Characterization of the Ca²⁺‐responsive signaling pathway in regulating the expression and secretion of cellulases in Trichoderma reesei Rut‐C30

Abstract

Calcium signaling plays pivotal roles in the hyphal growth, conidiation, and osmosis sensitivity of fungi through the Ca(2+) /calmodulin-calcineurin-dependent pathway. This study found that an appropriate extracellular Ca(2+) 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 reticulum-chaperones after Ca(2+) 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 Ca(2+) . A DNase I footprinting assay identified its putative binding consensus site (5'-[T/G]GGCG-3' or 5'-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 Ca(2+) involved in upregulation of cellulose degradation-related genes, reflecting progress in the study of Ca(2+) signaling in filamentous fungi. This study also provides insight that will facilitate further improvement of (hemi-)cellulase production by T. reesei.

CRZ1 regulator and calcium cooperatively modulate holocellulases gene expression in Trichoderma reesei QM6a

“…Once the role of Ca 2+ is clearly related to the CRZ1 regulatory role in T. reesei [26], we decided to investigate whether calcium transporters were target of modulation by CRZ1. After both SCB and cellulose growth, we observed that the deletion of crz1 elicited a loss in transcription of these genes in the presence of SCB (Figure 6B-C) while it boosted the expression of the same genes after cellulose induction ( Additional File 8: Figure S1 B-D ).…”

“…However, the influence of several stress conditions, such as the effect of ions concentration exposure in transcription, still remains poorly understood in the industrial workhorse T. reesei . Despite not completely elucidated, recent studies have demonstrated the balance mediated by signaling pathways to keep physiological homeostasis when T. reesei is exposed to Ca 2+ stress through Calmodulin-Calcineurin downstream activation steps [26].…”

“…In T. reesei , the role of Ca 2+ -CaM/CN/CRZ1 pathway is poorly studied and established in comparison to other fungal species. Yet, it is already known that Ca 2+ signaling pathway may exert effects on the modulation of holocellulases gene expression in the industrial strain of T. reesei RUT-C30 [26]. Therefore, Chen et al [26] showed that the regulation of relevant industrial genes, such as cbh1, eg1 and xyr1 genes were negatively modulated in the absence of CRZ1 when calcium was added to a cellulose-supplemented medium.…”

The Hybrid Strategy of Thermoactinospora rubra YIM 77501T for Utilizing Cellulose as a Carbon Source at Different Temperatures

“…The results of these regulations show that increased enzyme gene expression and up-regulated special enzyme genes at lower or higher temperatures may maintain biological functions of enzymes economically and efficiently for the microorganism at different temperatures. In addition to temperature, microorganisms can also change the expression of cellulase genes on other environmental factors, such as substrate, pH, and ions to utilize carbon source (Lynd et al, 2002; López-Contreras et al, 2004; Deng and Zhang, 2015; Hakkinen et al, 2015; Chen et al, 2016). The expression level of different cellulase genes can be changed by various substrates (López-Contreras et al, 2004).…”

“…Completely digestion of cellulose involves multiple cellulases, like endo-1, 4-β-glucanases (EC 3.2.1.4), cellobiohydrolases or exo-1, 4-β-glucanases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21; Lynd et al, 2002; Gilbert et al, 2008). For microorganisms, secretion of cellulases may be effected by substances, pH, ion, and temperature (Lynd et al, 2002; López-Contreras et al, 2004; Sohail et al, 2009; Deng and Zhang, 2015; Hakkinen et al, 2015; Chen et al, 2016). Temperature is one of the most important environmental factors (Lin et al, 2016), and it may impact utilization of cellulose as a carbon source from two aspects: (1) it could affect the activity and stability of cellulases, and (2) it might be a crucial factor for inducing some cellulases under special temperatures.…”