Mutagenesis Screening And Optimization Of Technological Conditions Of Industrial Abamectin Producing Strain

Avermectin,produced by Streptomyces avermectinus,has been widely known by the general public due to its effective anthelmintic activities,and mammals have been confirmed as a non-target organism.Furthermore,Avermectin has been exerted outstanding advantages based on its degradation characteristic for visible light compared with traditional pesticides.Under the background of increasing global market for Avermectin,it is urgent to improve the yield of Avermectin.However,the major bottlenecks to large-scale production of Avermectin is partly attributed to strain degradation,low yield,high production cost,and the resistance has also been highly concerned due to the extensive application of Avermectin.Consequently,strain breeding,culture medium optimization,fermentation conditions optimization,and extraction conditions optimization were investigated by lab-scale cultivation,and the interaction mechanism between Avermectin and glutamate-gated chloride channel was also studied by molecular simulation.In view of this,the relevant researches have been carried out from the following aspects:(1)A novel set of strains with higher Avermectin titer were obtained by physical and chemical mutagenesis.Specifically,twelve single colonies with high Avermectin titer were obtained by UV irradiation mutation,nitrosoguanidine mutation and UV-lithium chloride mutation,and the highest Avermectin titer(9060 μg /m L)was found,which was about 27.60%higher than the initial strain.(2)Streptomyces avermitilis was cultured in fermentation medium supplemented with different concentrations of carbon sources,including corn starch,dextrin,glucose,watersoluble starch,and dextrin may be a potential factor to increase the Avermectin titer.Also,to study the effects of exogenous nitrogen source on Avermectin titer,soybean cake powder,cottonseed cake powder,peanut cake powder and wheat germ powder were used as test objects,and the optimum nitrogen source was soybean cake powder.Finally,the optimization concentrations of different trace elements were investigated,and results were as follows: the optimal concentration of cobalt chloride was 0.02 g/L,the optimal concentration of manganese sulfate was 0.0023 g/L,the optimal concentration of sodium molybdate was 0.02 g/L and the optimal concentration of ammonium sulfate was 0.25 g/L.(3)The fermentation conditions of Streptomyces avermitilis were optimized by by single factor experiment and orthogonal experiment.Specifically,the highest Avermectin titer was found in cells treated with 44 h of seed age,40 m L of loading volume,220 r/min of shaking speed,5% of inoculation amount,7.5 of p H,28.5℃ of culture temperature,20 min of ultrasonic time,and 2 times of extraction time.(4)The molecular docking of avermectin and glutamate-gated chloride channel protein resulted in a binding energy of-8.8 kcal/mol,indicating that Avermectin＿B1a had the potential activity effect on glutamate-gated chloride channel.Meanwhile,Avermectin＿B1a binded to glutamate-gated chloride channel,and two hydrogen bonds were formed between Avermectin＿B1a and THR-251 in the polymer.In addition,hydrophobic interaction was formed with LEU-254,ALA-258 and ALA-261 in the ion channel,which may be the potential targets of glutamate-gated chloride channel protein.Meanwhile,molecular simulation showed that RMSD of both ligand and protein were stable within 8 ns,which provided potential significance for the follow-up study of abamectin pesticide resistance.