Strain Breeding And Exploration On Disruption Biosynthesis Information Stream Of Streptomyces Tenebrarius

Streptomyces tenebrarius was the producing strain of apramycin andtobramycin, which were two important aminoglycoside antibiotics. Inorder to increase its production of fermentation constantly,some methodshave been applied in this study.A high–producing strain was obtained byusing conventional selection and breeding. Then, it attempted to obtainsingle product mutant via blocking different component’s biosynthesisinformation stream. Taking knocking out tobramycin biosynthesis relatedgene tobS1(encoding2-deoxy-scyllo-inosose amino-transferase) and tobC(encoding-2-deoxy-scyllo-inosose synthase) as a goal, the vectors forsubsequent conjugations were constructed and preliminary research on anintergeneric conjugation system of S.tenebrarius were proceeded. Theresults were as follows:The original strain (Ts-228) was firstly selected by natural， thentreated by UV, after that breeded from plate containing self metabolite(tobramycin). Finally, a high yield mutant Tt-49was obtained throughseveral cyclic screening. Its productivity was detected in flask and thecurves of type fermentation were processed in50L fermenter. Itsfermentation unit increased to5865u/mL, which was1.8times higher thanoriginal strain Ts-228, the component ratio of tobramycin came up to68.5%. The fermentation age of Tt-49only need4days. The seedmycelium age was suggested in18h and the inoculation amount of beedswas10%. The combination of natural breeding and UV was an usefulmethod for tobramycin from S.tenebrarius. DO2was an importantparameter in fermentation regulation.Through sensitivity test， tetracyclin was determined as resistanceselection marker of gene knockout. Recombinant plasmid pIJ791, whichcontaining tetracyclin resistant gene was constructed based on pBR322、pUC19and pIJ773vector. The resistance gene with oriT were flanked by FRT sites (FLP recognition targets), which allowed FLP-mediatedexcision of the cassette. pIJ791plasmid, which lacked one SacI site, canbe used to gene replacement.Two fragments, which were respectively located upstream anddownstream of tobS1-tobC, were amplified by PCR from S.tenebrariusTt-49genomic DNA and were used as exchange arms. Tetracyclineresistant gene as a selection marker was inserted between these twofragments. And the new recombinant fragment was subseqnently clonedinto the MCS of pKC1139vector, then plasmid pKC1150for homologousrecombination was constructed. pKC1150was transformed to strainET12567containing the Tra+plasmid pUZ8002adopted by CaCl2andsubseqnently introducted into S.tenebrarius Tt-49by conjugal transfer.This laied a foundation for the further gene replacement.