The GATA-Type Transcriptional Factor Are1 Modulates the Expression of Extracellular Proteases and Cellulases in Trichoderma reesei

Abstract

Trichoderma reesei is a biotechnologically important filamentous fungus with the remarkable ability to secrete large amounts of enzymes, whose production is strongly affected by both the carbon and nitrogen sources. While the carbon metabolism regulators are extensively studied, the regulation of enzyme production by the nitrogen metabolism regulators is still poorly understood. In this study, the GATA transcription factor Are1, which is an orthologue of the Aspergillus global nitrogen regulator AREA, was identified and characterized for its functions in regulation of both protease and cellulase production in T. reesei. Deletion of the are1 gene abolished the capability to secrete proteases, and complementation of the are1 gene rescued the ability to produce proteases. Quantitative RT-PCR analysis revealed that the transcripts of protease genes apw1 and apw2 were also significantly reduced in the Δare1 strain when grown in the medium with peptone as the nitrogen source. In addition, deletion of are1 resulted in decreased cellulase production in the presence of (NH4)2SO4. Consistent with the reduction of cellulase production, the transcription levels of the major cellulase genes, including cbh1, cbh2, egl1, and egl2, were dramatically decreased in Δare1. Sequence analysis showed that all promoter regions of the tested protease and cellulase genes contain the consensus GATA elements. However, the expression levels of the major cellulase transcription activator Xyr1 and the repressor Cre1 had no significant difference between Δare1 and the parental strain QM9414, indicating that the regulatory mechanism deserves further investigation. Taken together, these results demonstrate the important role of Are1 in the regulation of protease and cellulase production in T. reesei, although these processes depend on the kind of nitrogen sources. The findings in this study contribute to the understanding of the regulation network of carbon and nitrogen sources in filamentous fungi.

Enhancement of Cellulase Production in Trichoderma reesei via Disruption of Multiple Protease Genes Identified by Comparative Secretomics

“…Here, tre123234 was found to be a serine protease at the late stage of fermentation and its gene deletion resulted in a 55% decrease in protease activity, demonstrating that tre123234 is probably the major serine protease related to cellulase degradation in T. reesei . In our previous study, the GATA transcription factor Are1 was shown to play an important role in the regulation of proteases (Qian et al, 2019). Especially, the protease tre123244 was found to be the target protease that was regulated by Are1, and there were the conserved Are1-binding sites in the promoter region of tre123244 .…”

Extracellular protease production regulated by nitrogen and carbon sources in Trichoderma reesei

“…Considering that the effects of nitrogen and carbon sources are exerted at the transcriptional level, further study need be done to illustrate the regulatory mechanism in T. reesei , which could contribute to precisely modulate protease production. Actually, it has been recently reported that the GATA‐type transcriptional factor Are1 could activate the expression of extracellular proteases in response to nitrogen limitation in T. reesei [8].…”

“…It has been reported that the expression of protease genes is prevented in the presence of preferred nitrogen sources, which is controlled by nitrogen catabolite repression (NCR) pathway [6]. Moreover, the regulation of protease production by nitrogen sources relies on GATA-type zinc finger transcription factors, such as areA in Aspergillus nidulans, are1 in Trichoderma reesei, and nit-2 in Neurospora crassa [7][8][9]. In addition, that carbon starvation leads to the increase of protease gene expression has been demonstrated in specific filamentous fungi, in which transcriptional factors creA and xprG are involved [3,10,11].…”

Glutamine involvement in nitrogen regulation of cellulase production in fungi

“…Nitrogen metabolism plays an important role in cellulase production in filamentous fungi like T. reesei , Aspergillus nidulans , and P. funiculosum . In T. reesei , deletion of the global nitrogen regulator Are1 decreased cellulase production when using (NH 4 ) 2 SO 4 as the nitrogen source, demonstrating a role of Are1 in the regulation of cellulase production [ 14 ]. In A. nidulans , cellulase production is impacted by both carbon and nitrogen source, and is under the regulation of the carbon regulators CreA, CreB, and CreC, as well as the nitrogen regulator AreA, either directly or indirectly [ 15 ].…”