Resina David, Bollók Mónika, Khatri Narendar K, Valero Francisco, Neubauer Peter, Ferrer Pau
Departament d'Enginyeria Química, Escola Tècnica Superior d'Enginyeria, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain.
Microb Cell Fact. 2007 Jul 16;6:21. doi: 10.1186/1475-2859-6-21.
The analysis of transcriptional levels of the genes involved in protein synthesis and secretion is a key factor to understand the host organism's responses to recombinant protein production, as well as their interaction with the cultivation conditions. Novel techniques such as the sandwich hybridization allow monitoring quantitatively the dynamic changes of specific RNAs. In this study, the transcriptional levels of some genes related to the unfolded protein response (UPR) and central metabolism of Pichia pastoris were analysed during batch and fed-batch cultivations using an X-33-derived strain expressing a Rhizopus oryzae lipase under control of the formaldehyde dehydrogenase promoter (FLD1), namely the alcohol oxidase gene AOX1, the formaldehyde dehydrogenase FLD1, the protein disulfide isomerase PDI, the KAR2 gene coding for the BiP chaperone, the 26S rRNA and the R. oryzae lipase gene ROL.
The transcriptional levels of the selected set of genes were first analysed in P. pastoris cells growing in shake flask cultures containing different carbon and nitrogen sources combinations, glycerol + ammonium, methanol + methylamine and sorbitol + methylamine. The transcriptional levels of the AOX1 and FLD1 genes were coherent with the known regulatory mechanism of C1 substrates in P. pastoris, whereas ROL induction lead to the up-regulation of KAR2 and PDI transcriptional levels, thus suggesting that ROL overexpression triggers the UPR. This was further confirmed in fed-batch cultivations performed at different growth rates. Transcriptional levels of the analysed set of genes were generally higher at higher growth rates. Nevertheless, when ROL was overexpressed in a strain having the UPR constitutively activated, significantly lower relative induction levels of these marker genes were detected.
The bead-based sandwich hybridization assay has shown its potential as a reliable instrument for quantification of specific mRNA species in P. pastoris cells grown in fed-batch cultures. As a proof-of-principle, the influence of the carbon and nitrogen sources, the specific growth rate, as well as the ROL overexpression on the transcriptional levels of a reduced set of bioprocess-relevant genes has been quantitatively studied, revealing that ROL overexpression and secretion seems to trigger the UPR in P. pastoris, resulting in a physiological bottleneck for the production process.
分析参与蛋白质合成和分泌的基因的转录水平是理解宿主生物体对重组蛋白生产的反应以及它们与培养条件相互作用的关键因素。诸如夹心杂交等新技术能够定量监测特定RNA的动态变化。在本研究中，使用在甲醛脱氢酶启动子（FLD1）控制下表达米根霉脂肪酶的X-33衍生菌株，在分批和补料分批培养过程中分析了一些与毕赤酵母未折叠蛋白反应（UPR）和中心代谢相关的基因的转录水平，即醇氧化酶基因AOX1、甲醛脱氢酶FLD1、蛋白二硫键异构酶PDI、编码BiP伴侣蛋白的KAR2基因、26S rRNA和米根霉脂肪酶基因ROL。
首先在含有不同碳源和氮源组合（甘油+铵、甲醇+甲胺和山梨醇+甲胺）的摇瓶培养物中生长的毕赤酵母细胞中分析所选基因集的转录水平。AOX1和FLD1基因的转录水平与毕赤酵母中已知的C1底物调控机制一致，而ROL的诱导导致KAR2和PDI转录水平上调，因此表明ROL过表达触发了UPR。这在以不同生长速率进行的补料分批培养中得到了进一步证实。在较高生长速率下，所分析基因集的转录水平通常较高。然而，当在UPR组成型激活的菌株中过表达ROL时，检测到这些标记基因的相对诱导水平显著降低。
基于磁珠的夹心杂交分析已显示出其作为可靠工具用于定量补料分批培养中生长的毕赤酵母细胞中特定mRNA种类的潜力。作为原理验证，已定量研究了碳源和氮源、比生长速率以及ROL过表达对一组减少的生物过程相关基因转录水平的影响，揭示ROL过表达和分泌似乎在毕赤酵母中触发了UPR，导致生产过程出现生理瓶颈。
