| Taxol, a highly derivatized diterpenoid, was firstly found from some species of Taxus. Due to its unique anticancer mechanism and the remarkable curative effect for various malignant tumors, it has been the focus to which people pay attention all the time. Taxol production by plant endophytic fungi fermentation has been the research hotspot in recent years. It is important to improve the Taxol production by fungi in order to meet the production requirement. Breeding of high yield strains and optimal cultivation conditions will be the effective ways to increase the yield of Taxol.The further research based the Fusarium mairei K178 from the protoplast mutation was investigated by optimization of biosynthesis precursor, breeding method and fermentation technology to increase Taxol yield. The main methods and results were as follows:1. The concentration of precursors for the production of Taxol with Fusarium mairei K178 was optimized by response surface methodology. Firstly, Plackett-Burman design was undertaken to evaluate the effects of eight factors. With statistic regression analysis, the significant factors affecting Taxol production were determined as follows: phenylalanine, sodium acetate and sodium benzoate. Box-Behnken design was used to optimize the three critical internal factors mentioned above, and we found out the optimum concentration levels and the relationships among these factors. By quadratic regression model equation with Design-Expert statistic methods, the optimal concentration of the variables were determined as: phenylalanine 2.8 mg/L, sodium acetate 3.3 g/L, sodium benzoate 31.8 mg/L. Under such conditions, the Taxol production was increased to 242.6μg/L, which was 15.6% higher than the maximum value in the single factor tests. The experiment values under the optimal conditions agreed with the predicted values, which indicated that the model was proper and effective.And the mechanism of the precursors synthesis of Taxol were also studied.2. Spores of Taxol-producing fungus Fusarium mairei K178 were treated with UV and nitrite, the mutants were selected in the plate with 40μg/mL nystatin. The results showed that 5 mutants exhibited an increase in Taxol production among the obtained 31 nystatin resistant mutants. The Taxol production of mutant Fusarium mairei UH23 was 259.8μg/L, which was 17.8% more than that of the starting strain.3. The fermentation conditions of endophytic fungus Fusarium maireiUH23 were investigated for Taxol production. The results showed that an inoculation volume of 5% into the 60 mL/250 mL Erlenmeyer flask at the condition of 26℃and initial pH 7.0 could reach the maximum Taxol production after incubation of 12 days. Glucose and ammonium nitrate were selected as the best carbon and nitrogen source in the further study. The results showed that the optimal concentrations were(g/L): glucose 60, NH4NO3 6, MgSO4 0.3, KH2PO4 0.5, ZnSO4 1×10-3, Cu(NO3) 2 1×10-3, FeCl3 2×10-3, VB1 5×10-2, phenylalanine 2.8×10-3, sodium benzoate 31.8×10-3, sodium acetate 3.3, L-Tyrosine 5×10-3. The Taxol yield reached 305.9μg/L under the optimized conditions. The expression of nitrate nitrogen and ammonium nitrogen are synergistic effects on the biosynthesis of Taxol. Under the condition of adding sucrose less than or equal to 10 g/L can promote the cell growth and improve the yield of Taxol after cutured for 6 days.
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