Studies On The Fermentation Conditions Of Saccharopolyspora Spinosa-Mar

Spinosad, a kind of macrocyclic lactones antibiotic, is the main secondary metabolite produced by Saccharopolyspora spinosa during fermentation. Due to its unique chemical structure and function, spinosad has both highly insecticidal activity of chemical pesticides and security of bio-pesticides. Spinosad has become the most international pospects for the development of biological pesticides. S. spionsa-Mar, build by combining Red/ET homologous recombination, transposition and conjugational transfer technique, has stability of genetic.In this study, the production levels of spinosad, the growth curves of S. spinosa-Mar and the original strain were compared, as well as the curves of reducing sugar and the variety of p H were compared in fermentation process. we found that the former growth slower than the latter, and the former showed a lower fermentation level of spinosad. Therefore, our study focused on optimizing the seed culture conditions and the fermentation medium of S. spinosa-Mar to improve the the fermentation level of spinosad.Seed quality is a very important part in the fermentation production. Culture medium, seed age, inoculation amount of dissolved oxygen, inoculum size and other factors have a great effect on seed quality. In this study, the CSM composite seed culture medium was selected to optimize. Results showed that the best inoculum size was 1.5%, and we obtained the optimal seed medium formula(g/L): TSB 60, yeast powder 12, glucose 10, MgSO4 1.4. The primary and secondary relationship of the various components of this medium was as follow: Glucose>yeast extractive>TSB>magnesium sulfate. The relative yield of spinosad reached 130.98% by using this formula, and was increased 30.98% when compared with the original.During the process of fermentation, the precursors play an important role in promoting the synthesis of target products, appropriatly adding precursor substances is likely to improve the fermentation titer. First, we used single factor experiment to eleven possible precursors. Then we used orthogonal design to the four promotive precursors. Results showed that the effect of sodium propionate, valine and n-butanol have are significant, and the effect of isoleucine is not significant. And then we used Box-Behnken Design to optimize the three significant factors. Results showed that the best components were sodium propionate(2.38g/L), valine(1.45g/L), n-butanol(0.45mL/L). The realive yield of spinosad reached 365.72% by using this formula, and was improved about 2.66 times. At the same time, we found that the best addition time of sodium propionate, valine, n-butanol was for 0h, 48 h and 36 h.Spinosad fermentation cycle was about 13 days, and it has lower yield, high cost of fermentation, the fermentation process has yet to be a breakthrough. This paper optimized the seed culture medium of S.spinosa-Mar, and studied the effects of the precursor concentration on the yield level of S. spinosa-Mar. And we further studied the effects of the adding time of precursors which showed a positive effect of spinosad biosynthesis. The results of our study provide some reference for spinosad fermentation process, and have certain application value.