossover. The main component of the mixture is 4″ isovaleryl spiramycin, the product of isovaleryltransferase which catalyzes the addition of isovaleryl onto 4″ position of 4″-OH spiramycin. At present, there is still unacylated spiramycin in the fermentation products as well as the acylated spiramycin, and we expect that the percentage of the 4″acylspiramycin will be increased. We choose the method of DNA shuffling to enhance the acylation ability of 4″-isovalayltrasferase in order to advance the percentage of biotechmycin among the fermentation products.DNA shuffling is a type of molecule-level directed evolution. It is based on the principle of sexual PCR, and it is easy to get the expected mutation by the reassembly of DNA fragments with the repeating template exchange.We cloned 6 ist genes from 6 strains with high output of biotechmycin. After shuffled, the finally obtained full-length genes were cloned into pSET152, an integrating Escherichia coli-Streptomyces shuttle plasmid. The 15 recombinant plasmids were obtained and then transferred into Streptomyces spiramyceticus B183. The 15 transformants were fermented after a generation grew on SCVM medium. Five of these strains were selected because their fermentation products contained obviously increased proportion of更多还原