Breeding And Fermentation Researches On Saccharopoly Sporaspinosa Producing Biological Pesticide Of Spinosad

Spinosad,a secondary metabolite produced by Saccharopolyspora spinosa,exhibits evident insecticidal activity.Spinosyn is macrocyclic lactone antibiotic and its most active components are spinosad A and D.Spinosad is widely used on pest control of vegetable,fruit crops,ornamentals and grain storage because of its toxic to many insects,but relatively nontoxic to mammals.In this paper,compound mutation breeding,optimization of culture medium,fed-batch fermentation based on two-stage pH regulation and effect of cloning and expression of glucose dehydrogenase gene?gdh?on synthesis of spinosad were studied using Saccharopolyspora spinosa ATCC 49460 as the original strain.1.The starting strain S.s 1-4 was obtained by natural selection.A bacterial strain S.s 3-37 producing high-level of spinosad with genetic stability was obtained by UV and NTG mutation combined with resistance factors of streptomycin,apramycin and rhamnose.The yield of S.s 3-37 reached 78.26 mg/L,which increased by 45.71%compared with the starting strain.The fermentation conditions were optimized by single factor experiment.The optimal culture condition of pH,inoculum size and loading volumn was 7.2,12%,40 mL/250 mL,respectively.2.The fermentation medium of mutant strain S.s 3-37 was optimized.First,the optimal carbon and nitrogen source were determined by single factor experiment.Then,7components of fermentation medium were selected by Plackett-Burman experimental design and 3 influential factors of glucose,dextrin and cottonseed protein were obtained.And the 3factors were optimized by Box-Behnken response surface experiment.At last,the optimal culture medium was obtained and its components were 54.3 g/L glucose,15.5 g/L dextrin,25.7 g/L cottonseed protein,3 g/L NaCl,1 g/L K₂HPO₄,0.05 g/L FeSO₄,1 g/L CaCO₃.The yield of spinosad reached 83.00 mg/L,which increased by 6.06%.3.The effects of pH control on fed-batch fermentation of spinosad were investigated in the 5 L fermenter.First,the pH of fermentation broth was maintained at 7.4,7.2,7.0,6.8,6.6,respectively.In the condition of constant pH,it was found that the optimum pH for cell growth was 7.2 and its cell concentration was 33.08 g/L.The optimum pH for synthesis of spinosad was 6.8 and its yield was 72.28 mg/L.The result showed that the optimum pH was different between synthesis of spinosad and cell growth.So,the two-stage pH regulation strategy was proposed.The pH was maintained at 7.2 at the beginning of fermentation.Then,the pH was slowly adjusted to 6.8 after a period of time.The effects of different regulation time on the fermentation of spinosad were systematically investigated by using this strategy.The pH 7.2 was maintained for 48 h,60 h,72 h,84 h,respectively.Then the pH was adjusted to 6.8 and maintained at 6.8 until the end of fermentation.The result showed that it was beneficial to the synthesis of the product when the pH 7.2 was reduced to 6.8 after 60 h and the production of spinosad reached 86.16 mg/L,which increased by 19.20%compared with that in the condition of constant pH.4.Based on the above pH regulation strategy,fed-batch fermentation was carried out.According to the consumption rate of glucose,different components were added to maintain the concentration of glucose at about 10 g/L.First,intermittent fed-batch fermentation was conducted by feeding with glucose,glucose and cottonseed protein,whole material every 8h,respectively.The production of spinosad reached 128.87 mg/L,135.89 mg/L and 139.43mg/L,respectively.It was found that the yield of spinosad were highest when feeding with whole material.Its cell concentration reached 48.89 g/L and productivity reached 15.49mg·L^-1·d^-1.Then,continuous fed-batch fermentation was carried out by feeding with whole material.The results showed that the cell concentration and yield of spinosad were further increased.They reached 52.89 g/L and 146.83 mg/L,which increased by 62.49%and 70.42%compared with batch fermentation.And its productivity reached 15.73 mg·L^-1·d^-1,which increased by 1.55%compared with intermittent fed-batch fermentation.5.Improving the expression of gdh in the rate-limiting step can release the influence of the rate-limiting step on the synthesis rate.First,strong promoter permE was inserted into pOJ260 plasmid.Target gene gdh was obtained by PCR amplification and it was linked to pMD19-T carrier.Then,it was induced into E.coli DH5?to clone.The plasmid was extracted and enzyme digestion was conducted.The enzyme-digested fragment was connected to the downstream of strong promoter of pOJ260 plasmid.The construction of the recombinant expression plasmid was completed and it was induced into E.coli S17-1.Conjugal transfer was applied to import the recombinant expression plasmid into Saccharopolyspora spinosa.The transformant was picked and verified at last.The maximum yield reached 89.47 mg/L by shaking flask fermentation,which increased by 14.32%compared with the starting strain.The results showed that the overexpression of gdh could promote the synthesis of spinosad,which layed a positive foundation for the construction of genetically engineered bacteria.