Effective expression of pIFN-α in recombinant Pichia pastoris was conducted in a 5 L fermentor. Ethanol accumulation during the late glycerol feeding period inhibited heterologous protein expression. Comparative transcriptome analysis was thus performed to compare the gene transcription profiles of Pichia pastoris KM71H in high and low ethanol concentration environments. The results showed that during the glycerol cultivation stage, 545 genes (265 up-regulated and 280 down-regulated) were differentially expressed with ethanol stress. These genes were mainly involved in protein synthesis, energy metabolism, cell cycle and peroxisome metabolism. During the methanol induction stage, 294 genes (171 up-regulated and 123 down-regulated) were differentially expressed, which were mainly related to methanol metabolism, amino acid metabolism and protein synthesis. Ethanol stress increased protein misfolding and reduced structural integrity of ribosome and mitochondria during cultivation stage, and led to the failure of endoplasmic reticulum stress removal and damaged amino acid metabolism during induction stage in Pichia pastoris.
在5 L 发酵罐中进行毕赤酵母发酵表达猪干扰素的实验，发现甘油培养末期乙醇的积累会抑制外源蛋白的表达。从转录组学角度系统分析不同浓度乙醇胁迫条件下，毕赤酵母甘油培养期和甲醇诱导期细胞的生理状态变化。研究结果表明，在甘油培养期，乙醇胁迫使得毕赤酵母细胞中的545 个基因发生了显著差异表达(265 个基因表达上调，280 个基因表达下调)，这些差异表达基因的功能主要涉及蛋白质合成、能量代谢、细胞周期和过氧化物酶代谢。乙醇胁迫增加了蛋白质错误折叠的情况，降低了核糖体和线粒体的结构完整性，使得甘油培养末期无法得到大量具有健全功能的酵母细胞。在甲醇诱导期，与甲醇代谢、蛋白质加工合成、氨基酸代谢等途径相关的294 个基因发生了显著差异表达 (171 个基因表达上调，123 个基因表达下调)，导致内质网胁迫不能被及时解除，破坏了细胞内的氨基酸正常代谢。.
Keywords: Pichia pastoris; ethanol stress; heterologous protein; transcriptome.