The Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Ave, Wuxi, 214122, China.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
World J Microbiol Biotechnol. 2018 Nov 21;34(12):180. doi: 10.1007/s11274-018-2562-0.
Heterologous proteins induction by methylotrophic recombinant Pichia pastoris is generally implemented at high cells density condition. Methanol concentration (MeOH) and dissolved oxygen concentration (DO) are two crucial operating parameters controlling proteins production. It is difficult to control MeOH/DO at their desired levels simultaneously due to the extremely high oxygen consumption features. Methanol utilization plus (Mut) and slow (Mut) strains are the two typical phenotypes of recombinant P. pastoris with quite different dynamic characteristics. Therefore, different MeOH/DO combinational control strategies or sub-optimal induction strategies could be adopted. Environments of "high MeOH/low DO" and "high DO/low MeOH" are the realistic induction strategies. In this study, we summarized our own experimental results (using Mut/Mut strains to produce human serum albumin-human granulocyte colony stimulating factor-HSA-GCSF/porcine interferon-α-pIFN-α), and compared to data from the literature using the above mentioned two induction strategies. The results suggested that, heterologous proteins production by Mut strains favors "high DO/low MeOH (DO ~ 10%, MeOH ~ 0 g/L)" induction condition, while proteins production by Mut strains prefers "high MeOH/low DO (MeOH 5-10 g/L, DO ~ 0%)" induction environment. Thus, based on the P. pastoris types, the corresponding sub-optimal induction strategies should be applied accordingly. The related metabolic analysis indicating methanol utilizing efficiency and the transcriptional analysis reflecting gene up- or down-regulations involved in several key routes in methanol and sorbitol metabolism were implemented. The analysis results strongly supported the conclusions of using the proposed sub-optimal induction strategies for different heterologous proteins production by Mut and Mut strains.
甲醇营养型重组毕赤酵母生产异源蛋白通常在高细胞密度条件下进行。甲醇浓度（MeOH）和溶解氧浓度（DO）是控制蛋白质生产的两个关键操作参数。由于耗氧率极高，很难同时将 MeOH/DO 控制在所需水平。甲醇利用增强（Mut）和缓慢（Mut）菌株是重组毕赤酵母的两种典型表型，具有截然不同的动态特性。因此，可以采用不同的 MeOH/DO 组合控制策略或次优诱导策略。“高 MeOH/低 DO”和“高 DO/低 MeOH”是现实的诱导策略。在这项研究中，我们总结了自己的实验结果（使用 Mut/Mut 菌株生产人血清白蛋白-人粒细胞集落刺激因子-HSA-GCSF/猪干扰素-α-pIFN-α），并与文献中的数据进行了比较，使用了上述两种诱导策略。结果表明，Mut 菌株生产异源蛋白时有利于“高 DO/低 MeOH（DO10%，MeOH0 g/L）”诱导条件，而 Mut 菌株生产蛋白时则偏好“高 MeOH/低 DO（MeOH 5-10 g/L，DO~0%）”诱导环境。因此，应根据毕赤酵母的类型，相应地应用次优诱导策略。进行了相关的代谢分析，以指示甲醇利用效率，以及转录分析，以反映甲醇和山梨醇代谢中几个关键途径的基因上调或下调。分析结果强烈支持了使用所提出的次优诱导策略来生产 Mut 和 Mut 菌株不同异源蛋白的结论。
