Breending Of Spinosad Producing Strains Using Compound Mutagenesis And Characterization Study

Spinosad is a macrolide biopesticide produced by the aerobic fermentation of Saccharopolyspora spinosa.Spinosyns A and D are the main active components,which account for about 85%and 15%,respectively.Spinosad is a broad-spectrum insecticide.It has a high insecticidal effect against Lepidoptera,Thysanoptera,and Homoptera,and is non-toxic to non-target beneficial insects,mammals,and birds.Spinosad is also one of the safest and most effective insecticide so far.It has the characteristics of short half-life,complete biodegradation,and environmental friendliness.At present,the yield of spinosad is still too low to meet the requirements of industrial production.In this study,the high-yield strain of spinosad was bred by composite mutagenesis,and the culture conditions were optimized.The physiological characteristics of the mutated strain were studied by analyzing the morphological characteristics of mycelia,cell viability,and respiratory activity.Finally,the effect of butyl oleate on the production of spinosad by high-yield strains was studied to further improve its yield.The research contents of this paper include the following:1.S.spinosa AEG-6 with 100.06 mg/L spinosad was obtained by screening and used as the original strain.S.spinosa AEG-6 was mutated and evaluated using the following four methods:ultraviolet(UV),atmospheric pressure and room temperature plasma(ARTP),nitrosoguanidine(NTG),and ethyl methanesulfonate(EMS)mutation.By using ARTP mutation,the yield of spinosad was increased to 127.51 mg/L,which was 27.43%higher than that of the original strain.The positive mutation rate of EMS mutation was54.00%,which was higher than that of other mutation methods.Then,the minimum inhibitory concentration(MIC)of streptomycin and gentamicin were obtained to be 0.2mg/L and 2.5 mg/L by ribosomal engineering.The 2MIC of streptomycin was the optimum for the mutation of S.spinosa AEG-6.The triple composite mutagenesis of"ARTP+EMS+str~r"was conducted,and the mutant strain S.spinosa AES-8 was obtained.Its yield was 186.92 mg/L,which was 86.81%higher than that of the original strain and was genetically stable.2.Subsequently,the culture conditions for S.spinosa AES-8 were optimized.The optimized conditions were as follows,the optimum seed age of 60 h,the inoculation amount of 10%,the liquid volume of 40 m L/250 m L,and the initial p H value of 7.2.The kinetic curves in shake flasks were compared between the two strains.It was found that S.Spinosa AES-8 changed in a direction that was more favorable to the growth of bacteria and the secondary metabolism of spinosad.The yield of spinosad reached 205.54 mg/L.The colony morphology of S.spinosa AES-8 is"crater"type.The mycelial morphology of S.spinosa AES-8 in the fermentation broth was observed by a biological microscope.The mycelial morphology of the two strains was similar in the early stage of fermentation,and the main difference appeared in the middle and late stage of fermentation.During the middle and late stages of fermentation,the mycelia of the mutant strain were more vigorous,longer,and less autolytic.The cell viability and cell respiratory activity of mutant strain observed by laser confocal microscope were stronger than S.spinosa AEG-6.In batch fermentation of mutant strain in a 5-L fermentor,the biomass and spinosad yield reached 23.44 g/L and 221.57 mg/L,respectively,which was 5.30%and7.80%higher than those of shake flasks.3.The results in shaking flasks showed that camellia oil,walnut oil,methyl oleate,and butyl oleate could promote the production of spinosad,respectively,and butyl oleate was the best.However,oleic acid and linoleic acid inhibited the spinosad production of S.spinosa AES-8.Adding 0.5 g/L of butyl oleate at 120 h was the best,the yield of spinosad reached 270.06 mg/L,which was 30.27%higher than that of the control.Simultaneously,the lipase activity was 26.96 U/m L with an increase of 31.06%compared with the control.Finally,the yield of S.spinosa AES-8 was increased to 370.12 mg/L by fed-batch fermentation combined with the addition of butyl oleate on the 5th day in the 5-L fermentor.By adding butyl oleate from the 4th to 6th day of fermentation,the production reached 391.43 mg/L,which was 5.76%higher than that of one-time addition.The above results would provide technical support for the industrial production of spinosad.