Studies On The Ansamitocin P-3Produced By Microgrganism Fermentation

Ansamitocions are extraordinarily eukaryotic cell potent toxicity and antitumor maytansinoids from microorganisms sources,therein ansamitocin P-3(AP-3)has an excellent activity.As an ideal toxicity of targeting drug,ansamitocions attracted much attention in the antibody-drug conjugates research. The antibody drug conjugate Kadcyla of Roche was approved for the treatment of HER-2patients with advanced metastatic breast cancer by FDA in2013. In addition, there are SAR3419, BT062, BAY94-9343and other ADC of maytansinoids in phase I-II clinical trials, which achieved significant clinical effects. At present, the low level fermentation and the expensive market price of ansomitocions, severely limits its further pharmacological and applications studies, so there must be a great practical significance if a research on improving production of AP-3through fermentation process optimization and regulation is carried out.In this thesis Actinosynnema pretiosum spp.auranticum APSP-Olwas chosen as starting strain, and the medium and the culture conditions of shaking-flask fermentation were optimized and the amplification study of fermentation was discussed, then the fermentation kinetics model was established and the best batch culture strategy was determined.The main results were taken as follows:Monofactorial experiments and uniform design were applied to optimize the medium of AP-3. The medium composition such as carbon sources, organic nitrogen sources and ammonium saltsnitrogen were investigated through monofactorial experiments. Then the uniform experimental design of U*15(157) were taken to further optimization. The experimental results showed that the optimum medium composition were glucose4.22%, maltose0.75%, corn steep liquor3.42%, ammonium acetate0.41%, isobutanol0.43%and the AP-3reached (51.86+1.33) mg/L,which was consistent with the maximum predicted value.The research above provided a practical basis for the subsequent experiments.The optimization of fermentation conditions for ansamitocin AP-3production by the Actinosynnema pretiosum spp.auranticum. To optimize the fermentation conditions, monofactorial experiment and the Box-Behnken experimental design were combined. The response surface was applied to analyze experiment data by SAS8.1software, the quadratic regression fitting equation is obtained. P<0.01,regression model was very significant. The correlation coefficient R2=0.9696, the fitting degree of model was better. The optimal fementation conditions were:inoculum concentration13%,220r/min, pH7.4, fementation time8d. It has been verified by test, the experimental values very close to predicted values, the maximum mass of product of AP-3was82mg/L,10.6%higher than before.The amplification study of AP-3fermentation by the strain APSP-01was carried out in a5L bioreactor and the batch fermentation kinetics were established. Firstly the metabolic characteristics of the fermentation process were analyzed and determined it’s non-coupling model. According to the Logistic equation and Leudeking-piret equation, the parameter containing microbial biomass, the synthesis of AP-3and the residual sugar content were estimated based on genetic algorithm.Then the batch fermentation of strains producing AP-3fungus growth, AP-3kinetic model and parameters and substrate consumption were obtained through the further nonlinear regression.The model described the dynamic process of batch fermentation of AP-3very well and provide reference for the fed-batch culture.The effect of feeding time and feeding pattern on fed-batch of AP-3was studied. Experimental results established that the feeding strategies:in the time about84h when the residual sugar concentration down to20g/L, with the pattern of constant speed flow.Under the optimum conditions, the eventually yield of AP-3reached92.24mg/L in216h,which was28.11%higher than before.