Search Results (53)

Thefilamentous fungus Trichoderma reesei is renowned for its exceptional ability to secrete cellulolytic enzymes, which play a crucial role in the hydrolysis of lignocellulose biomass. The expression of fungal cellulases is meticulously regulated at the transcriptional level, depending on the carbon source available in the medium. To obtain new insights into the transcriptional network controlling cellulase expression in T. reesei, we analyzed RNA-Seq data and identified and characterized a new transcription factor, Rme1, that regulates the expression of cellulolytic genes. Combining functional genomics and protein-DNA interaction assay, we showed that Rme1 acts as a repressor of cellulase production in T. reesei by directly regulating two critical genes involved in cellulose degradation: the cellobiohydrolase cel7a and the carbon catabolite repressor cre1. This is the first report of a transcription factor regulating Cre1. This study contributes to a better understanding of the complex regulation of the cellulolytic system of T. reesei and may be useful for the genetic modification of strains for the biorefinery industry. Full article

The β-glucosidase CEL1B has been linked to regulating cellulase expression in Trichoderma reesei, yet its inducer-specific functions and broader regulatory roles remain poorly characterized. In this study, CRISPR-Cas9-mediated gene knockout was applied in the industrial high-producing T. reesei Rut C30 to investigate CEL1B function without the confounding effects of KU70 deletion. Unlike previous studies focused solely on cellulose or lactose induction, transcriptomic analysis of the CEL1B knockout strain revealed its regulatory roles under both lactose- and sophorose-rich conditions, with sophorose representing the most potent natural inducer of cellulase expression. Under lactose induction, CEL1B deletion resulted in a 52.4% increase in cellulase activity (p < 0.05), accompanied by transcriptome-wide upregulation of β-glucosidase genes (CEL3A: 729%, CEL3D: 666.8%, CEL3C: 110.9%), cellulose-sensing receptors (CRT1: 203.0%, CRT2: 105.8%), and key transcription factors (XYR1: 2.7-fold, ACE3: 2.8-fold, VIB1: 2.1-fold). Expression of ER proteostasis genes was significantly upregulated (BIP1: 3.3-fold, HSP70: 6.2-fold), contributing to enhanced enzyme secretion. Conversely, under sophorose induction, CEL1B deletion reduced cellulase activity by 25.7% (p < 0.05), which was associated with transcriptome profiling showing significant downregulation of β-glucosidase CEL3H (66.6%) and cellodextrin transporters (TrireC30_91594: 79.3%, TrireC30_127980: 76.3%), leading to reduced cellobiohydrolase expression (CEL7A: 57.8%, CEL6A: 67.8%). This first transcriptomic characterization of the CEL1B knockout strain reveals its dual opposing roles in modulating cellulase expression in response to lactose versus sophorose, providing new strategies for optimizing inducer-specific enzyme production in T. reesei. Full article

Trichoderma reesei is a well-established industrial enzyme producer and has been the subject of extensive research for various applications. The basis of many research studies is the analysis of gene expression, specifically with RT-qPCR, which requires stable reference genes for normalization to yield reliable results. Yet the commonly used reference genes, act1 and sar1, were initially chosen based on reports from the literature rather than systematic validation, raising concerns about their stability. Thus, properly evaluated reference genes for T. reesei are lacking. In this study, five potentially new reference genes were identified by analyzing publicly available transcriptome datasets of the T. reesei strains QM6a and Rut-C30. Their expression stability was then evaluated under relevant cultivation conditions using RT-qPCR and analyzed with RefFinder. The two most stable candidate reference genes were further validated by normalizing the expression of the well-characterized gene cbh1 and comparing the results to those obtained using act1 and sar1. Additionally, act1 and sar1 were normalized against the new reference genes to assess the variability in their expression. All five new reference genes exhibited a more stable expression than act1 and sar1. Both in silico and RT-qPCR analysis ranked the so far uncharacterized gene, bzp1, as the most stable. Further, we found that act1 and sar1 have strain- and condition-dependent expression variability, suggesting that they are unsuitable as universal reference genes in T. reesei. Based on these results, we propose to use the combination of bzp1 and tpc1 for the normalization in RT-qPCR analysis instead of act1 and sar1. Full article

