Breeding Strains Of Streptomyces Avermitilis With High-Yield

Avermectins,a series of 16-membered macrocyclic lactones with potent anthelmintic and insecticidal activity,were produced through fermentation of Streptomyces avermitilis. They are the most effective agricultural pesticides and antiparasitic agents,and used widely in medical,veterinary and agricultural fields.This paper had studied the optimization on the industrial Streptomyces avermitilis strain by genetic modification.orfX,aveR,afsR,afsQ1,afsQ2 were four positive regulatory genes discovered in the genome of Streptomyces.The four regulator genes were introduced into Streptomyces avermitilis industrial strain Z1 through conjugal transfer and analyzed the avermectins yield of recombination strains.The result showed that the avermectins yield in strain Z1 increased by 30%after the regulatory gene orfX.The aveR showed little effect.The afsR and afsQ₁Q₂ genes decreased the avermectins production in strain Z1.The protoplast preparation and fusion of the industrial Streptomyces avermitilis were studied in this paper.Results showed that conditions for protoplast preparation for Streptomyces avermtitlis were following.The 35 mL YEME media(plus 0.5%glycine) in 250mL flask inoculated with spore suspension and incubated at 28℃for 40h on a rotary shaker at 200rpm.Mycelia were pelletted through centrifugation at 3000g and washed with 10.3%sucrose.Mycelia were resuspended with P buffer(contain 4mg/mL lysozyme) and incubated at 32℃for 50min to release protoplast.50%PEG1000 was suit for protoplast fusion.The strain OLM(with apramycin resistance gene,apr) and Z1-spc(with spectromycin resistance gene,spc) were used for recursive fusion.After five round recursive fusion,the fusion rate reached 41.6%and the system were stable.Four strains of Streptomyces avermitilis,as initial strains with deferent yield and source,were used for five round recursive genome shuffling.The ratios of strains with high avermectin production increase with the further shuffling..Through the five round recursive genome shuffling,two high yield strains F5-64 and F5-170were selected.The avermectin B1a titres were 3322 ug/mL and 3328 ug/mL respectively.