类转运蛋白受体CRT1在瑞氏木霉纤维素酶基因表达中的信号感应传递机制

Signal sensing and transduction play a key role in microbes coping with the extreme changes in the availability of nutrients including carbon sources in their living environment. Filamentous fungi Trichoderma reesei is characterized by its outstanding ability to rapidly initiate the launching of cellulase biosynthesis in the presence of cellulose substrates which otherwise are kept silent. This important biological characteristic is also of industrial relevance as T. reesei has been developed into one of the workinghorses for protein production. Unfortunately, the precise mechanism underlying the inducer recognition and the ensuing signal transduction is not clear yet. Receptors on the plasma membrane are usually the first choice to initiate the signal sensing and transducing. Understanding the detailed mechanisms by which membrane receptors are involved in regulating the induced synthesis of cellulases in T. reesei will provide deeper insights into the regualtion system for T. reesei cellulases . The present application aims to focus on our recently identified sugar transceptor Crt1, which is essential for the induced cellulase gene expression. Systematic studies will be carried out to elucidate how it recognizes the inducer and transduces the activating signal. Attempts will first be made to screen for and verify downstream effectors interacting with Crt1 using yeast screening system for membrane proteins. In combination with structural bioinformatics, interactions between Crt1 and the inducer ligand as well as the effectors would be thoroughly dissected to provide structural information for inducer recognition and pathways for signal transduction. The effects of change in Crt1-inducer interaction on the Crt1-effector interaction and on the final induced expression of cellulase genes will be analyzed systematically. The results are anticipated not only to extend the structural and functional understanding on eukaryotic transceptors, but also to help design highly efficient inducing strategy, or to genetically engineer T. reesei strains to improve their productivity of cellulases/hemicellulases, thus lowering the cost of biomass conversion.

信号感应传递在微生物应对环境营养物变化过程中发挥着重要作用。丝状真菌瑞氏木霉具有在纤维素条件下快速大量合成纤维素酶的重要生物学特性，但其精确的诱导物感应与传导机制并不清楚。细胞膜蛋白受体是起始信号感应传递的重要组分，深入阐释膜蛋白受体在纤维素酶诱导合成中的作用机理有助于全面深化对瑞氏木霉纤维素酶调控体系的认识。本项申请拟围绕前期鉴定的瑞氏木霉纤维素酶基因诱导表达必需的类转运蛋白受体Crt1，系统研究其信号感应与传递机制。利用酵母膜蛋白杂交体系筛选鉴定与Crt1相互作用的下游效应因子；解析二者相互作用的结构基础；结合Crt1与诱导物配体识别的结构特征探究，系统分析下游效应因子及其与Crt1相互作用的变化对纤维素酶靶基因转录表达的影响及影响机制，明晰Crt1信号传递的相关途径机理。研究结果将不仅拓展真核生物类转运蛋白受体的结构功能研究，还将为构建高产纤维素酶菌株或提高酶生产效率提供理论依据。

信号感应传递在微生物应对环境营养物变化过程中发挥着重要作用。丝状真菌里氏木霉具有在纤维素条件下快速大量合成纤维素酶的重要生物学特性，但其精确的诱导物感应与传导机制并不清楚。本项目围绕里氏木霉纤维素酶基因诱导表达必需的类转运蛋白受体Crt1，系统研究其信号感应与传递机制，取得了一系列研究成果：1）利用荧光检测、免疫金电镜检测及细胞分级分离等多种手段对Crt1的细胞定位进行了较为深入的研究，发现Crt1与典型的细胞膜蛋白不同，其不但在细胞膜上分布，在诱导过程中更多的定位于细胞核膜上。且在碳源转换过程中其能够从细胞膜上转移到细胞核膜上；2）对Crt1参与的信号传递途径进行了解析。对Crt1与早期内吞体标记蛋白Rab5进行共定位检测发现，Crt1在诱导的中后期能够进入内吞途径，进一步对内吞途径中end3进行缺失说明了抑制内吞过程并不改变Crt1的细胞定位，但引起了里氏木霉纤维素酶表达的缺陷；证明了Sec61复合物参与了Crt1的定位形成及功能发挥过程；通过Co-IP实验钓取了可能与Crt1相互作用的蛋白；3）通过研究Crt1与Xyr1之间的转录调控关系，发现了Xyr1对Crt1的转录是必要且分充分的，证明了Xyr1是Crt1的下游调控靶点之一；4）对Crt1的转录特点进行了研究，通过启动子单杂交筛选实验，钓取了结合在crt1启动子上蛋白，其中Tr108357及Rce1可能参与了对crt1的转录调控；5) 对Crt1的N端及C端进行突变，证明了N端及C端在Crt1的功能发挥中起关键作用，并其C端的赖氨酸可能参与了信号传递的过程；6）在里氏木霉中鉴定到了Hog1类型MAPK的上游调控途径——Sho1支路及Sln1支路，发现了Sho1支路在纤维素酶的完全表达过程中是必需的，而Sln1支路在里氏木霉纤维素酶表达过程中发挥抑制作用。上述创新型研究结果极大得丰富了里氏木霉的碳源感应机制研究。