黑曲霉蛋白分泌途径中脂类压力诱导未折叠蛋白应答机制的研究

Aspergillus niger has received increasing attention as a host for Food enzymes production due to its capacity to secrete large amounts of proteins. But production levels of non-fungal recombinant proteins are generally lower than those of homologous proteins with likely bottlenecks at protein quality control mechanisms in the endoplasmic reticulum(ER) of the secretory pathway. The expression of heterologous proteins and the chemicals stimulation result in the accumulation of unfolded proteins to induce the unfolded protein response (UPR). And UPR can detects misfolded proteins in the ER and activates transcription of genes that facilitate proper protein folding. Recent, UPR also upregulates key phospholipid synthesis genes, thus providing a potential mechanism to respond to lipid stress. And the proteins secretory pathway consists of organelles with membranes such as ER, Golgi apparatus and vesicles. It is the important roles in the functions of the protein secretory pathway that the equilibrium are controlled between the lipid and the protein homeostasis. This project want to answer the following scientific questions the UPR mechanism induced by the lipid stress of ER and its regulation mechanism to control the lipid and the protein homeostasis in the filamentous fungi A. niger. Based on our studies on the transcription regulation of phospholipid synthesis in A. niger, we will build the function gene defective strains to change the equilibrium of lipids to induce UPR independently. Furthermore, we want to explore the lipid-dependent regulation of UPR to control the equilibrium between the lipid and the protein homeostasis with the improvement of the expression of heterologous food enzymes.

黑曲霉高效分泌蛋白，是生产食品酶的理想宿主菌。但非真菌来源的食品酶产量却相当低，其瓶颈在于分泌途径内质网中蛋白的折叠和修饰。异源蛋白表达、化学物刺激等导致分泌途径中非折叠蛋白积累而形成内质网压力，并诱导UPR信号机制，激活与蛋白分泌途径相关的基因表达，加强细胞蛋白折叠能力。近年的研究显示UPR信号机制具有应答脂类代谢信号调控的功能，蛋白分泌途径由有膜细胞器如内质网、高尔基体、囊泡等组成，内质网是脂类合成的主要场所，脂类动态平衡与蛋白质动态平衡控制对蛋白分泌途径有着同样的重要作用。本项目针对黑曲霉中脂类成分平衡失调而产生脂类压力，并单独诱导UPR信号机制及其如何调控脂类合成和蛋白质折叠质量的机制等科学问题而提出，本项目基于完善黑曲霉磷脂合成途径调控机制的基础上，构建磷脂合成功能缺陷株，改变脂类成分平衡形成脂类压力，研究其单独诱导UPR机制以及对蛋白分泌途径的调控机制，并促进异源食品酶生产。

内质网是蛋白质折叠、脂类合成的主要场所，脂类动态平衡与蛋白质动态平衡控制对蛋白分泌途径有着同样的重要作用。本项目针对黑曲霉中脂类成分平衡失调而产生脂类压力，诱导UPR信号机制及其如何调控脂类合成和蛋白质折叠质量的机制等科学问题而提出。.项目首先建立黑曲霉CRISPR基因组编辑系统、单碱基编辑系统以及首次适合丝状真菌的ATAC-seq技术平台，为本项目在基因组水平研究转录因子的调控机制提供了研究工具。本项目按计划（1）鉴定解析黑曲霉中调控磷脂合成的碱性螺旋-转角-螺旋家族转录因子ino2，解析ino2的调控作用机制，即与自身结合形成同源二聚体，调控具有UASINO顺式成分启动子靶向基因，从而调控磷脂合成相关基因的转录，ino2基因的缺失会导致黑曲霉磷脂酰肌醇PI的失衡。基于ino2缺陷型菌株Δino2、外源添加肌醇菌株INO以及野生菌WT转录组差异分析在基因组水平阐明黑曲霉磷脂合成途径转录调控机制；（2）构建黑曲霉磷脂酰乙醇胺PE连续合成磷脂酰胆碱PC途径的关键基因cho2和opi3以及磷脂酸PA合成重要基因dgk1的缺陷株，改变脂类成分平衡形成脂类压力，进而导致细胞内质网蛋白稳态失衡，诱导UPR机制及其对蛋白分泌途径的调控机制，阐明了黑曲霉中磷脂稳态失衡以及内质网稳态失衡的互相作用；（3）建立黑曲霉宿主异源蛋白表达研究模型，比较研究了蛋白分泌途径中UPR信号通路、ERAD途径、囊泡运输途径以及本项目研究脂类压力信号机制的关键基因对异源蛋白在黑曲霉宿主中表达的影响，过表达转录因子ino2和磷脂酰胆碱（PC）合成途径关键基因opi3的可促进异源蛋白Monellin的表达水平。基于本项目建立的黑曲霉表达系统已应用于葡萄糖氧化酶、氨肽酶、核酸酶和海藻糖酶等食品酶制剂的表达。