Selection And Breeding Of High-yielding Avermectins Strains And The Cloning And Expression Of Vitreoscilla Hemoglobin Gene In Streptomyces Avermitilis

Streptomyces avermitilis is a kind of microbe that can produce avermectin. Its fermentation broth is so mucous that it cannot dissolve too much oxygen, So DO (dissolved oxygen) becomes one of the restricted factor affecting the increase of avermectin yield. This study was carried out to improve the genetically engineered bacteria's ability to transport and absorb oxygen, better its growth, as well as strengthen its synthesis of protein and secondary metabolites, by expression of Vitreoscilla hemoglobin gene in Streptomyces avermitilis .In this work, conditions for preparation and regeneration of protoplasts of Streptomyces avermitilis were optimized. Medium R₂YE appeared to be more suitable than YMB, SM and YEME for protoplast formation. The mycelia of Streptomyces avermitilis was cultivated in the form of secondary culture , namely cultivation at 28℃ for 48h in R₂YE culture, then transfered to medium R₂YE added 0.25% glycine, continues cultivation at 28℃ for 20～24h. The washed mycelia were incubated in P buffer containing 2mg·mL^-1 lysozyme at 30℃ for 30min. The final concentration and regeneration rate of protoplasts reached 6.3×10⁸·mL^-1 and 1.98%respectively.The inactivated parental strain protoplasts fusion method was used to select high avermectin-producing strain. The total titer of Streptomyces avermitilis F32 was up to 3 904μg·mL^-1 , and the titer of effective composition B_1a was 1 016μg·mL^-1, increased by 117.1% compared with that of S. avermitilis 620.An E.coli-streptomyces shuttle plasmid pSY702 was constructed and transformed into the protoplasts of Streptomyces avermitilis. Transformants were screened with the thiostrepton resistance. Expression of VHb protein in transformants and the bioactivity of the expressed VHb were verified by the analysis of a CO difference spectra experiments. It was shown by HPLC analysis that avermectin was produced by VHb-expressing transformants in flask fermentation. Under low dissolved oxygen conditions, the S. avermitilis T26 transformant produced higher yields of avermectin compared with the control strain in flask fermentation experiment. These results demonstrated that VHb expression in S. avermitilis can improve the productivity of avermectin even under the conditions that aeration was reduced.Several factors affecting avermectin production were studied. The highest avermectin...