毕赤酵母转录因子Fhl1p功能鉴定及其促进外源蛋白表达的机理

Pichia pastoris has been widely used as the cellular factory for the efficient expression of heterogeneous proteins. The effects of the transcription, post-translational modification, transportation and secretion on the expression of heterogeneous proteins have been surveyed deeply. However, little research analyzes the relationship between translation and heterogeneous proteins expression. Ribosome is the site of protein synthesis and the translation efficiency of target mRNA significantly affects the final production. The sharp increase of target mRNA abundance results in the translation stress and limits the protein production. Although overexpression of the transcription factor Fhl1p enhances the production of phytase, the mechanism remains unclear. Combining RNAi，function recovery and CHIP-Seq, we aim to identify the function of Fhl1p and the binding sites. The activation of rRNA synthesis and ribosomal protein genes transcription is also analyzed through RNA-Seq. Besides, the rate of protein synthesis is quantified by Ribosome profiling. We analyze the regulation of ribosome biosynthesis and translation efficiency and further investigate the mechanism of enhancement of heterogeneous protein expression mediated by Fhl1p, offering theory basis for rationally controlling the ribosome biosynthesis and translation efficiency in Pichia pastoris.

毕赤酵母是高效分泌表达外源蛋白的细胞工厂，目的基因转录、翻译后修饰、转运及分泌过程对外源蛋白表达的影响已有深入的研究。然而鲜有文献分析毕赤酵母翻译过程与外源蛋白表达的关系。核糖体是蛋白质的合成场所，目的基因mRNA在核糖体上的翻译效率显著影响其最终产量。前期研究发现目的基因mRNA的丰度增加到一定程度会产生翻译压力而限制产量的提高。过表达核糖体蛋白基因的转录因子Fhl1p可促进植酸酶的表达，然而其机理不明确。本课题拟通过RNAi技术、功能异源回补及CHIP-Seq技术对Fhl1p进行功能鉴定并分析其结合位点；采用RNA-Seq技术分析Fhl1p对rRNA合成及核糖体蛋白基因转录的激活作用；结合核糖体图谱技术在翻译水平对毕赤酵母蛋白合成速率进行定量，分析Fhl1p对核糖体生物合成及蛋白翻译过程的调控，探讨其促进外源蛋白表达的机理；为毕赤酵母核糖体生物合成及蛋白翻译效率的理性调控提供理论依据。

毕赤酵母是高效分泌表达外源蛋白的细胞工厂，实现外源蛋白高效表达涉及目的基因的转录、目的基因mRNA 的翻译、翻译后修饰、目的蛋白转运及分泌等过程。本项目主要针对FHL1基因功能及其促进外源蛋白表达的机理进行研究，具有研究内容为：FHL1基因敲除和过表达、免疫共沉淀分析转录因子Fhl1p的结合位点、转录组测序研究Fhl1p 对rRNA 合成及核糖体蛋白基因转录的激活效应、核糖体图谱分析Fhl1p对翻译过程的调控。FHL1基因敲除后，菌株生长急剧下降，rRNA合成受阻，但其对于雷帕霉素敏感性增加，而抗逆性有所提升。FHL1基因过表达对菌株生长无明显影响，外源蛋白果胶酶的表达量提高约60%。通过激光共聚焦显微镜观察发现Fhl1p定位于细胞核。RNA-Seq分析结果显示rRNA加工相关基因及核糖体蛋白基因显著上调。核糖体谱图分析进一步表明过表达Fhl1p能明显促进翻译活性的上升。本项目在鉴定FHL1基因功能的基础上，发现毕赤酵母转录因子Fhl1p过表达可促进核糖体生物合成及蛋白翻译过程，提升外源蛋白产量；从而为在翻译速率层面进行调控进而提高外源蛋白表达提供了新的研究策略。