Effect Of Genes Disruption Of AveR1, AveR2 On Avermections Production In Streptomyces Avermitilis And Studies On The Fermention Condition

Avennectins are a series of 16-membered macrocyclic lactones produced by Streptomyces avermitilis with potent anthelmintic and insecticidal activity. They are the most effective agricultural pesticides and antiparasitic agents, and used widely in medical, veterinary and agricultural fields.The regulatory genes of avermection biosynthesis, aveR1 and aveR2, in Streptomyces avermitilis ATCC31267(wild strain),B-l-7 and D73-12(high avermection producing strains) were disrupted separatedly by deletion vector pXF05(pKC1139::0.8kb Rl+0.8kb R2+1.7 kb.Hyg). The disruptants were genetically stable because aveR1 and aveR1 genes in the chromosome were displaced by deletion allele on the plasimid via double crossover. The deletion mutants were confirmed by PCR. Shaking flask experiments showed that titer of the mutant from ATCC31267 improved more than three times. But the titer of the mutants from B-1-7 and D73-12 had no change. We will have a further research on this phenomenon.For D-3 (the mutant of D73-12 aveR1,aveR2 disruption) the fermentation conditions of avennectins were studied.The influence of nitrogen sources, carbon sources, inorganic salts and trace elements on avermectin production were evaluated. In the medium the optimum conditions were : KC1 6g/l, MgSO4-7H₂O 0.9g/l, K₂HPO₄-3H₂O 0.9g/l, MnCl₂·4H₂O2mg/l, CoCl₂·6H₂O5mg/l, (NH₄)₂SO₄0.05g/l, initial pH 7.2.Through orthogonal experiment, the avermectin yield 1936.09 μg/ml was obtained.