毕赤酵母DGKpp基因在内质网增生和促进异源蛋白表达中的分子功能研究

Pichia pastoris as one of the most widely used expression system has successfully expressed thousands of proteins, but the productivities of most target genes were very low. Using the molecular technique to modify of the Pichia host cell to improve the heterologous protein expression is becoming a newly research hotspot. Endoplasmic reticulum act as the major place to fold and modify of nascent secreted proteins, and expansion of endoplasmic reticulum was thought as a new breakthrough to enhance the heterologous protein production, however, rare study about the relationship between endoplasmic reticulum and protein expression had been conducted in Pichia. In our previous study, we analyzed the transcriptome of two high expression strains and obtained a phospholipid metabolism related gene DGKpp, and which could enhance the production of heterologous production by more than 50%. On the basis of existing work, in this study, we will use the biochemical and molecular techniques, combining the transcriptome, quantitative proteomics and fluorescent labelling method to investigate the function of DGKpp on expansion of endoplasmic reticulum and heterologous protein expression. The research results will provide us new ideas and technical support for the modification of Pichia and other host cells.

毕赤酵母作为应用最广泛的表达系统之一，已经成功实现了数千个蛋白的表达，然而大多数蛋白表达量都很低，如何改造宿主细胞，提高异源蛋白表达水平是毕赤酵母表达系统研究的热点。内质网是蛋白质折叠加工等过程的主要场所，通过分子改造促进内质网增生是增加异源蛋白表达的新突破口，但目前这方面研究还鲜见报导。本课题前期对两株蛋白高效表达毕赤酵母菌株进行了转录组分析，发现内质网脂膜代谢相关基因DGKpp显著影响蛋白表达，过表达该基因可以提高异源蛋白的表达水平50%以上。本课题将在已有工作基础上，采用生物化学与分子生物学的技术，并结合组学手段和荧光标记技术深入研究DGKpp在内质网增生与促进异源蛋白表达过程中的分子功能，为毕赤酵母乃至其它表达系统宿主细胞的改造提供新的思路和技术支撑。

毕赤酵母是蛋白表达的高效平台，然而对不同蛋白，其表达水平差异较大，此外在蛋白表达过程中会产生非折叠蛋白响应现象。为了构建高效的通用的蛋白表达平台菌株，本项目构建了一系列高产毕赤酵母工程菌株，在此基础上通过截短表达和融合表达获得了一个促表达的融合肽。进一步通过转录组学分析，发现了高效的启动子元件，同时发现高表达菌株中调节脂类代谢的关键基因DGKpp可能会影响内质网增生从而影响目标蛋白合成。在此基础上通过构建CRISPR毕赤酵母基因编辑技术，对OPI基因进行了敲除，对DGKpp基因进行了过表达。通过RT-PCR对DGKpp过表达菌株和OPI敲除菌株进行转录组水平定量分析，发现脂类代谢相关基因都呈现下调表达，说明这两个基因的确影响脂类代谢，然而和酿酒酵母相比，这两个基因呈现了相反的调控作用。通过Sec63-GFP标记的荧光显微分析，发现OPI敲除菌株的细胞核变得无规则了，而在细胞核周围的荧光标记的内质网减少。进一步通过透射电子显微镜进行超微结构分析，发现DGKpp过表达菌株和GS115相比，其液泡数目少，细胞器更完整，而OPI敲除菌株其液泡数目增加，细胞器发生凋亡，可能发生了早衰现象。本项目研究证实DGKpp和OPI基因可以调控脂类代谢相关基因，同时也影响了细胞器的完整性抑或细胞周期，为构建高效的具有鲁棒性的毕赤酵母蛋白表达平台菌株提供了很好的理论依据。