pUC118-ski/DH5α工程菌发酵工艺的优化

Optimization of fermentation procedure for recombinant E.coli pUC118-ski/DH5α

目的优化pUC118-ski/DH5α工程菌的发酵工艺。方法筛选高拷贝质粒工程菌,通过优化培养基组分、发酵温度、补料成分和补料方式及培养时间,确定最佳发酵参数;以最佳发酵参数在50 L发酵罐中连续发酵3批,验证优化的发酵工艺的重复性。结果最佳发酵参数为:以甘油为碳源的培养基,发酵温度25℃,以50%Glycerol梯度恒速流加方式补料,培养时间为12 h;以最佳发酵参数在50 L发酵罐中连续发酵3批工程菌,菌体湿重和质粒含量分别达52.7～60.3 g/L和1.79～1.95 mg/g湿菌,质粒拷贝数为1012copies/μl,超螺旋质粒的比例达90%以上。结论优化了pUC118-ski/DH5α工程菌的发酵工艺,为重组质粒的规模化生产及下游纯化奠定了基础。

Objective To optimize the fermentation procedure for recombinant E. coli pUC118-ski/DH5ct. Methods Re- combinant E. coli pUCll8-ski/DH5cL with high copy number of plasmid was screened, of which the fermentation parameters, in- cluding component of medium, temperature for fermentation, matrix component and mode for fed batch and time for culture, were optimized. Three consecutive batches of recombinant E. coli pUC118-ski/DH5oL were fermented in 50 L fermenter under the opti- mized condition, based on which the reproducibility of the optimized procedure was verified. Results The fermentation parameters Were optimized as follows： the recombinant E. coli pUC118-ski/DH5cx was iA6culated to the m^un~＇/iS^ng glycerol as carbon source and fermented at 25~C, supplemented with 50% glycerol by gradient feeding at a constant rate and cultured for 12 h. The wet weights and plasmid contents of three batches of recombinant E. coli pUC118-ski/DH5o~ fermented under the optimized condition reached 52. 7 N 60. 3 g/L and 1.79 N 1.95 mg/g wet bacteria respectively, while the plasmid copy numbers were 1022 copies/txl, and the proportion of supercoiled plasmid were more than 90%. Conclusion The fermentation procedure for recombinant E. coli pUC118-ski/ DH5a was optimized, which laid a foundation of large-scale production and downstream purification of recombinant plasmid.