Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China.
J Proteomics. 2013 Oct 8;91:58-72. doi: 10.1016/j.jprot.2013.06.031. Epub 2013 Jul 11.
The methylotrophic yeast Pichia pastoris is an attractive platform for a plethora of recombinant proteins. There is growing evidence that host cells producing recombinant proteins are exposed to a variety of cellular stresses resulting in the induction of the unfolded protein response (UPR) pathway. At present, there is only limited information about the cellular reactions of the host cells at the level of the proteome, especially with regard to recombinant protein secretion. Here we monitored xylanase A secretion from Bacillus halodurans C-125 (xynA) in P. pastoris, using strains containing different copy numbers of the gene encoding xylanase A and co-overexpressing the gene encoding the UPR-regulating transcription factor HAC1 by applying a quantitative proteomics approach (iTRAQ-LC-MS/MS). Many important cellular processes, including carbon metabolism, stress response and protein folding are affected in the investigated conditions. Notably, the analysis revealed that strong over-expression of xynA can efficiently improve protein production but simultaneously cause an unfolded protein burden with a subsequent induction of the UPR. This limits the further improvement of protein production levels. Remarkably, constitutive expression of the gene encoding HAC1 lessens the unfolded protein burden by attenuating protein synthesis and increasing ER protein folding efficiency which is beneficial for protein secretion.
Pichia pastoris expression systems have been successfully used for over 20years in basic research and in the biotechnology industry for the production and secretion of a wide range of recombinant proteins. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. It has become obvious that many protein products can lead to severe stress on the host cell when being over-expressed, thus limiting the potential yield. Detailed understanding of the physiological responses to such stresses gives rise to engineering of host cells that can better cope with the stress factors. Therefore, the regulatory mechanism of heterologous protein secretion by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavor in improving protein annotation. Many important cellular processes, including carbon and amino acid metabolism, stress response and protein folding are affected in the over-expression strains. This data represent a first step towards a systems wide approach to assess the response with recombinant protein induced stress in P. pastoris.
甲醇营养酵母毕赤酵母是一个极具吸引力的平台，可用于生产大量重组蛋白。越来越多的证据表明，生产重组蛋白的宿主细胞会受到各种细胞应激的影响，从而诱导未折叠蛋白反应（UPR）途径。目前，关于宿主细胞在蛋白质组水平上的细胞反应，特别是关于重组蛋白分泌，只有有限的信息。在这里，我们使用含有不同拷贝数的木聚糖酶 A 基因的毕赤酵母菌株监测木聚糖酶 A 从嗜盐杆菌 C-125（xynA）的分泌，同时通过应用定量蛋白质组学方法（iTRAQ-LC-MS/MS）共过表达编码 UPR 调节转录因子 HAC1 的基因。许多重要的细胞过程，包括碳代谢、应激反应和蛋白质折叠，在研究条件下受到影响。值得注意的是，分析表明，xynA 的过表达可以有效地提高蛋白质的产量，但同时也会导致未折叠蛋白的负担增加，从而随后诱导 UPR。这限制了蛋白质产量水平的进一步提高。值得注意的是，组成型表达编码 HAC1 的基因通过减弱蛋白质合成和增加内质网蛋白质折叠效率来减轻未折叠蛋白的负担，这有利于蛋白质的分泌。
毕赤酵母表达系统已成功使用了 20 多年，用于基础研究和生物技术工业中广泛的重组蛋白的生产和分泌。特别是，重组蛋白的分泌仍然是使用毕赤酵母的主要原因之一。很明显，当许多蛋白质产物被过表达时，会对宿主细胞造成严重的压力，从而限制了潜在的产量。详细了解对这些压力的生理反应会促使对宿主细胞进行工程改造，使其能够更好地应对应激因素。因此，通过定量质谱（MS）蛋白质组学研究异源蛋白分泌的调控机制是一个不断发展的领域，也是提高蛋白质注释的重要努力。许多重要的细胞过程，包括碳和氨基酸代谢、应激反应和蛋白质折叠，在过表达菌株中受到影响。这些数据代表了朝着全面评估毕赤酵母中重组蛋白诱导应激的响应系统方法迈出的第一步。
