Research On Breeding Strains With High Yield Spinosyn Based On ARTP And RNA Scaffold

Spinosyn is a product produced by secondary metabolism in cells,which is generally produced by Saccharopolyspora spinosa during aerobic fermentation Spinosyn products as biological pesticides can be used to kill pests in the field and control pests in stored grains.In particular,it has obvious destructive effects on grassland greedy night'e.These scenes are very common in China.Therefore,spinosyn will undoubtedly have a wide range of application prospects in China Spinosyn has strong targeting and has a strong target for mammals.There is almost no impact on life and health,and there is no carcinogenicity,deformity promoting,mutagenicity,and neurotoxicity.SPA can greatly inhibit the proliferation of breast cancer cells in vivo and in vitro,especially for highly malignant,refractory triple-negative breast cancer.SPA and its derivatives are effective tumor suppressorsIn this study,the fermentation medium of recombinant Streptomyces albicans was first optimized,and the decomposition was refined to optimize the design of the carbon source and the nitrogen source respectively.In this experiment,the carbon source was finally determined to be glycerol,and the nitrogen source was determined to be safe.Qi Yeast Dip Powder FM808 and Angel Soy Peptone FP410,specially designed and optimized fermentation medium,are not only more suitable for the growth and reproduction of Streptomyces albicans but also greatly reduce the cost,and the yield of spinosyn has been increased by about 2 times.We explored the relationship between the fermentation culture time of recombinant Streptomyces albicans and the yield of spinosyn,and we found that it is not linear.On the seventh day of fermentation culture,the yield of synthetic spinosyn from recombinant Streptomyces albicans was the highest.Through the optimization of the whole chain,the production of spinosyn high-yielding strains from the pre-stage,saving time and cost.Then,the strains were mutagenized and screened by plasma mutagenesis.The operating conditions of the ARTP instrument are fixed.The power supply is often 100W.The radio frequency power is 11.5w mutagenized at a distance of 2.0mm.The plasma temperature is 28 degrees Celsius,and the ventilation volume is about 10.0SLM.The processing time of the instrument was optimized.When the processing time was 150S,the lethality rate of recombinant Streptomyces albicans reached 90%Atmospheric pressure and room temperature plasma causes very abundant DNA damage,causing large fragment damage to the genome.When the fatality rate reaches 90%,a relatively large number of mutation libraries are finally obtained,which is conducive to obtaining a large number of positive mutations;after five consecutive mutagenesis,The yield of spinosyn has been greatly improved,and the morphology of the strain has undergone tremendous changes,which is conducive to subsequent cultivation and breeding.The synthetic pathway of spinosyn involves multiple functional genes and various enzyme molecules expressed.For recombinant strains,the recombinase molecules are randomly distributed inside the cell,causing crosstalk of various intermediate products in the catalytic pathway,which will inevitably affect the metabolic process of the recombinant bacteria,thereby affecting the output rate of the target product.For this reason,this article adopts synthetic biology As a means to solve this problem,we construct an RNA scaffold in the cell body to anchor the recombinase at a specific site,shorten the distance between the enzymes involved in the cascade reaction,reduce the diffusion interval of intermediate products,and increase the production of spinosyn.Because there are too many enzymatic reactions involved in the synthesis of spinosyns,which is not conducive to the verification of the RNA scaffold,therefore,this study first used the reaction of psicose to synthesize allol to verify that the RNA scaffold anchored the recombinase to improve the enzymatic reaction The research results show that one-dimensional and two-dimensional RNA scaffolds can increase the rate of enzymatic reactions,and the role of anchoring recombinase on the triangular mesh scaffold is more obvious.In order to further anchor multiple recombinases,it is used to increase spinosyn Lay the foundation for production.