里氏木霉中诱导纤维素酶表达的碳源信号转导途径及其机制

Trichoderma reesei is capable of synthesizing a massive amount of extracellular proteins that are primarily composed of cellulases, therefore making it one of the best industrial strains for the conversion of lignocellulosics and one of the best platform organisms for the heterologous production of proteins. The synthesis of cellulases in T. reesei is induced by carbon signals, but the current understanding on this process is limited to the types of extracellular inducing signals and the nuclear transcription factors that regulate the expression of cellulase genes, while relatively less is known about how the carbon source signals are transferred from outside the cell to inside the nucleus for the induction of cellulases. In our previous work, our group has discovered that the Tmk3-containing MAPK signal transduction pathway is involved in the transduction of inducing carbon source signals. In this project, we plan to perform further investigations based on this discovery, to identify the composition and mechanisms of signal transduction pathways that transfer extracellular carbon source signals to the Tmk3 pathway, to identify the downstream effector and signal transduction mechanisms of the Tmk3 pathway, and to understand the mechanism by which carbon signal enters the nucleus for the induction of cellulase genes. These investigations will solve the problem of a fragmented and unsystematic understanding on how the cellulase-inducing carbon source signals regulate cellulase synthesis, elucidate the composition and mechanisms of the cellulase-inducing carbon source signal transduction pathways, and provide theoretical guidance for further engineering T. reesei for higher production of cellulases and heterologous proteins.

里氏木霉能够合成大量的以纤维素酶为主的胞外蛋白，因而成为木质纤维素降解转化的最佳工业菌株和异源蛋白生产的最佳底盘生物之一。里氏木霉中纤维素酶合成受碳源信号的诱导，但是人们对该过程的了解限于胞外诱导信号的种类和核内调控纤维素酶基因表达的转录因子，而碳源信号如何从胞外传至核内并诱导纤维素酶的表达尚未有深入研究。在前期工作中，本项目组发现包含有Tmk3的MAPK信号转导途径参与了诱导碳源信号的传递。在本项目中，我们拟在该发现基础上深入研究，揭示传递外界碳源信号至Tmk3途径的信号转导途径及其机制，Tmk3途径下游的效应器及信号转导机制，以及碳源信号进入细胞核并诱导纤维素酶基因表达的机制。上述研究将突破当前人们对碳源诱导纤维素酶表达机制的认识不完整、不系统的障碍，完整地阐明诱导纤维素酶表达的碳源信号转导途径及其机制，并为改造里氏木霉以提高纤维素酶和异源蛋白的生产提供理论指导。

里氏木霉能够产生高水平的纤维素酶，能够对秸秆等木质纤维素进行降解，所产生的糖类物质可以作为平台化合物进一步转化成为其他化学品。因此，纤维素酶的合成及其活性是木质纤维素利用技术的核心。项目组发现了里氏木霉中Tmk3途径能够介导纤维素酶的诱导，并在本项目中对里氏木霉中的Tmk3途径诱导纤维素酶的机制进行了研究。项目组构建了Tmk3组成型表达菌株，鉴定了Tmk3的正确表达和表达的时间特征，并对Tmk3信号转导的上下游因子进行了鉴定。研究表明，Tmk3同蛋白翻译相关因子、代谢相关蛋白、核糖体相关蛋白和部分信号转导相关蛋白之间具有相互作用，从而表明Tmk3通过磷酸化核糖体蛋白S6来降低核糖体活性，并通过磷酸化翻译起始因子5A和翻译延长因子来抑制全局蛋白的合成；通过磷酸化糖酵解、三羧酸循环相关蛋白促进葡萄糖代谢并促进葡萄糖利用；同时发现Tmk3可以接受来自Ras GTPase的信号以调控纤维素酶合成。项目组同时对里氏木霉纤维素酶CBHI的糖基化进行了深入的研究，发现了里氏木霉CBHI催化域的O-糖基化广泛存在，且在N45和N64位上具有之前未发现过的富甘露糖形式的N-糖基化。通过进一步结构分析表明，纤维素酶CBHI的糖基化和其同底物的结合密切相关，因此纤维素酶的糖基化可能通过影响和底物的结合影响纤维素酶的活性。上述研究工作为进一步遗传改造里氏木霉以提高纤维素酶的合成水平或者活性奠定了理论基础，也对高效利用木质纤维素提供了理论依据。