Construction of Streptomyces-Eschetichia coli bifunctional cosmid and cloning of antibiotic biosynthetic genes.

1.链霉菌E.利用pUR222：：CoS构建了两种双功能粘粒载体pUR222：：CoS，该粘粒含有大肠杆菌载体pURe222和噬菌体基因组0.47kb的结合区。双功能粘粒I型由链霉菌多拷贝载体pIJ703和pUR222：：CoS组成。双功能粘粒II型由天蓝色链球菌A3(2)、pUR222：：CoS和硫链菌耐药基因2的单拷贝质粒SCP的复制区组成。重组质粒转导条件及稳定性的研究：将在特定位置切割的每个双功能粘粒与链霉菌染色体DNA以1：3~3：1的摩尔比连接，并将连接后的DNA转导到大肠杆菌中。载体与染色体DNA摩尔比为3：1的连接DNA转导效果最好。来自双功能粘粒I和II的重组质粒均能在大肠杆菌中稳定繁殖。但缺失了I型双功能粘粒重组质粒的一部分区域。3.聚酮类抗生素生物合成基因的克隆：卡拉芬净产生菌、纳霉素甲酯产生菌基因组文库。用双功能粘粒II构建了OM-173和阿维菌素产生菌。每个克隆都是以卡拉津相关化合物之一的放线菌素生物合成缩合步骤的基因为产物，通过菌落杂交的方法进行筛选。利维链霉菌的转化子从卡拉芬净和纳诺霉素甲酯的文库克隆中导入重组质粒，产生了抗菌化合物。

1. Construction of Streptocyces-E. coli bifuctional cosmid.Two kinds of bifunctionsl cosmid vectors have been constructed by using the cosmid pUR222::cos,which contains e. coli vector pURe222 and 0.47kb cohesive region of lambda phage genome. Bifunctional cosmid type I consists of Streptomyces multi-copy vector pIJ703 and pUR222::cos. Bifunctional cosmid type II consists of the replication region of a single-copy plasmid SCP of s. coelicolor A3 (2),pUR222::cos and thiostrepton resistance gene.2. Studies of transduction condition and stability of recombinant plasmids.Each bifunctional cosmid nector cut at a unique site was ligated with Sau3A-cut Streptomyces chromosomal DNA at the molar ratio from 1:3 to 3:1 and e. coli was tranxduced by the ligated DNA. The highest transduction was performed by the ligated DNA of 3:1 molar ratio of vector and chromosomal DNA. Both recombinant plasmids derived from bifunctional cosmid type I and II were stably propagated in e. coli. However,a part of region of recombinant plasmid derived from difunctionl cosmid type I was deleted.. 3. Cloning of polyketide-antibiotic biosynthetic genes.Genomic libraries of kalafungin producer S. tanashiensis,nanaomycin methylester producer Streptomyces sp. OM-173 and avermectin producer S. avermitilis were constructed by using bifunctinal cosmid type II. Each clone was selected by colony hybridization method using a gene for the condensation step of actinorhodin biosyntheses,which was one of related compounds of kalajungin,as prode. Transformants of S. lividans introduced recombinant plasmids from clones of libraties of kalafungin and nanaomycin methylester procuders produced antimicrobial compounds.