Rab GTPase 在里氏木霉早期内吞体介导的纤维素酶高效分泌路径中的作用机制研究

解析里氏木霉高效分泌纤维素酶的分子机制是里氏木霉重组蛋白细胞工厂构建的关键理论基础。Rab GTPase调控的分泌囊泡的发生、运输与融合被认为是蛋白高效分泌的重要因素。里氏木霉早期内吞体介导的分泌路径中Rab GTPase调控机制及其在纤维素酶高效分泌中的功能还不清楚。本项目拟围绕早期内吞体介导的分泌路径中的重要调控因子Rab14、Rab5和Rab4，首先通过敲除和活性突变体构建及荧光共定位分析，研究它们在纤维素酶主要组分CBH1高效分泌中的功能；其次系统解析Rab14、Rab5与Rab4的级联调控模式，阐明囊泡运输方向的调控机制；同时利用酵母双杂交、GST pull-down和基于蛋白亲近的生物素标记等技术全面鉴定Rab GTPase的效应因子并对它们进行功能表征；最后利用基于RUSH的脉冲示踪技术研究CBH1在关键分泌节点的运输过程，阐明早期内吞体介导的CBH1高效分泌的分子机制。

At present, the market size of biopharmaceutical proteins and industrial enzymes in the world reaches hundreds of billions and tens of billions of dollars, respectively. Continuously increasing the protein production capacity of microbial cell factories has always been the focus of biotechnology industry. Filamentous fungi are known for their high capacity of degrading enzyme production, representing novel potentials as microbial cell factory for recombinant protein production. Dissecting the molecular mechanism of the secretory pathway of filamentous fungi is expected to find new functional gene elements for the rational design and construction of recombinant protein cell factories. .Trichoderma reesei is extraordinary for its capability of secreting large amounts of cellulase enzymes, especially cellobiohydrolase CBH1. However, the molecular machineries of CBH1 secretion in T. reesei remain largely unknown, limiting its application for protein production in industry. Based on our preliminary research and literature reports, we pose a hypothesis that early endosome is an important node for efficient secretion of CBH1 in T. reesei. The newly synthesized CBH1 proteins are transported in secretory vesicle from trans-Golgi network to early endosome and then to plasma membrane for secretion. This pathway is probably regulated by Rab GTPases, namely Rab14, Rab5 and Rab4, which are the master regulators of vesicular trafficking in cells. Our present study aim to clarify that CBH1 efficient secretion is achieved by early endosome mediated secretion pathway as proposed and to elucidate the regulatory mechanism of its vesicle trafficking. Firstly, Rab GTPases and their function in CBH1 secretion will be characterized by gene knockout, active mutant construction and fluorescence co-localization analysis. Secondly, the interaction of Rab14 and Rab5 GEF as well as Rab5 and Rab4 GEF will be evaluated by yeast two-hybrid assay and GST pull-down to study the cascade regulation of Rab GTPase and prove the proposed direction of vesicle transport. Thirdly, effectors of Rab GTPase will be systematically identified using yeast two-hybrid assay, GST pull-down and proximity-dependent biotinylation labeling technique and their functions in CBH1 secretion will be analyzed. Finally, the transport kinetics of CBH1 in this secretory pathway will be investigated by RUSH (retention using selective hooks) pulse chase technique. This project will elucidate the early endosome mediated CBH1 efficient secretion pathway and reveal its molecular mechanism of vesicle trafficking regulation, thus shedding novel lights on further enhancing cellulase production and increasing heterologous protein expression at the secretory level through genetic engineering of T. reesei.