Pichia pastoris (syn. Komagataella phaffii) is an important yeast system for heterologous protein expression. A robust P. pastoris mutant with oxidative and thermal stress cross-tolerance was acquired in our previous study. The robust mutant can express a 2.5-fold higher level of lipase than its wild type (WT) under methanol induction conditions. In this study, we found that the robust mutant not only can express a high level of lipase, but also can express a high level of other heterogeneous proteins (e.g., green fluorescence protein) under methanol induction conditions. Additionally, the intracellular reactive oxygen species (ROS) levels in the robust mutant were lower than that in the WT under methanol induction conditions. To figure out the difference of cellular response to methanol between the WT and the robust mutant, RNA-seq was detected and compared. The results of RNA-seq showed that the expression levels of genes related to antioxidant, MAPK pathway, ergosterol synthesis pathway, transcription factors, and the peroxisome pathway were upregulated in the robust mutant compared to the WT. The upregulation of these key pathways can improve the oxidative stress tolerance of strains and efficiently eliminate cellular ROS. Hence, we inferred that the high heterologous protein expression efficiency in the robust mutant may be due to its enhanced oxidative stress tolerance. Promisingly, we have indeed increased the expression level of lipase up to 1.6-fold by overexpressing antioxidant genes in P. pastoris. This study demonstrated the impact of methanol on the expression levels of genes in P. pastoris and emphasized the contribution of oxidative stress tolerance on heterologous protein expression in P. pastoris. Our results shed light on the understanding of protein expression mechanism in P. pastoris and provided an idea for the rational construction of robust yeast with high expression ability.

中文翻译：

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氧化应激耐受性有助于毕赤酵母中的异源蛋白质生产

Pichia pastoris (syn. Komagataella phaffii) 是用于异源蛋白质表达的重要酵母系统。在我们之前的研究中获得了具有氧化和热应激交叉耐受性的强大 P. pastoris 突变体。在甲醇诱导条件下，稳健的突变体可以表达比其野生型 (WT) 高 2.5 倍的脂肪酶水平。在这项研究中，我们发现稳健的突变体不仅可以高水平表达脂肪酶，而且在甲醇诱导条件下也可以高水平表达其他异质蛋白（例如绿色荧光蛋白）。此外，在甲醇诱导条件下，稳健突变体的细胞内活性氧 (ROS) 水平低于 WT。为了找出 WT 和稳健突变体之间细胞对甲醇反应的差异，检测和比较 RNA-seq。RNA-seq结果表明，与WT相比，健壮突变体中与抗氧化剂、MAPK途径、麦角甾醇合成途径、转录因子和过氧化物酶体途径相关的基因表达水平上调。这些关键通路的上调可以提高菌株的氧化应激耐受性并有效消除细胞内的 ROS。因此，我们推断稳健突变体中的高异源蛋白表达效率可能是由于其增强的氧化应激耐受性。令人鼓舞的是，通过在毕赤酵母中过表达抗氧化基因，我们确实将脂肪酶的表达水平提高了 1.6 倍。该研究证明了甲醇对 P. 基因表达水平的影响。Pastoris 并强调了氧化应激耐受性对 P. pastoris 中异源蛋白表达的贡献。我们的研究结果阐明了对毕赤酵母蛋白质表达机制的理解，并为合理构建具有高表达能力的健壮酵母提供了思路。