Understanding the molecular mechanisms of cellobiohydrolase I (CBHI), a key enzyme in cellulase complexes, is crucial for developing efficient enzymes for the degradation of lignocellulosic biomasses (LCB). Building on our previous discovery that Chaetomium thermophilum CBHI (C-CBH) exhibits significantly higher specific activity than Trichoderma reesei CBHI (T-CBH), systematic domain-swapping experiments were conducted to elucidate the structural determinants of catalytic efficiency in CBHI. Herein, the carbohydrate-binding modules (CBM) of the CBHIs from Trichoderma reesei (T-CBH) and Chaetomium thermophilum (C-CBH) were interchanged and to obtain two chimeric mutants TC-CBH and CT-CBH. These four CBHs were expressed in T. reesei, and the enzyme properties were analyzed. Comparative characterization revealed that while module exchange preserved native temperature/pH adaptability, it significantly altered substrate specificity and catalytic performance. The CT-CBH variant was identified as the most efficient biocatalyst, exhibiting four key advantages over T-CBH: (1) protein expression levels that far exceed those of T-CBH, (2) specific activity enhanced by 2.6-fold (734.5 U/μM vs. 282.5 U/μM on MU-cellobiose), (3) superior degradation capacities for filter paper (1.6-fold) and xylan, and (4) improved binding affinity for crystalline cellulose. These findings establish cross-species domain engineering as a viable strategy for creating high-performance cellulases, providing both mechanistic insights and practical solutions for lignocellulose degradation. Full article

The efficient hydrolysis of lignocellulosic biomass relies on the action of enzymes, which are crucial for the development of economically feasible cellulose bioconversion processes. However, low hydrolysis efficiency and the inhibition of cellulase production by carbon catabolite repression (CCR) have been significant obstacles in this process. The aim of this study was to identify the patterns of cellulose degradation and related genes through the genome analysis of a newly isolated lignocellulose-degrading fungus Flavodon sp. x-10. The whole-genome sequencing showed that the genome size of Flavodon sp. x-10 was 37.1 Mb, with a GC content of 49.48%. A total of 11,277 genes were predicted, with a total length of 18,218,150 bp and an average length of 1615 bp. Additionally, 157 tRNA genes responsible for transporting different amino acids were predicted, and the repeats and tandem repeats accounted for only 0.76% of the overall sequences. A total of 5039 genes were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, representing 44.68% of all genes, and 368 metabolic pathways were involved. Of the 595 genes annotated in the carbohydrate-active enzyme (CAZy) database, 183 are associated with plant cell wall-degrading enzymes (PCWDEs), surpassing those of Aspergillus niger (167), Trichoderma reesei (64), and Neurospora crassa (86). Compared to these three fungi, Flavodon sp. x-10 has a higher number of enzyme genes related to lignin degradation in its genome. Transporters were further identified by matching the whole-genome sequence to the Transporter Classification Database (TCDB), which includes 20 sugar transporters (STs) closely linked to sugar utilization. Through the comprehensive exploration of the whole-genome sequence, this study uncovered more vital lignocellulase genes and their degradation mechanisms, providing feasible strategies for improving the strains to reduce the cost of biofuel production. Full article

Wood blue staining is one of the most common wood defects, which commonly occurs in rubberwood and Masson pine. It not only affects the appearance of the wood, but also its properties. In this study, rubberwood from Xishuangbanna was examined. During the process, blue-stain fungi and endophytic fungi were isolated and identified. The antagonism of biocontrol strains against blue-stain fungi was studied, and their inhibitory effects were evaluated by inoculating the biocontrol strains on non-blue-stained rubberwood. The morphology and distribution of the strains on the wood were observed using polarized light and fluorescence microscopy, infrared spectroscopy, scanning electron microscopy (SEM–EDS), and X-ray diffraction (XRD). The effects of biocontrol strains on the blue stain of the wood were then evaluated. The results showed that at room temperature, the fungus causing the blue stain in rubberwood was identified as Lasiodiplodia theobromae, and the biocontrol strains of endophytic fungi isolated were Trichoderma koningii and Trichoderma reesei. It was found that T. reesei was more effective than T. koningii in inhibiting the development of L. theobromae. Based on these findings, T. reesei was suggested as a biocontrol strain for preventing and controlling blue stain in rubberwood. Full article

