Study on the Screening of Doramectin-producing Strains by Nitrogen Ion Implantation and Optimization of Fermentation
|School||Wuhan Polytechnic University|
|Course||Microbial and Biochemical Pharmacy|
|Keywords||Doramectin Streptomyces avermitilis mutation optimization of fermentation medium precursor|
Doramectin,is new macrolide antibiotics and an avermectin derivative with higher and wider antiparasitic activity in veterinary medicine and pesticides,which was biosynthesis by feeding cyclohexanecarboxylic acid in the fermentation process of Streptomyces avermitilis with bkdFGH gene deletion.It is one of the most effective agricultural pesticides and antiparasitic agents with a potential development . In this paper , we studied the screening methods of high doramectin-producing strains with 96-well plates , and selected high doramectin-producing strain with precursor and end-products resistance experiments and N+ mutation . Then , we optimized the fermentation medium.Meanwhile,the effect of precursor on doramectin biosynthesis were researched.The main results were as follows:1.According to screening strain by the precursor resistance experiment,a high doramectin-producing mutant was achieved,which was 60.62% higher than the initial strain.And The minimum inhibiting concentration of CHC was 300μg/μL for this strain.2.According to the mutagenic effects of the high doramectin-producing strain by nitrogen ion implantation with different dosage,the two- summit survival curves was obtained.3.According to the studied on the growth of Streptomyces avermitilis in both solid medium and liquid medium,we found that growth were both good.However,CHC and CaCO3 were insoluble and irreplaceable in the liquid fermentation medium,so use 96-well plates to screen high doramectin-producing strains we must overcome this difficulty.4.Initial studies to optimize carbon and nitrogen sources,inorganic nitrogen sources , inorganic salts and trace elements the doramectin production were performed by single factor experiments and two factor experiment of carbon and nitrogen sources.And the fermentation medium for the strain of XJ-CHC150-17 was optimized by response surface analysis.The results showed that optimum fermentation medium is（g/L）:corn starch 75,dry yeast 18,KCl 1.35,MgSO4 0.16, K2HPO4·3H2O 1.8, FeSO4·7H2O 0.012, Na2Mo4·2H2O 0.045, NH4Cl 0.09,CoCl2·6H2O 0.005,CaCO3 2,pH7.0,and the doramectin yield in the optimum fermentation medium was 103.54μg/mL that increased 1.80 times compared with that using the original medium.5.According to the study of fermentation conditions, the results showed that 30mL medium volume in 250mL shaking flask,initial pH 7.0,seed age 72h,seed inoculum 6% without glass beard,the doramectin production was maximum.6.According to effect of precursor on doramectin biosynthesis,it was suitable for cultivating 48h feeding 300μg/mL CHC.And we found that amino acids inhibited the doramectin biosynthesis obviously meanwhile among low -grade fatty acids sodium propionate as a precursor is superior to other precursor for promoting doramectin biosynthesis . By feeding of 0.1% sodium propionate into the fermentation broth at the 4th day of cultivation,the doramectin production was 139.89μg/mL,38.20% higher than that in the condition without any addition of precursor.7.According to orthognnal design of sodium acetate anhydrous,sodium propinonate and trisodium citrate dehydrate, doramectin yield was increased 9.39%.