The Mutational Biosynthesis Of Doramectin And Studies On The Fermentation Condition

Doramectin,25-cyclohexyl-5-O-demethyl-25-de(1-methylpropyl)-avermectin,is an avermectin derivative with higher and wider antiparasitic activity in veterinary medicine and pesticides. It is one of the most effective agricultural pesticides and antiparasitic agents with a potential development.A second cluster of genes encoding the E1αsubunit of branched-chain a-keto acid dehydrogenase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microorganism which produces anthelmintic avermectins. The bkdFGH gene cluster mutant strain was incapable of producing avermectins.Gene deletion vector pLJ04(pKC1139::△bkdF+△bkdH) was used to disrupt bkdFGH in Streptomyces avermitilis 76-02-e, an industrial producer of anthelmintic avermectin. The disruptants were confirmed by PCR. Shake flask experiment and HPLC analysis showed that the mutant lost the ability to produce avermectins. As we expected, the mutant, named S.avermitilis bkd76-3, could restore the ability of producing avermectins when the feeding of methylbutyric acid and isobutyric acid to its fermentations was carried out. The addition of cyclohexanecarboxylic acid (CHC) into fermentations of the S.avermitilis bkd76-3 allowed for production of four components, two of which was confirmed as CHC-B1 and CHC-A2 by LC/MS analysis, respectively.The optimization of the fermentation conditions on the mutant bkd76-3 was studied.Through single factor test and double factor test, the influence of nitrogen sources, carbon source, inorganic salts, and trace elements on doramectin production were evaluated. Through orthogonal experiment, the optimal compositon of the fermentation medium were soluble amylum115g/L, soybean meal 30g/L, peanut protein11g/L, NH4NO31g/L, MgSO4.7H2O 1.5g/L, KCl 5g/L,CoCl2.6H2O25mg/L, the yield of doramectin was 68.51ug/mL, increased339.73 by %.