Recent research has revealed the calcium signaling significance in the production of cellulases in Trichoderma reesei. While vacuoles serve as the primary calcium storage within cells, the function of vacuolar calcium transporter proteins in this process remains unclear. In this study, we conducted a functional characterization of four vacuolar calcium transport proteins in T. reesei. This was accomplished by the construction of the four mutant strains ∆trpmc1, ∆tryvc1, ∆tryvc3, and ∆tryvc4. These mutants displayed enhanced growth when subjected to arabinose, xylitol, and xylose. Furthermore, the mutants ∆trpmc1, ∆tryvc1, and ∆tryvc4 showed a reduction in growth under conditions of 100 mM MnCl₂, implying their role in manganese resistance. Our enzymatic activity assays revealed a lack of the expected augmentation in cellulolytic activity that is typically seen in the parental strain following the introduction of calcium. This was mirrored in the expression patterns of the cellulase genes. The vacuolar calcium transport genes were also found to play a role in the expression of genes involved with the biosynthesis of secondary metabolites. In summary, our research highlights the crucial role of the vacuolar calcium transporters and, therefore, of the calcium signaling in orchestrating cellulase and hemicellulase expression, sugar utilization, and stress resistance in T. reesei. Full article

The spectrum of enzymes required for complete lignocellulosic waste hydrolysis is too diverse to be secreted by a single organism. An alternative is to employ fungal co-cultures to obtain more diverse and complete enzymatic cocktails without the need to mix enzymes during downstream processing. This study evaluated the co-cultivation of Aspergillus brasiliensis and Trichoderma reesei RUT-C30 in different conditions using sugarcane bagasse as the carbon source. The resulting enzymatic cocktails were characterized according to the impact of strain inoculation time on enzymatic activities and proteome composition. Data revealed that the profile of each enzymatic extract was highly dependent on the order in which the participating fungi were inoculated. Some of the co-cultures exhibited higher enzyme activities compared to their respective monocultures for enzymes such as CMCase, pectinase, β-glucosidase, and β-xylosidase. Analysis of the T. reesei RUT-C30 and A. brasiliensis co-culture secretome resulted in the identification of 167 proteins, with 78 from T. reesei and 89 from A. brasiliensis. In agreement with the enzymatic results, proteome analysis also revealed that the timing of inoculation greatly influences the overall secretome, with a predominance of T. reesei RUT-C30 proteins when first inoculated or in simultaneous inoculation. Full article

Trichoderma erinaceum is a filamentous fungus that was isolated from decaying sugarcane straw at a Brazilian ethanol biorefinery. This fungus shows potential as a source of plant cell wall-degrading enzymes (PCWDEs). In this study, we conducted a comprehensive multiomics investigation of T. erinaceum to gain insights into its enzymatic capabilities and genetic makeup. Firstly, we performed genome sequencing and assembly, which resulted in the identification of 10,942 genes in the T. erinaceum genome. We then conducted transcriptomics and secretome analyses to map the gene expression patterns and identify the enzymes produced by T. erinaceum in the presence of different substrates such as glucose, microcrystalline cellulose, pretreated sugarcane straw, and pretreated energy cane bagasse. Our analyses revealed that T. erinaceum highly expresses genes directly related to lignocellulose degradation when grown on pretreated energy cane and sugarcane substrates. Furthermore, our secretome analysis identified 35 carbohydrate-active enzymes, primarily PCWDEs. To further explore the enzymatic capabilities of T. erinaceum, we selected a β-glucosidase from the secretome data for recombinant production in a fungal strain. The recombinant enzyme demonstrated superior performance in degrading cellobiose and laminaribiose compared to a well-known enzyme derived from Trichoderma reesei. Overall, this comprehensive study provides valuable insights into both the genetic patterns of T. erinaceum and its potential for lignocellulose degradation and enzyme production. The obtained genomic data can serve as an important resource for future genetic engineering efforts aimed at optimizing enzyme production from this fungus. Full article

