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.
Highlights
Filamentous fungi have the excellent capacity to secrete a wide range of enzymes, such as cellulase and protease, involved in the degradation and recycling of complex biopolymers from organisms [1,2]
The phylogenetic analysis of AREA/NIT2-related orthologues from different fungal species showed that the T. reesei Are1 is most close to NIT2 of N. crassa, while Are2 and Are3 are most similar to AREB of A. nidulans (Figure 1A)
The predicted protein Are1 shares extensive regions of homology with the known positive nitrogen regulatory proteins AREA of A. nidulans and NIT2 of N. crassa, which are highly conserved fungal GATA-type zinc finger proteins. ∆are1 was unable to grow on medium containing skim milk as the sole nitrogen source, which was similar to the AreA mutant of A. nidulans and the NIT2 mutant of N. crassa [16,17], suggesting that Are1 is required for utilization of the non-preferred nitrogen in T. reesei
Summary
Filamentous fungi have the excellent capacity to secrete a wide range of enzymes, such as cellulase and protease, involved in the degradation and recycling of complex biopolymers from organisms [1,2]. These hydrolytic enzymes play an important role in nutrition intake for fungi by releasing carbon and nitrogen locked in insoluble macromolecules [3]. Two recent findings have suggested that among the over 400 CAZy genes in T. reesei, only a few genes appear to be directly regulated by Xyr and Cre1 [13,14] According to these reports, it can be inferred that additional regulatory factors that regulate the expression of cellulase genes should be found in the genome of T. reesei
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