Selection And Breeding Of Good Avermectins Strains And The Cloning And Expressing Of Integrative Vitreoscilla Hemoglobin Gene In Streptomyces Avermitilis

Avermectin produced by Streptonmyces avermitilis is a type of 16-membered ring macrolide oleandrose disaccharide derivatives with structural similitude.In the fermentation of large scale and high density,oxygen supply is one of the key factors affecting cell growth and products yield.Therefore,the dissolved oxygen becomes a limiting factor to increase the production of Avermectin.This research is to integrate Vitrescilla globin gene(vgb)to genome of Streptomyces avermitilis,aimed at increasing oxygen uptake rate and improving cell growing condition in order to increase the production of avermectin.S.avermitilis62 and S.avermitilis 64 screened by natural separation were stable with high ability of producing antibiotics.Their B_latiter were respectively 547μg·mL^-1and 512μg·mL^-1.The inactivated parental stain protoplasts fusion method was used to select high avermectin-producing stain.The total titer of Streptomyces avermitilis R08 was up to 2889μg·ml^-1,and the titer of effective composition B_lawas 716μg·ml^-1,increased respectively by 30.9%and 39.8%compared with those of S.avermitilis 62 and S.avermitilis 64.An E.coli-streptomyces stochastic integrated plasmid pLH1001 was constructed by streptomyces stochastic integrated plasmid pSET152 and E.coli plasmid pUC19-vgb with vgb and transformed into Streptomyces avermitilis by conjugation method.Transformants were screened with the Apramycin 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,and there is an absorption peak at 420nm.S.avermitilis(contains or non-contains gene vgb)was cultured for 24h by secondary fermentation method,then transferred to shaking culture of fermentation in inoculum concentration of 7%(v/v).Seven days later,by evaluating its titer,the titer of transformant is lower than before.The possible reason of the low production is the random integratation of the objective fragment to genome of StreptomycesTo optimize the fermentation medium of transformant S.avermitilisT3,Comprehensive studies were carried out about the fermentation composions.The results are as follows:Corn flour 10g/100ml,Soy flour 0.4g/100ml,Peanut flour 0.6g/100ml,Yeast powder 0.8g/100ml.Yeast extract paste 0.20g/100ml,Corn steep liquor 0.30g/100ml.The avermectin producing strain S.avermitilisT3 with the total avermectin titer was 3298μg·mL^-1,and the B_latiter was up to987μg·mL^-1,increased by 56.7%.