吸水链霉菌Streptomyces hygroscopicus 17997中噬菌体基因转移系统的建立及其应用  

Establishment of Gene Transduction System in Geldanamycin Producer—streptomyces hygroscopicus 17997 and its Application for Gene Disruption Experiment

作　　者：高慧英[1] 王以光[1] 高群杰[1] 尚广东[1] 孙桂芝[1] 杨樱[1] 

机构地区：[1]中国医学科学院中国协和医科大学医药生物技术研究所,北京100050

出　　处：《生物工程学报》2003年第4期407-411,共5页Chinese Journal of Biotechnology

基　　金：973基础研究重大项目前期研究专项基金资助 (No .2 0 0 1CCA0 0 5 0 0~~

摘　　要：由吸水链霉菌Streptomyceshygroscopicus 17997产生的格尔德霉素geldanamycin(GA)属安莎类抗生素 ,具有良好的抗肿瘤和抗病毒活性。本文应用链霉菌温和噬菌体C3 1衍生的KC5 15载体 ,在吸水链霉菌S .hygroscopicus17997中建立并优化了S .hygroscopicus 17997的基因转染体系。利用所建立的基因转染体系 ,以基因阻断技术从S .hygroscopicus 17997基因文库含有多组PKS基因柯斯质粒中 ,鉴定了与GAPKS生物合成相关基因的柯斯质粒 。Streptomyces hygroscopicus 17997 produces the antiviral and antitumor ansamycin antibiotic, geldanamycin. Studies on geldanamycin biosynthetic pathway will provide good tools for genetic manipulation of the antibiotic-producing strain to improve the productivity or to facilitate making novel geldanamycin analogs. The structural similarities between geldanamycin and ansamycins such as rifamycin or ansatrienin suggest that both geldanamycin and ansamycins has a closely related pathways of biosynthesis and that biosynthetic system for geldanamycin is similar to the one of type I polyketide synthase (PKS) enzyme system. To explore the possible PKS genes involved in geldanamycin biosynthesis, the degenerate primers were designed according to the conserved sequence of KS-AT region from erythromycin and oleandomycin type I PKS genes. Cosmids containing multiple PKS genes (pCGBK2,4,6,10,11,18) were obtained by hybridization with the PCR products, which were amplified from S. hygroscopisus 17997 genomic DNA. The designed primers above were used for PCR. Development of a Streptomyces temperate phage ΦC31-derivative KC515(tsr R) transduction system was carried out for identification of cosmids containing the PKS gene related to biosynthesis of geldanamycin. Several factors, mainly the Ca 2+ and Mg+ concentrations in different culture media affecting the frequency of gene transfection, were optimized .Transfection efficiency could reach up to 10 3/μg DNA on YMG medium supplemented with 10mmol/L MgSO 4. Reversely, the transfection efficiency decreased when YMG medium was supplemented with 30mmol/L MgSO 4. Gene transfection system based on the integration-defective phage KC515 had been established for S. hygroscopicus17997. Recombinant phages (ph111, 258, 287, 116, 105)were constructed by insertion of the homologous to PKS gene fragments into the KC515 phage vector. Gene disruption experiments were performed by transduction of recombinant phages into S.hygroscopicus 17997 genome, and disruption of geldanamycin pr

关 键 词：吸水链霉菌 噬菌体基因转移系统 应用 基因阻断 格尔德霉素 生物合成基因 

分 类 号：Q78[生物学—分子生物学] Q933