Research On Nsda Homologues & Application Of A New Breeding System On S.clavuligerus

This thesis comprises two parts as summaried following:(1) Research on the similarity of nsdA homologues in StreptomycesThe global regulatory gene nsdA negatively regulates antibiotics production in Streptomyces coelicolor. The goal of this study is to sequence nsdA homologous gene of different Streptomyces species, test whether this gene is conserved in Streptomyces, and whether it has the potential to be exploited in strain improvement.Based on the published genome sequences of S.coelicolor and S.avermitilis, primers were designed to PCR amplify nsdA homologous gene of S.hygroscopicus 5008, S.albus JA3453, S.aureofaciem 211, S.lividans ZX64, S.spp.FR-008 derivative DX600, S.hygroscopicus 10-22, S.qingfengmyceticus A553 and Micromonospora 40027. PCR products were purified and sequenced. Sequences of seven nsdA homologues had been acquired and were submitted to GenBank data base, except strain 40027. nsdA homologues shown high similarity in Streptomyces, indicating that this gene is conserved in Streptomyces.(2) Application of a new system in improving yield of clavulanic acid Antibiotic breeding system utilizing report gene has been first established by our laboratory and applied in improving the avermectins production. This study applies this breeding system to S.clavuligerus, in order to improve the clavulanic acid production.Firstly, in order to mark ORF7, ORF11 and the regulator gene claR in the clavulanic acid gene cluster with a neo report gene cassette, gene replacement plasmids pHL557, pHL558 and pHL559 were constructed and conjugated into S.clavuligerus NRRL3585, respectively. Three labeled strains YZ4, YZ5 and YZ6, were obtained and mutagenized by NTG Reasulting mutants were screened by kanamycin selection. HPLC analysis of fermentation culture indicated that four mutants produced more clavulanic acid than original strains did.Further analyze indicated that some mutants from the strains YZ4 and YZ5, in which the structure gene ORF7 and ORF11 was labeled respectively, increased clavulanic acid production. In another word, this report gene assistant breeding system had an effect on the clavulanic acid improvement. However, for the regulator labeled strain YZ6 no mutant produced high CA than wild type has been screened by kanamycin selsection yet. Possible reasons were discussed.