Quantitative Site-Specific Phosphoproteomics of <i>Trichoderma reesei</i> Signaling Pathways upon Induction of Hydrolytic Enzyme Production

The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed.丝状真菌里氏木霉（Trichoderma reesei）被广泛应用于工业生产分泌型酶类，其中包括碳水化合物活性酶（carbohydrate active enzymes），如纤维素酶与半纤维素酶。里氏木霉所生产的多数此类酶类，其合成水平受其生长所处碳源的调控，而水解酶基因的调控系统涉及多条信号通路。本研究将里氏木霉置于山梨醇（sorbitol）——一种无法诱导纤维素酶与半纤维素酶产生的碳源——中进行培养，随后将其分别暴露于槐糖（sophorose）或谷粒残留提取物（spent-grain extract），这两种物质均为高效的酶生产诱导剂。本研究基于质谱（MS）分析框架，探究了与纤维素酶、半纤维素酶合成相关的磷酸化位点特异性变化。在碳源切换后的诱导早期阶段（0、2、5和10分钟），研究人员对全蛋白质组范围内的磷酸化修饰展开了分析。实验流程依次包括分步胰蛋白酶酶解、TiO₂富集以及使用Q Exactive质谱仪进行质谱检测。本研究共鉴定并定量到1721个磷酸化位点。对数据的分析揭示了诱导后激活的复杂信号网络，该网络涉及与光介导纤维素酶诱导、渗透压调节以及碳源感知相关的组分。研究还检测到糖酵解通路中的蛋白质磷酸化变化，表明在添加槐糖后10分钟、以及添加谷粒残留提取物后最早2分钟时，葡萄糖分解代谢受到抑制。此外，研究还观察到与碳储存、细胞内运输、细胞骨架以及纤维素酶基因调控相关的因子出现了差异磷酸化修饰。