BackgroundThe 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.ResultsWe 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.ConclusionsOur 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.

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.

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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.

Background Trichoderma reesei is the most important industrial producer of lignocellulolytic enzymes. These enzymes play an important role in biomass degradation leading to novel applications of this fungus in the biotechnology industry, specifically biofuel production. The secretory pathway of fungi is responsible for transporting proteins addressed to different cellular locations involving some cellular endomembrane systems. Although protein secretion is an extremely efficient process in T. reesei , the mechanisms underlying protein secretion have remained largely uncharacterized in this organism. Results Here, we report for the first time the isolation and characterization of T. reesei extracellular vesicles (EVs). Using proteomic analysis under cellulose culture condition, we have confidently identified 188 vesicular proteins belonging to different functional categories. Also, we characterized EVs production using transmission electron microscopy in combination with light scattering analysis. Biochemical assays revealed that T. reesei extracellular vesicles have an enrichment of filter paper (FPase) and β-glucosidase activities in purified vesicles from 24, 72 and 96, and 72 and 96 h, respectively. Furthermore, our results showed that there is a slight enrichment of small RNAs inside the vesicles after 96 h and 120 h, and presence of hsp proteins inside the vesicles purified from T. reesei grown in the presence of cellulose. Conclusions This work points to important insights into a better understanding of the cellular mechanisms underlying the regulation of cellulolytic enzyme secretion in this fungus. Electronic supplementary material The online version of this article (10.1186/s13068-019-1487-7) contains supplementary material, which is available to authorized users.

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.

BackgroundTrichoderma reesei is a saprophytic fungus implicated in the degradation of polysaccharides present in the cell wall of plants. T. reesei has been recognized as the most important industrial fungus that secretes and produces cellulase enzymes that are employed in the production of second generation bioethanol. A few of the molecular mechanisms involved in the process of biomass deconstruction by T. reesei; in particular, the effect of sugar transporters and induction of xylanases and cellulases expression are yet to be known.ResultsIn our study, we characterized a novel sugar transporter, which was previously identified by our group through in silico analysis of RNA-seq data. The novel T. reesei 69957-sugar transport system (Tr69957) is capable of transporting xylose, mannose, and cellobiose using a T. reesei 69957-sugar transport system in Saccharomyces cerevisiae. The deletion of Tr69957 in T. reesei affected the fungal growth and biomass accumulation, and the sugar uptake in the presence of mannose, cellobiose, and xylose. Molecular docking studies revealed that Tr69957 shows reduced protein–ligand binding energy for interactions towards disaccharides in comparison with monosaccharides. Furthermore, the deletion of Tr69957 affected the gene expression of cellobiohydrolases (cel7a and cel6a), β-glucosidases (cel3a and cel1a), and xylanases (xyn1 and xyn2) in the cultures of parental and mutant strains in the presence of cellobiose and sugarcane bagasse (SCB).ConclusionThe transporter Tr69957 of T. reesei can transport cellobiose, xylose, and mannose, and can affect the expression of a few genes encoding enzymes, such as cellulases and xylanases, in the presence of SCB. We showed for the first time that a filamentous fungus (T. reesei) contains a potential mannose transporter that may be involved in the degradation of cellulose.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1084-1) contains supplementary material, which is available to authorized users.