State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Mol Microbiol. 2021 Nov;116(5):1298-1314. doi: 10.1111/mmi.14825. Epub 2021 Oct 19.
The filamentous fungus Trichoderma reesei is widely used for industrial cellulase production. In T. reesei, cellulase gene expression is tightly controlled by a regulatory network involving multiple transcription factors. Here, we isolated a novel protein, Rce2, using a pull-down assay and mass spectrometry analysis, from a partial carbon catabolite de-repression mutant, T. reesei Rut-C30, cultured under glucose-repressing conditions. Deletion and overexpression of Rce2 in T. reesei wild-type QM6a and mutant Rut-C30 revealed that Rce2 acts as a repressor of cellulase gene expression. DNase I footprinting assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Rce2 was located in the nucleus and bound to the consensus sequences 5'-(T/A)NNNNCCG-3' and 5'-CGGNNNN(T/A)-3' in the promoters of cellulase-related genes to repress their transcription. Additionally, Rce2 antagonized Ace3 binding to the cbh1 promoter to repress its transcription. However, Rce2 was not involved in Cre1-mediated carbon catabolite repression. These results demonstrate the mechanism through which Rce2 represses the expression of cellulase genes and provide novel insights into the regulatory system of cellulases and methods that can be used for the regulation of gene expression in T. reesei.
里氏木霉是一种广泛用于工业纤维素酶生产的丝状真菌。在里氏木霉中，纤维素酶基因的表达受到涉及多个转录因子的调控网络的严格控制。在这里，我们使用下拉测定和质谱分析，从在葡萄糖抑制条件下培养的部分碳分解代谢去阻遏突变体里氏木霉 Rut-C30 中分离到一种新型蛋白 Rce2。在里氏木霉野生型 QM6a 和突变体 Rut-C30 中敲除和过表达 Rce2 表明，Rce2 作为纤维素酶基因表达的抑制剂发挥作用。DNase I 足迹测定、电泳迁移率变动分析和染色质免疫沉淀分析表明，Rce2 位于细胞核内，并与纤维素酶相关基因启动子中的保守序列 5'-(T/A)NNNNCCG-3'和 5'-CGGNNNN(T/A)-3'结合，从而抑制其转录。此外，Rce2 拮抗 Ace3 结合 cbh1 启动子以抑制其转录。然而，Rce2 不参与 Cre1 介导的碳分解代谢抑制。这些结果表明了 Rce2 抑制纤维素酶基因表达的机制，并为纤维素酶的调控系统以及在里氏木霉中调节基因表达的方法提供了新的见解。
