Cloning And Expression Of Vitreoscilla Globin Gene(Vgb) In E.Coli And Streptomyces

In this work, cloning and expression of Wlreoscilla globin gene (Vgb) with its nativeoxygen-regulated promoter in StroptOmyces aureofaciens and of the ntreoscilla globinstructural gene transcribed by a tetracycline promoter in Ecoli were studied.The conditions of preparing and regenerating protopIasts of Stroptomyces aureOfaCiens wereoptimized, which included the concentrations of sugar and glycine in the medium, the age ofcuIture, the treating time of lysozyme and the composition of the solid regeneration medium.StrePtOmyCes aureOfaciens couldn't grow well in the YEME liquid medium and regenerate onthe R2YE or R3YE solid medium which were generaIIy used media in the DNA operationtechnology of StroptomyCes. In our experiments, the optimized conditions including theconcentration of sugar and glycine in the medium l2% and 0.5%, the age of cuIture 36h, theconcentration of lysozyme 3mg/ml, the treating time of lysozyme lh were helpfuI to preparehigh qualities of protoplastS and for the transformation. The bran medium creatively used in ourexperiments was beneficia1 to the regeneration oftransformants.The transformillg conditions including themolecular weight and concentration of PEG were optimized. The optimized conditions werePEG l000 and its concentration 33 .3%.Based on the successful construction of the recombinant plasmid pUCl9-Vgb, anE.coli-stroptomyces shuttle plasmid pIJ699-pUCl9-Vgb was constructed and then transformedinto the protOplasts of mpmyCes aureOfaciens. Through screening with the thiostrepton (thefinal concentration 5 P g/ml) and auremycin (the final concentration 3lmg/ml) resistance, therewere three kinds of recombinant strains screened out and the three kind was successful. Underthe cu1ture conditions of the higher medium loading (60ml/250ml) and limited oxygen provided,the successful transfOrmant could express VHb and the final concentration of mycelia andaureomycin were higher than the fermentative leveI of the originaI StroptomyCes aureOfaciensunder the conditions of the normal medium loading (30ml/250ml) and the full oxygen provided.The recombinant plasmid pBR322-vgb I containing a tetracycline promoter wasconstructed creativeIy and transformed into Ecoli JM109. The successful recombinant strainswere screened out with ampicil1in resistance. The results showed that tetracycline could induceNtreoscthe globin structure gene more validIy than native promoter to express VHb.The study broadened the application of Vgb and became the base of further studying thecharacteristics of cloning and expression of Vgb in Stroptomyces aureofaciens. At the same time,the study had crucial theoretical and practicaI direction function on genetically modification ofother antibiotics-producing stains especially Stroptomyces.