Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions.
Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions.
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DOI：
10.1186/1471-2164-8-179
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发表时间：
2007-06-19
期刊：
影响因子：
4.4
通讯作者：
Mattanovich D
中科院分区：
文献类型：
--
作者：
Gasser B;Maurer M;Rautio J;Sauer M;Bhattacharyya A;Saloheimo M;Penttilä M;Mattanovich D
很明显，宿主细胞通过各种代谢和内在应激（例如未折叠蛋白反应（UPR）途径）对重组蛋白产生做出反应。此外，生长温度等环境条件可能对细胞生理学和比生产力产生强烈影响。然而，关于宿主细胞在基因组水平上的分子反应的信息很少，特别是在重组蛋白分泌方面。我们首次监测了常见生产宿主毕赤酵母中一部分标记基因的转录调控，以深入了解蛋白质生产条件下细胞的一般生理状态，主要关注分泌应激相关基因。利用 UPR 激活转录因子 Hac1p 的过表达来鉴定毕赤酵母中的 UPR 靶基因，并将响应与已知的酿酒酵母响应进行比较。大多数折叠/分泌相关基因在两种酵母中显示出相似的调节模式，而与一般应激反应相关的基因则受到不同的调节。抗体 Fab 片段的分泌导致毕赤酵母中 UPR 靶基因的诱导，但程度与 Hac1p 过量产生的程度不同。酿酒酵母蛋白二硫键异构酶 (PDI1) 的过度表达可将 Fab 分泌率提高 1.9 倍，但不能缓解 UPR 应激。将培养温度从 25°C 降低至 20°C 导致恒化培养中特定产物分泌率增加 1.4 倍，尽管产物基因（Fab 轻链和重链）的转录水平在较低温度下显着降低。折叠相关基因的子集似乎在降低的温度下下调，而与内质网降解和分泌运输相关的成分的转录则增强。使用转录分析方法 TRAC 监测巴斯德毕赤酵母中标记基因的基因组调控，揭示了与酿酒酵母相比反应的相似性和差异。因此，我们的结果强调了在实际生产条件下分析个体宿主的重要性，而不是从模型生物中得出结论。培养温度对比生产力有显着影响，不仅与热力学效应有关，而且强烈影响特定基因的调控。
It has become evident that host cells react to recombinant protein production with a variety of metabolic and intrinsic stresses such as the unfolded protein response (UPR) pathway. Additionally, environmental conditions such as growth temperature may have a strong impact on cell physiology and specific productivity. However, there is little information about the molecular reactions of the host cells on a genomic level, especially in context to recombinant protein secretion. For the first time, we monitored transcriptional regulation of a subset of marker genes in the common production host Pichia pastoris to gain insights into the general physiological status of the cells under protein production conditions, with the main focus on secretion stress related genes. Overexpression of the UPR activating transcription factor Hac1p was employed to identify UPR target genes in P. pastoris and the responses were compared to those known for Saccharomyces cerevisiae. Most of the folding/secretion related genes showed similar regulation patterns in both yeasts, whereas genes associated with the general stress response were differentially regulated. Secretion of an antibody Fab fragment led to induction of UPR target genes in P. pastoris, however not to the same magnitude as Hac1p overproduction. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1) enhances Fab secretion rates 1.9 fold, but did not relief UPR stress. Reduction of cultivation temperature from 25°C to 20°C led to a 1.4-fold increase of specific product secretion rate in chemostat cultivations, although the transcriptional levels of the product genes (Fab light and heavy chain) were significantly reduced at the lower temperature. A subset of folding related genes appeared to be down-regulated at the reduced temperature, whereas transcription of components of the ER associated degradation and the secretory transport was enhanced. Monitoring of genomic regulation of marker genes with the transcriptional profiling method TRAC in P. pastoris revealed similarities and discrepancies of the responses compared to S. cerevisiae. Thus our results emphasize the importance to analyse the individual hosts under real production conditions instead of drawing conclusions from model organisms. Cultivation temperature has a significant influence on specific productivity that cannot be related just to thermodynamic effects, but strongly impacts the regulation of specific genes.
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影响因子：
9.8
作者：
Patil CK;Li H;Walter P
通讯作者：
Walter P
影响因子：
4.1
作者：
Hohenblum, H;Borth, N;Mattanovich, D
通讯作者：
Mattanovich, D
影响因子：
4.8
作者：
Brodsky, JL;Werner, ED;McCracken, AA
通讯作者：
McCracken, AA
影响因子：
4.1
作者：
Jahic, M;Gustavsson, M;Enfors, SO
通讯作者：
Enfors, SO
影响因子：
6.4
作者：
Maurer, Michael;Kuehleitner, Manfred;Mattanovich, Diethard
通讯作者：
Mattanovich, Diethard