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

1.链球菌-E. coli双功能粘粒，利用pUR 222：：cos质粒构建了两种双功能粘粒载体。coli载体pURe 222和λ噬菌体基因组0.47kb粘性区。I型双功能粘粒由链霉菌多拷贝载体pIJ 703和pUR 222：：cos组成。双功能粘粒II型由S的单拷贝质粒SCP的复制区组成。coelicolorA 3（2）、pUR 222：：cos和硫链丝菌素抗性基因。重组质粒转导条件及稳定性的研究：将每个双功能粘粒连接子与Sau 3A切割的链霉菌染色体DNA以1：3 ~ 3：1的摩尔比连接，并将两个连接子连接在一起。将连接的DNA导入大肠杆菌中。最高的转导是通过载体和染色体DNA的3：1摩尔比的连接DNA进行的。来自I型和II型双功能粘粒的重组质粒在大肠杆菌中稳定繁殖。杆菌而来自I型双功能粘粒的重组质粒部分区域缺失。3.聚酮抗生素生物合成基因的克隆。tanashiensis、纳米霉素甲酯产生菌链霉菌OM-173和阿维菌素产生菌S.用II型双功能粘粒构建阿维菌素。以卡拉青苷相关化合物之一的放线菌红素生物合成缩合步骤的基因为探针，通过菌落杂交法筛选每个克隆。转化子S. Lividans从卡拉芬净和纳米霉素甲酯的文库克隆中引入重组质粒，产生抗微生物化合物。

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.