Characterization of the Ca2+-responsive signaling pathway in regulating the expression and secretion of cellulases in Trichoderma reesei Rut-C30
Characterization of the Ca2+-responsive signaling pathway in regulating the expression and secretion of cellulases in Trichoderma reesei Rut-C30
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里氏木霉 Rut-C30 中 Ca2 响应信号通路调节纤维素酶表达和分泌的表征
DOI：
10.1111/mmi.13334
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发表时间：
2016-05-01
影响因子：
3.6
通讯作者：
Zhou, Zhihua
中科院分区：
文献类型：
--
作者：
Chen, Ling;Zou, Gen;Zhou, Zhihua
钙信号通过钙/钙调素-钙调神经磷酸酶依赖的途径在真菌的菌丝生长、分生孢子形成和渗透敏感性中起关键作用。本研究发现，适量的胞外钙离子浓度显著促进了里氏木霉RUT-C30纤维素酶高产菌株的菌丝生长、纤维素酶产量和总蛋白分泌。转录分析表明，Ca~(2+)加入后，不仅纤维素酶和转录激活因子XYR1的编码基因上调，内质网伴侣蛋白的编码基因也上调。CRZ1是里氏锥虫钙调神经磷酸酶反应的锌指转录因子1，通过基因中断进一步鉴定其功能。凝胶迁移率改变分析(EMSA)结合染色质免疫沉淀(CHIP)证实CRZ1可以直接结合到编码区的上游区域。DNase I足迹分析确定了其假定的结合共识位置(5‘-[T/G]GGCG-3’或50-GGGC[G/T]-3‘)。EMSA证实CRZ1与另一个转录因子ACE1竞争了xyr1启动子的位置。这些结果揭示了钙调神经磷酸酶下游响应细胞外钙离子参与纤维素降解相关基因上调的可能信号通路，反映了丝状真菌钙信号转导的研究进展。这项研究也为进一步提高里氏木霉(T.reesei)的(半)纤维素酶产量提供了依据。
Calcium signaling plays pivotal roles in the hyphal growth, conidiation, and osmosis sensitivity of fungi through the Ca2+/calmodulin-calcineurin-dependent pathway. This study found that an appropriate extracellular Ca2+ concentration markedly stimulated the hyphal growth, cellulase production, and total protein secretion of the cellulase hyper-producing strain, Trichoderma reesei Rut-C30. Transcription analysis revealed upregulation of not only encoding genes of cellulases and the transcriptional activator XYR1 but also several genes encoding endoplasmic reticulumchaperones after Ca2+ addition. The function of CRZ1, T. reesei calcineurin-responsive zinc finger transcription factor 1, was further characterized by gene disruption. Electrophoretic mobility shift assays (EMSAs) in combination with chromatin immunoprecipitation (ChIP) verified that CRZ1 could bind directly to the upstream regions of xyr1 and cbh1 (cellobiohydrolase I-encoding gene) in response to Ca2+. A DNase I foot-printing assay identified its putative binding consensus site (5'-[T/G] GGCG-3' or 50-GGGC[G/T]-3'). EMSAs confirmed that CRZ1 competed for occupancy of the xyr1 promoter with another transcription factor, ACE1. These results revealed putative signaling pathways downstream of calcineurin in response to extracellular Ca2+ involved in upregulation of cellulose degradation-related genes, reflecting progress in the study of Ca2+ signaling in filamentous fungi. This study also provides insight that will facilitate further improvement of (hemi-)cellulase production by T. reesei.