The cellulose present in the cell wall of vegetables prevents the greater release of nutrients to the animal. Therefore, the use of the cellulase enzyme is a viable strategy as it is capable of breaking cellulose bonds, releasing nutrients such as glucose, increasing dietary energy, and thus improving the productive performance of birds. Trichoderma reesei is efficient in the production of cellulase, which is produced via submerged fermentation followed by purification, formulation, and drying. Therefore, an experiment was carried out using 240 male broilers of the Cobb-500^® lineage to verify the effects resulting from the addition of powdered (500 g/t and 1000 g/t) and liquid (500 mL/t) cellulase over a period of 1 to 21 days. A completely randomized experimental design was used, consisting of four treatments with six replications and ten birds per replication that were housed in an experimental cage. It was observed that performance and digestibility results were significantly different with cellulase supplementation. Also, the relative weight of the large intestine in the period between one and seven days increased when cellulase was added at 1000 g/t. In the period of between eight and 14 days of life, the birds that consumed only the basal diet obtained higher levels of liver protein than those that received the treatments with the addition of the enzyme. However, 15 and 21 days, the consumed feed effect did not occur between thus, it is not conclusive whether hepatotoxicity occurs with the addition of cellulase. For the blood parameters, at 21 days, the diets with added cellulase were not significantly different regarding electrolytes. It was concluded that this cellulase produced by Trichoderma reesei can be included in the animals’ diet. Full article

The small GTPases of the Rho family are known to regulate various biological processes in filamentous fungi. In this study, we investigated the impact of deleting Rho proteins on the growth and cellulase production of Trichoderma reesei. Our findings revealed that deletion of cdc42 led to the most severe growth defect and impaired cellulase production. Conversely, overexpression of cdc42 resulted in a hyperbranched phenotype, significantly enhancing cellulase production. Furthermore, the cdc42-overexpressing (OCdc42) strain showed an increased expression of multiple cellulase genes and Rho GTPase genes. Analysis of the secretome in the OCdc42 strain unveiled an increased abundance and diversity of extracellular proteins compared to the parent strain. These discoveries provide valuable insights into the functionality of Rho GTPases in T. reesei and offer potential targets for engineering fungi to improve plant biomass deconstruction in biorefineries. Full article

Cassava bagasse has great potency as a substrate in the biorefinery industry. This paper proposes the valorisation of cassava bagasse into organic acids by cellulase through the co-cultivation of Aspergillus violaceofuscus and Trichoderma reesei RUT-C30 at the ratio 1:1. The optimised conditions for β-glucosidase production under submerged fermentation were pH 4.50, a tween 80 concentration of 0.05% (v/v), and a spore concentration of 7.18 × 10⁷ spores·mL⁻¹. We found base steam cassava bagasse (BSCB) to have high cellulose content, making it possible to replace avicel phosphoric acid swollen cellulose (PASC) as a substrate. The co-cultivation with the BSCB substrate had higher levels of β-glucosidase (1.72-fold), cellobiohydrolase (2.83-fold), and endoglucanase (2.82-fold) activity compared to that of the avicel PASC substrate. Moreover, acetic acid (7.41 g·L⁻¹), citric acid (3.54 g·L⁻¹), gluconic acid (0.30 g·L⁻¹), and malic acid (0.37 g·L⁻¹) were detected in the BSCB crude extract. These results demonstrate the considerable prospects of the A. violaceofuscus and T. reesei RUT-C30 co-cultivation approaches in the biorefinery industry. Full article

