Recombinant Strain Of Streptomyces Avermitilis Breeding

With increasing of the consumers of the international market and domestic to the quality of agricultural products, people attach great importance to hazards from the agricultural pollution by tocic chemicals.As a kind of biological pesticides, avermectin is the most effective antibiotics to insecticidal and acaricidal so far. The process of fermentation of streptomyces which can product the avermectin is an aerobic process. The traditional fermentation in industry uses the high-density and deeply fermentation.Dissolved oxygen always become an important limiting factors affecting the increase of avermectin yild. This study was carride 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, byexpression of Vitreoscilla hemoglobin gene in Streptomyces avermitilis.In this article, conditions for preparation and regeneration of protoplasts of Streptomyces avermitilis were sthdied. First, use the cell dry weight method to get the growth curves of Streptomyces by sampling in point at the process of fermentation of S.avermitilis. Order to it, determined the best incubation time of mycelia. Concentration in the medium with glycine 0.25 g/ml. The concentration of lysozyme was 2 %. The temperature of incubation was 34℃. The time of hydrolysis was 30min. Under these conditions, the amount of protoplast formation was 6.4×10⁸ per ml. To spread on protoplast prepared regeneration medium with 0.2 %çš„MgCl₂·6H₂O of buffer by overlayer coating. The density of protoplast for each plate coated less than 1, 000. Under these conditions, the regeneration rate was 20.6 %.Successfully constructed the shuttle vector from E.coli to Streptomyces. Vitreoscilla hemoglobin gene that can be successfully transformed into S.avermitilis.And, derived the lowest concentration of thiostrepton which can inhibitory avermitilis protoplast was 0.5 slightly per ml. Comparing with the fermentation titer of strain, concluded the conclusion that vitreoscilla hemoglobin successful expressed in Strptomyces. Therefore, the capacity of growth and antibiotic production is effectively improved. Makes the avermectionrermentation titer increased by 32 % at the oxygen limited conditions.