烟曲霉TMS-26产紫杉醇补料分批发酵条件初步探索

内生真菌发酵法是解决紫杉醇药源短缺问题的有效途径之一。本研究以摇瓶分批发酵为基础,进行摇瓶补料分批发酵研究,探究了苯丙氨酸、甘氨酸、苯甲酸钠乙酸钠混合液、3,5-二硝基水杨酸、H₂O₂、CuSO₄在发酵周期（13d）中,不同添加时间点对TMS-26菌体量及紫杉醇产量的影响,发现在第8天添加苯丙氨酸、甘氨酸、3,5-二硝基水杨酸时,其产量分别达到了(664.80±40.34)µg/L、(628.72±30.44)µg/L、(641.36±19.62)µg/L;在第9天添加CuSO₄时,其产量达到了(697.46±15.76)µg/L;在第10天添加H₂O₂、苯甲酸钠乙酸钠混合液,其产量分别达到了(615.78±36.28)µg/L、(792.54±10.04)µg/L。在摇瓶补料分批发酵研究结果的基础上,进行了5L罐发酵工艺放大研究,探究了前体和诱导子通过进行一次补加和恒速补加的方式对Aspergillus fumigatus TMS-26菌体量及紫杉醇产量的影响,结果表明恒速补加苯丙氨酸乙酸钠混合液,紫杉醇产量达到了746.17µg/L。通过本次研究,优化了TMS-26产紫杉醇摇瓶补料分批发酵和5L罐发酵工艺,为后续实现紫杉醇工业化生产奠定基础。

A preliminary exploration of fed-batch fermentation of Aspergillus fumigatus TMS-26 for taxol production

Fermentation of endophytic fungi is a potential and feasible approach for taxol production. Fed-batch way was performed in batch fermentation of Aspergillus fumigatus TMS-26 for determining the optimization adding time of phenylalanine, glycine, sodium benzoate and sodium acetate mixture, 3,5-dinitrosalicylic acid, H₂O₂, and CuSO₄ within the fermentation period (13d) and the effect of different adding time on the taxol yield was also observed. The results showed that using phenylalanine, glycine and 3,5-dinitrosalicylic acid as additives, the yield of taxol can reach (664.80±40.34)µg/L, (628.72±30.44)µg/L, (641.36±19.62)µg/L respectively on day 8. Adding CuSO₄, the yield of taxol can reach (697.46±15.76)µg/L on day 9, while adding H₂O₂, sodium benzoate and sodium acetate mixture, the yield of taxol can reach (615.78±36.28)µg/L, (792.54±10.04)µg/L on day 10. The scale-up experiment with 5L fermenter was conducted on the basis of flash fed-batch fermentation. The effect of precursor and eliclitor on the yield of taxol was investigated by single feeding and constant rate feeding strategies. The results showed that when phenylalanine and sodium acetate mixture was added under the condition of constant rate feeding strategy, the yield of taxol can reach 746.17µg/L. This study optimized the technology of flash fed-batch and 5L fermenter fermentation of Aspergillus fumigatus TMS-26 producting taxol, which laid a foundation for the industrialization of taxol production.