The fungus Trichoderma reesei is an essential producer of enzymes that degrade lignocellulosic biomass to produce value-added bioproducts. The cellulolytic system of T. reesei is controlled by several transcription factors (TFs) that efficiently regulate the production of these enzymes. Recently, a new TF named Azf1 was identified as a positive regulator of cellulase expression. Here, we investigated novel regulatory functions of Azf1 by its overexpression. In the mutant strain OEazf1, overexpression of azf1 was achieved under both repression and induction conditions. Although azf1 was more abundant in transcript and protein, overexpression of this TF did not activate transcription of the cellulase gene in the presence of the repressor glucose, suggesting that Azf1 may be subject to posttranslational regulation. In cellulose, the expression of swo, encoding the accessory protein swollenin, and the β-glucosidases cel1a, cel1b, cel3b, and cel3g increases in the early stages of cultivation. The increased production of these β-glucosidases increases the hydrolysis rate of cellobiose and sophorose, which activates carbon catabolite repression (CCR) and causes repression of cellulase genes and the regulator Xyr1 in the later stages of cultivation. Moreover, overexpression of azf1 led to increased cellulase activity in T. reesei during long-term cultivation in cellulose and sugarcane bagasse. Our results provide new insights into the mechanisms regulating Azf1 and novel genes that are important targets of this TF. This work contributes to a better understanding of the complex mechanisms regulating cellulase expression in T. reesei. It will contribute to the development of strains with higher production of these essential enzymes. Full article

The conversion of lignocellulosic biomass to second-generation biofuels through enzymes is achieved at a high cost. Filamentous fungi through a combination of oxidative enzymes can easily disintegrate the glycosidic bonds of cellulose. The combination of cellobiose dehydrogenase (CDH) with lytic polysaccharide monooxygenases (LPMOs) enhances cellulose degradation in many folds. CDH increases cellulose deconstruction via coupling the oxidation of cellobiose to the reductive activation of LPMOs by catalyzing the addition of oxygen to C-H bonds of the glycosidic linkages. Fungal LPMOs show different regio-selectivity (C1 or C4) and result in oxidized products through modifications at reducing as well as nonreducing ends of the respective glucan chain. T. reesei LPMOs have shown great potential for oxidative cleavage of cellobiose at C1 and C4 glucan bonds, therefore, the incorporation of heterologous CDH further increases its potential for biofuel production for industrial purposes at a reduced cost. We introduced CDH of Phanerochaete chrysosporium (PcCDH) in Trichoderma reesei (which originally lacked CDH). We purified CDH through affinity chromatography and analyzed its enzymatic activity, electron-donating ability to LPMO, and the synergistic effect of LPMO and CDH on cellulose deconstruction. The optimum temperature of the recombinant PcCDH was found to be 45 °C and the optimum pH of PcCDH was observed as 4.5. PcCDH has high cello-oligosaccharide k_cat, K_m, and k_cat/K_m values. The synergistic effect of LPMO and cellulase significantly improved the degradation efficiency of phosphoric acid swollen cellulose (PASC) when CDH was used as the electron donor. We also found that LPMO undergoes auto-oxidative inactivation, and when PcCDH is used an electron donor has the function of a C1-type LPMO electron donor without additional substrate increments. This work provides novel insights into finding stable electron donors for LPMOs and paves the way forward in discovering efficient CDHs for enhanced cellulose degradation. Full article

Recently, a genetically modified microorganism (GMM) detection strategy using real-time PCR technology was developed to control fermentation products commercialized in the food and feed chain, allowing several unexpected GMM contaminations to be highlighted. Currently, only bacterial strains are targeted by this strategy. Given that fungal strains, like Trichoderma reesei, are also frequently used by the food industry to produce fermentation products, a novel real-time PCR method specific to this fungal species was developed and validated in this study to reinforce the GMM detection strategy. Designed to cover a sequence of 130 bp from the translation elongation factor alpha 1 (Tef1) gene of T. reesei, this real-time PCR method, namely TR, allows for the screening of commercial fermentation products contaminated with T. reesei, genetically modified or not, which is one of the major fungal species used as an industrial platform for the manufacturing of fermentation products. The developed real-time PCR TR method was assessed as specific and sensitive (LOD_95% = eight copies). In addition, the developed real-time PCR TR method performance was confirmed to be in line with the “Minimum Performance Requirements for Analytical Methods of GMO Testing” of the European Network of GMO Laboratories. The validated real-time PCR TR method was also demonstrated to be applicable to commercial microbial fermentation products. Based on all these results, the novel real-time PCR TR method was assessed as valuable for strengthening the current GMM detection strategy regarding major fungal species used by the food industry to produce microbial fermentation products. Full article