Deletion of pH Regulator pac-3 Affects Cellulase and Xylanase Activity during Sugarcane Bagasse Degradation by Neurospora crassa.

Microorganisms play a vital role in bioethanol production whose usage as fuel energy is increasing worldwide. The filamentous fungus Neurospora crassa synthesize and secrete the major enzymes involved in plant cell wall deconstruction. The production of cellulases and hemicellulases is known to be affected by the environmental pH; however, the regulatory mechanisms of this process are still poorly understood. In this study, we investigated the role of the pH regulator PAC-3 in N. crassa during their growth on sugarcane bagasse at different pH conditions. Our data indicate that secretion of cellulolytic enzymes is reduced in the mutant Δpac-3 at alkaline pH, whereas xylanases are positively regulated by PAC-3 in acidic (pH 5.0), neutral (pH 7.0), and alkaline (pH 10.0) medium. Gene expression profiles, evaluated by real-time qPCR, revealed that genes encoding cellulases and hemicellulases are also subject to PAC-3 control. Moreover, deletion of pac-3 affects the expression of transcription factor-encoding genes. Together, the results suggest that the regulation of holocellulase genes by PAC-3 can occur as directly as in indirect manner. Our study helps improve the understanding of holocellulolytic performance in response to PAC-3 and should thereby contribute to the better use of N. crassa in the biotechnology industry.

Understanding the Role of the Master Regulator XYR1 in Trichoderma reesei by Global Transcriptional Analysis.

We defined the role of the transcriptional factor-XYR1-in the filamentous fungus Trichoderma reesei during cellulosic material degradation. In this regard, we performed a global transcriptome analysis using RNA-Seq of the Δxyr1 mutant strain of T. reesei compared with the parental strain QM9414 grown in the presence of cellulose, sophorose, and glucose as sole carbon sources. We found that 5885 genes were expressed differentially under the three tested carbon sources. Of these, 322 genes were upregulated in the presence of cellulose, while 367 and 188 were upregulated in sophorose and glucose, respectively. With respect to genes under the direct regulation of XYR1, 30 and 33 are exclusive to cellulose and sophorose, respectively. The most modulated genes in the Δxyr1 belong to Carbohydrate-Active Enzymes (CAZymes), transcription factors, and transporters families. Moreover, we highlight the downregulation of transporters belonging to the MFS and ABC transporter families. Of these, MFS members were mostly downregulated in the presence of cellulose. In sophorose and glucose, the expression of these transporters was mainly upregulated. Our results revealed that MFS and ABC transporters could be new players in cellulose degradation and their role was shown to be carbon source-dependent. Our findings contribute to a better understanding of the regulatory mechanisms of XYR1 to control cellulase gene expression in T. reesei in the presence of cellulosic material, thereby potentially enhancing its application in several biotechnology fields.

The impact of chromatin remodelling on cellulase expression in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging. RESULTS: Comparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1. CONCLUSIONS: The regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

Defining the genome-wide role of CRE1 during carbon catabolite repression in Trichoderma reesei using RNA-Seq analysis.

The ascomycete Trichoderma reesei is one of the most well-studied cellulolytic fungi and is widely used by the biotechnology industry in the production of second generation bioethanol. The carbon catabolite repression (CCR) mechanism adopted by T. reesei is mediated by the transcription factor CRE1. CCR represses genes related to cellulase production when a carbon source is readily available in the medium. Using RNA sequencing, we investigated CCR during the synthesis of cellulases, comparing the T. reesei Δcre1 mutant strain with its parental strain, QM9414. Of 9129 genes in the T. reesei genome, 268 genes were upregulated and 85 were downregulated in the presence of cellulose (Avicel). In addition, 251 genes were upregulated and 230 were downregulated in the presence of a high concentration of glucose. Genes encoding cellulolytic enzymes and transcription factors and genes related to the transport of nutrients and oxidative metabolism were also targets of CCR, mediated by CRE1 in a carbon source-dependent manner. Our results also suggested that CRE1 regulates the expression of genes related to the use of copper and iron as final electron acceptors or as cofactors of enzymes that participate in biomass degradation. As a result, the final effect of CRE1-mediated transcriptional regulation is to modulate the access of cellulolytic enzymes to cellulose polymers or blocks the entry of cellulase inducers into the cell, depending on the glucose content in the medium. These results will contribute to a better understanding of the mechanism of carbon catabolite repression in T. reesei, thereby enhancing its application in several biotechnology fields.

Comparative metabolism of cellulose, sophorose and glucose in Trichoderma reesei using high-throughput genomic and proteomic analyses.

BACKGROUND: The filamentous fungus Trichoderma reesei is a major producer of lignocellulolytic enzymes utilized by bioethanol industries. However, to achieve low cost second generation bioethanol production on an industrial scale an efficient mix of hydrolytic enzymes is required for the deconstruction of plant biomass. In this study, we investigated the molecular basis for lignocellulose-degrading enzyme production T. reesei during growth in cellulose, sophorose, and glucose. RESULTS: We examined and compared the transcriptome and differential secretome (2D-DIGE) of T. reesei grown in cellulose, sophorose, or glucose as the sole carbon sources. By applying a stringent cut-off threshold 2,060 genes were identified as being differentially expressed in at least one of the respective carbon source comparisons. Hierarchical clustering of the differentially expressed genes identified three possible regulons, representing 123 genes controlled by cellulose, 154 genes controlled by sophorose and 402 genes controlled by glucose. Gene regulatory network analyses of the 692 genes differentially expressed between cellulose and sophorose, identified only 75 and 107 genes as being specific to growth in sophorose and cellulose, respectively. 2D-DIGE analyses identified 30 proteins exclusive to sophorose and 37 exclusive to cellulose. A correlation of 70.17% was obtained between transcription and secreted protein profiles. CONCLUSIONS: Our data revealed new players in cellulose degradation such as accessory proteins with non-catalytic functions secreted in different carbon sources, transporters, transcription factors, and CAZymes, that specifically respond in response to either cellulose or sophorose.