Trichoderma reesei CRE1-mediated Carbon Catabolite Repression in Re-sponse to Sophorose Through RNA Sequencing Analysis.

Carbon catabolite repression (CCR) mediated by CRE1 in Trichoderma reesei emerged as a mechanism by which the fungus could adapt to new environments. In the presence of readily available carbon sources such as glucose, the fungus activates this mechanism and inhibits the production of cellulolytic complex enzymes to avoid unnecessary energy expenditure. CCR has been well described for the growth of T. reesei in cellulose and glucose, however, little is known about this process when the carbon source is sophorose, one of the most potent inducers of cellulase production. Thus, we performed high-throughput RNA sequencing to better understand CCR during cellulase formation in the presence of sophorose, by comparing the mutant ∆cre1 with its parental strain, QM9414. Of the 9129 genes present in the genome of T. reesei, 184 were upregulated and 344 downregulated in the mutant strain ∆cre1 compared to QM9414. Genes belonging to the CAZy database, and those encoding transcription factors and transporters are among the gene classes that were repressed by CRE1 in the presence of sophorose; most were possible indirectly regulated by CRE1. We also observed that CRE1 activity is carbon-dependent. A recent study from our group showed that in cellulose, CRE1 repress different groups of genes when compared to sophorose. CCR differences between these carbon sources may be due to the release of cellodextrins in the cellulose polymer, resulting in different targets of CRE1 in both carbon sources. These results contribute to a better understanding of CRE1-mediated CCR in T. reesei when glucose comes from a potent inducer of cellulase production such as sophorose, which could prove useful in improving cellulase production by the biotechnology sector.

Expression pattern of cellulolytic and xylanolytic genes regulated by transcriptional factors XYR1 and CRE1 are affected by carbon source in Trichoderma reesei.

Trichoderma reesei is the most important fungus for the industrial production of enzymes to biomass deconstruction. Most of the genes encoding cellulases and hemicellulases are regulated by the transcription factors CRE1 and XYR1. In this work, the regulation of 22 genes of cellulases and xylanases by these transcription factors was investigated under three different carbon sources. Analysis of gene expression and enzymatic profiles of CMCase, ß-glucosidase, and xylanases showed different regulation that was depended of the carbon source in both Δxyr1 and Δcre1 mutants. In the presence of glucose, the majority of genes evaluated (82%) showed increased expression levels in the Δcre1 mutant compared to the parental QM9414 strain. In the Δxyr1 mutant, it was observed that expression of cellulase and xylanase genes was reduced compared to the parental QM9414 strain, when cultured in the presence of cellulose or sophorose. Interesting, in the presence of glucose, approximately 60% of the analyzed genes had increased expression in the Δxyr1 mutant compared to parental strain. Furthermore, no correlation between gene expression and the number of putative binding sites of XYR1 and CRE1 to promoter region of cellulolytic and xylanolytic studied genes was observed. Therefore, these results demonstrated that the regulation of cellulase and xylanase by the transcription factors CRE1 and XYR1 is influenced by different carbon sources.

Deciphering the cis-regulatory elements for XYR1 and CRE1 regulators in Trichoderma reesei.

In this work, we report the in silico identification of the cis-regulatory elements for XYR1 and CRE1 proteins in the filamentous fungus Trichoderma reesei, two regulators that play a central role in the expression of cellulase genes. Using four datasets of condition-dependent genes from RNA-seq and RT-qPCR experiments, we performed unsupervised motif discovery and found two short motifs resembling the proposed binding consensus for XYR1 and CRE1. Using these motifs, we analysed the presence and arrangement of putative cis-regulatory elements recognized by both regulators and found that shortly spaced sites were more associated with XYR1- and CRE1-dependent promoters than single, high-score sites. Furthermore, the approach used here allowed the identification of the previously reported XYR1-binding sites from cel7a and xyn1 promoters, and we also mapped the potential target sequence for this regulator at the cel6a promoter that has been suggested but not identified previously. Additionally, seven other promoters (for cel7b, cel61a, cel61b, cel3c, cel3d, xyn3 and swo genes) presented a putative XYR1-binding site, and strong sites for CRE1 were found at the xyr1 and cel7b promoters. Using the cis-regulatory architectures nearly defined for XYR1 and CRE1, we performed genome-wide identification of potential targets for direct regulation by both proteins and important differences on their functional regulons were elucidated. Finally, we performed binding site mapping on the promoters of differentially expressed genes found in T. reesei mutant strains lacking xyr1 or cre1 and found that indirect regulation plays a key role on their signalling pathways. Taken together, the data provided here sheds new light on the mechanisms for signal integration mediated by XYR1 and CRE1 at cellulase promoters.