| Spinosad, a series of macrolide antibiotics produced by Saccharopolysporaspinosa, are excellent in insecticidal effect and environment friendly characteristics.Metabolomics sampling process for Saccharopolyspora spinosa was optimized.Cold methanol inaction, ground in liquid nitrogen and cold methanol extraction wereadapted for S.spinosa metabolomics studies.The global metabolomics analysis on three S. spinosa strains (wild type strain WT,ultraviolet mutant strain WH124and metabolic engineering strain LU104) wasperformed. The cluster analysis showed that, in WH124, a large number of metabolicintermediates such as the primary intermediate metabolites of the central carbonmetabolism intermediates, amino acids, showed the highest level in the three strainsduring the early stage of fermentation, followed by WT and LU104orderly. The resultsuggested that WH124and LU104had two different mechanisms on higher spinosadproduction. PCA analysis revea l9biomarkers, they are PSA, glumate, glumaine,valine, succinic acid, α-ketoglutarate,ATP, acetyl-CoAand ectoine.The macroporous resin method was established for spinosad separation andpurification from fermentation broth. HDP100was screened from several kind ofmacroporous adsorption resin. pH9-11could benefit spinosad adsorption on the resin.In dynamic adsorption process of fermentation broth, optimum flow rate was selectedas60mL/h. And then gradient elution experiment was performed as follows:8mldeionized water,8mL30%(v/v) acetone,8mL60%acetone and finally with95%acetone (2mM ammonium) or ethyl acetate washed off. Ultimately, the spinosadproduct yield was91.23%and93.4%.Base on that, membrane separation and water phase crystallization method wereperformed for further purifying the concentrated solution obtained form themacroporous resin method. Finally, the purity of spinosad reached to95.43%and93.23%, respectively. The total yield of the separation and purification process were87%and79.8%, respectively. |
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