The Research Of Fermentation And Metabolic Regulation Of Spinosad And Transcriptional Analysis Of Some Related Genes In Biosynthetic Pathway

Spinosad,a novel macrolide antibiotic utilized in agriculture,produced by Saccharopolyspora spinosa,was a promising and prominent member of biological pesticides.The major research contents,including the growth characteristics of S.spinosa in agar slant and liquid medium,the optimization of fermentation medium,the functional mechanism of stimulative factors in spinosad synthetic process,the regulation of lipid metabolism and catabolism repression in spinosad synthesis,were investigated in this dissertation.The growth characteristics and inoculation standard of S.spinosa were inquired in this article.The results showed that the optimal cultivation time of slant strains was 7 days,the wrinkled surface of thicker lawns was pink colouring and covered with abundant spores.The optimized inoculum age of liquid seed was 28-36 h,the stronger mycelium of S.spinosa with majority of branch points was full of vitality in the logarithmic phase and was the desirable seed for fermental cultivation.Rapeseed oil,hydrolysis sugar of maize meal,CoCl₂ were screened by single factor experiment which could promote spinosad synthesis significantly and the yield of spinosad was improved by 4.5 folds.The optimal medium composition was determined by Plackeet-Burman design and response surface method.The spinosad production was increased by21.15%when the optimized medium was applied in fermentation.The functional mechanism of stimulative factors in spinosad synthetic process were studied.The results indicated that the effects of different vegetable oils on the growth of S.spinosa and production of spinosad varied significantly.The rapeseed oil was conducive to the transcriptional intensity of lipase gene and spinosad synthetic genes and enhanced lipase expression,the growth of S.spinosa and spinosad synthesis.The lipase activity,the biomass and spinosad yield were improved by 310.09%,8.97%and 33.94%,respectively.In addition,spinosad would be biosynthesized efficiently when the fermentation broth was supplemented frequently with a small amount of rapeseed oil or the proportion of glucose and rapeseed oil was adjusted to 35:45.The screening of non-repressible carbon and nitrogen substances for S.spinosa was investigated.The repression effects caused by carbon and nitrogen metabolites could be relieved in some extent with adding sucrose and beef extract during spinosad fermentation process.The spinosad production was enhanced by 12.29%and 9.75%when feeding 1.5%sucrose on third day and 0.3%beef extract on fourth day,respectively.The batch fermention process in shake flasks was also inquired in a 5 L fermentor and the titer of spinosad were at the same level.