甲醇/山梨醇共混流加诱导改变毕赤酵母生产猪α干扰素过程的代谢产能途径强化发酵性能

Enhanced porcine interferon-α production by Pichia pastoris by methanol/sorbitol co-feeding and energy metabolism shift

摘要:

旨在探讨毕赤酵母生产猪α干扰素过程的代谢产能规律及其对发酵性能的影响。在10 L罐下，开展了不同诱导条件下的毕赤酵母高效发酵生产猪α干扰素过程的代谢酶学和能量再生分析研究。结果表明：甲醇单独诱导条件下、将诱导温度从30 ℃降低到20 ℃，胞内醇氧化酶 (AOX)、甲醛脱氢酶 (FLD) 和甲酸脱氢酶 (FDH) 的比活性增加显著，细胞的甲醇代谢和甲醛异化产能能力、猪α干扰素抗病毒活性大幅提高，最高抗病毒活性达到1.4×106 IU/mL，约为30 ℃诱导条件下的10倍。30 ℃、甲醇/山梨醇共混流加下，主要供能途径由甲醇单独诱导时的甲醛异化代谢转向TCA循环，甲醛异化供能途径被弱化、毒副产物甲醛的生成积累得到抑制，走向目标蛋白合成途径的甲醇分配比例得到提高。此时，最高抗病毒活性达到1.8×107 IU/mL，是30 ℃甲醇单独诱导下最高活性的100倍以上。更加重要的是，共混流加诱导可以在常温、使用空气供氧的条件下进行，发酵成本明显下降、整体发酵性能改善显著。

Abstract:

Porcine interferon-α (pIFN-α) fermentative production by recombinant Pichia pastoris was carried out in a 10-L bioreactor to study its metabolism changes and effects on fermentation under different inducing strategies, by analyzing the change patterns of the corresponding metabolism and energy regeneration. The results show that the specific activities of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) largely increased when reducing temperature from 30 °C to 20 °C under pure methanol induction, leading significant enhancements in methanol metabolism, formaldehyde dissimilatory energy metabolism and pIFN-α antiviral activity. The highest pIFN-α antiviral activity reached 1.4×106 IU/mL, which was about 10-folds of that obtained under 30 °C induction. Using methanol/sorbitol co-feeding strategy at 30 °C, the major energy metabolism energizing pIFN-α synthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle, formaldehyde dissimilatory pathway was weakened and accumulation of toxic intermediate metabolite-formaldehyde was relieved, and methanol flux distribution towards to pIFN-α synthesis was enhanced. Under this condition, the highest pIFN-α antiviral activity reached 1.8×107 IU/mL which was about 100-folds of that obtained under pure methanol induction at 30 °C. More important, enhanced pIFN-α production with methanol/sorbitol co-feeding strategy could be implemented under mild conditions, which greatly reduced the fermentation costs and improved the entire fermentation performance.