Construction Of The Genomic Library From The Streptomyces Cat-tleya A520 And Study On The Biosynthesis Genes Of Thienamycin

The genomic library from the Streptomyces cattleya A520 was established in E.coli DH1 using pKC505,a E.coli/ Streptomyces cosmid vector ,by in vitro packing. Foreign DNA fragments of insertion in recombinant plasmids was 20-30kb in size. The probability of finding a specific gene from the library composed of 4000 colonies was over 99.9%.The Stringency of hybridization conditions were studied first for the three DNA probes with different homologous levels. 32 positive colonies, pKWlOl-pKW132/E.coli DH1,hybridized with the 1.5kb fragment upstream the presumptive thienamycin tcs gene from S.cattleya A520, one positive colony ,pKW201/E.coli DH1 hybridized with the IPNS gene from S.lipmanii and one positive colony pKW301/E.coli DH1, hybridized with cs2 gene from S.clavuligerus were obtained.Two colonies pKW102,pKW105/S.lividans TK24 produced an active compounds against an Am resistant and penicillin G resistant Serratia marcescens AG4410 strain after screening over 4000 transformants and fermentation in different media.Southern analysis revealed that 2.7kb, 4.3kb and 1.9kb BamHI-BamHI fragments from pKW201 were homologous with IPNS, tcs and cs2 genes, respectively. Restriction enzyme physical map of pKW201 was constructed. Several BamHI-BamH I fragments were ligated to plasmid pUC18 resulting in construction of recombinant plasmids pKWGl-pKWG8. 1.9kb,2.7kb and 4.3kb three fragments locating in plasmids pKWG6,pKWG4 and pKWG3, respectively, were linked together in the order of 4.3kb-2.7kb-1.9kb.Part of the 2.7kb DNA fragment homologous to the IPNS gene was se-quenced and analysed. The result showed the homologous DNA fragment with the IPNS from the S.lipmanii is real IPNS gene , the direction and termination...