Department of Bioengineering, Nagaoka University of Technology, 1603-1, Kamitomioka, Nagaoka, 940-2188, Japan.
Advanced Course, KOSEN (National Institute of Technology), Nagaoka College, 888, Nishikatakai, Nagaoka, Niigata, 940-8532, Japan.
Curr Microbiol. 2020 Aug;77(8):1506-1517. doi: 10.1007/s00284-020-01955-y. Epub 2020 Apr 1.
The secretome of Trichoderma reesei contains a mixture of cellulases, hemicellulases, amylases, proteases, and lipases that synergistically degrade plant biomass. Trichodermapepsin (TrAsP), the most prominent protease of T. reesei, affects the stability of cellulases. Similar to cellulase production, TrAsP production also depends on carbon and nitrogen sources. Unlike the cellulase mechanism, the regulatory mechanism of TrAsP remains unknown. Therefore, this study aimed to determine the effect of the main cellulase regulator Xyr1 and nitrogen regulator Are1 on trasp regulation. Cellulase inducer Avicel and TrAsP inducer galactose were used as carbon sources. qRT-PCR analysis revealed that Xyr1 and Are1 acted as a repressor and an activator for trasp expression, respectively. Compared to Avicel, relative expression was higher in galactose. The binding motifs of Xyr1 and Are1 were located in upstream of the trasp promoter. From promoter deletant analysis using the β-glucuronidase reporter gene, the area from - 870 bp to - 670 bp was identified as the only region for positive regulation and there were both binding motifs of Xyr1 and Are1. Reporter assay of mutants confirmed functions of downregulation of Xyr1 and upregulation of Are1. Electrophoretic mobility shift assay demonstrated the binding ability of Xyr1 and Are1 to the particular binding motifs and their functionality was confirmed. Further, this study demonstrated that Cre1, Xpp1, and Pac1 downregulate trasp expression similar to that in cellulase regulation mechanism. These results demonstrate that transcriptional regulators of cellulase control trasp expression and suggest the possibility of the existence of specific protease regulators in T. reesei.
里氏木霉的分泌组包含了纤维素酶、半纤维素酶、淀粉酶、蛋白酶和脂肪酶的混合物，这些酶协同作用降解植物生物质。里氏木霉胃蛋白酶（TrAsP）是里氏木霉中最主要的蛋白酶，它影响纤维素酶的稳定性。与纤维素酶的产生类似，TrAsP 的产生也依赖于碳源和氮源。与纤维素酶机制不同，TrAsP 的调控机制尚不清楚。因此，本研究旨在确定主要纤维素酶调控因子 Xyr1 和氮调控因子 Are1 对 trasp 调控的影响。纤维素酶诱导剂微晶纤维素和 TrAsP 诱导剂半乳糖被用作碳源。qRT-PCR 分析表明，Xyr1 和 Are1 分别作为 trasp 表达的阻遏物和激活物。与微晶纤维素相比，半乳糖中的相对表达量更高。Xyr1 和 Are1 的结合基序位于 trasp 启动子的上游。通过β-葡聚糖酶报告基因的启动子缺失分析，确定了从-870 bp 到-670 bp 的区域是正调控的唯一区域，并且存在 Xyr1 和 Are1 的结合基序。突变体的报告基因实验证实了 Xyr1 的下调和 Are1 的上调功能。电泳迁移率变动分析证明了 Xyr1 和 Are1 与特定结合基序的结合能力，并且证实了它们的功能。此外，本研究表明 Cre1、Xpp1 和 Pac1 与纤维素酶调控机制类似，下调 trasp 表达。这些结果表明纤维素酶的转录调控因子控制 trasp 的表达，并暗示了里氏木霉中可能存在特定的蛋白酶调控因子。
