Project description:Lactose (1,4-0-M-CM-^_-d-galactopyranosyl-d-glucose), a by-product from cheese manufacture or whey processing industries, is known to induce the formation of plant biomass hydrolyzing enzymes needed for the biorefinery industry in the fungus Trichoderma reesei, but the reason for this induction and the underlying mechanism are not fully understood. Here, we used systems analysis of the Trichoderma reesei transcriptome during utilization of lactose. We found that the respective CAZome encoded glycosyl hydrolases specifically tailored for the attack of monocotyledon xyloglucan. In addition, genes for a high number of putative transporters of the major facilitator superfamily were also induced. Systematic knock out of them identified a gene whose knock-out completely impaired lactose utilization and cellulase induction in Trichoderma reesei. These data shed new light on the mechanism by which Trichoderma reesei metabolizes lactose and illuminate the key role of M-CM-^_-D-galactosides in habitat specificity of this fungus. We used two biological replicas of Trichoderma reesei growing on lactose, glucose and glycerol
Project description:Lactose (1,4-0-ß-d-galactopyranosyl-d-glucose), a by-product from cheese manufacture or whey processing industries, is known to induce the formation of plant biomass hydrolyzing enzymes needed for the biorefinery industry in the fungus Trichoderma reesei, but the reason for this induction and the underlying mechanism are not fully understood. Here, we used systems analysis of the Trichoderma reesei transcriptome during utilization of lactose. We found that the respective CAZome encoded glycosyl hydrolases specifically tailored for the attack of monocotyledon xyloglucan. In addition, genes for a high number of putative transporters of the major facilitator superfamily were also induced. Systematic knock out of them identified a gene whose knock-out completely impaired lactose utilization and cellulase induction in Trichoderma reesei. These data shed new light on the mechanism by which Trichoderma reesei metabolizes lactose and illuminate the key role of ß-D-galactosides in habitat specificity of this fungus.
Project description:The disruption of vacuolar protein sorting related genes vps13 and vps21 in filamentou fungi Trichoderma reesei resulted in a dramtic increase in cellulase transcription especially in the late Avicel induction phase, relieving the RESS (REpression under Secretion Stress) effect. Great interest arisen concered about the reason and underlying mechanism of vps13 and vps21 disruption for increased cellulase transcription. Thus, transcriptome analysis through RNA-Seq could give a deep insight of the potential pathways involved in enhanced cellulase transcription in late Avicel induction.
Project description:MicroRNAs (miRNAs) are small non-coding RNAs capable of negatively regulating gene expression. Trichoderma reesei is an industrial filamentous fungus that can secrete abundant hydrolases for cellulosic biofuels. Recently, microRNA-like RNAs (milRNAs) were discovered in several filamentous fungi rather than T. reesei. The purpose of this study was to explore the presence of milRNA in T. reesei, to characterize the differential expression of T. reesei milRNA under cellulose induction, and to reveal the target genes of milRNA involved in cellulase production. Two small RNA libraries of cellulose induction (IN) or non-induction (CON) were generated and sequenced using Solexa sequencing technology. A total of 664,463 and 529,545 unique sequences, representing 1,271 and 1,021 unique small RNAs, were obtained from the IN and CON samples, respectively. Thirteen milRNAs were finally identified in T. reesei using the hairpin structure analysis. The milRNAs profiles obtained in deep sequencing were validated by RT-qPCR assay. The miRanda program predicts a number of potential targets for T. reesei milRNAs, including several hydrolases and carbon catabolite repressor Cre1.The presence and differential expression of T. reesei milRNAs, along with their predicted targets indicate that milRNAs might play a regulatory role in cellulase induction. This work lays foundation for further functional study of fungal milRNAs and their industrial application.
Project description:The ascomycete Trichoderma reesei is an industrial producer of cellulolytic and hemicellulolytic enzymes and also serves as a model for investigations on these enzymes and their genes. The strain QM9978 has a cellulase negative phenotype and therefore presents a valuable tool for understanding the mechanisms underlying cellulase regulation. A transcriptomic analyses of the cellulase negative strain QM9978 and the original strain QM6a have been performed to identify the genetic differences between QM6a and QM9978 leading to the cellulase-negative phenotype
Project description:The filamentous fungus Trichoderma reesei is the main industrial cellulytic enzymes producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome sequencing, transcriptome) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG14 and RUT C30. In this follow-up work, we examined how these mutations affect phenotype at the level of the transcriptome and cultivation behavior. We performed kinetic transcriptome analysis of the NG14 and RUT C30 strains of early cellulase production induced by lactose using bioreactor cultivations close to industrial conditions. RUT C30 exhibited both earlier onset of cellulase production and higher steady-state productivity. A rather low number of genes were regulated, most of them being specific to the NG14 strains. Clustering of these genes highlighted similar behavior for some functional categories, and allowed to distinguish between induction-related genes and productivity-related genes. The lower number of genes regulated in RUT C30 could not formally be linked to the relief in catabolic repression that is characteristic of this strain. Cross-comparison of our transcriptome data with mutations previously identified revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting these clusters contain genes playing a role in strain performance. This is the first kinetic transcriptome study carried out in industry-relevant conditions with two related strains of T. reesei showing distinctive performances. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggest that the induction mechanism is essentially intact and that there is room for further improvement of T. reesei. We also provide some potential target for further genetic improvement of these strains.
Project description:The filamentous fungus Trichoderma reesei is the main industrial cellulytic enzymes producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome sequencing, transcriptome) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG14 and RUTM-BM- C30. In this follow-up work, we examined how these mutations affect phenotype at the level of the transcriptome and cultivation behavior.M-BM- We performed kinetic transcriptome analysis of the NG14 and RUTM-BM- C30 strains of early cellulase production induced by lactose using bioreactor cultivations close to industrial conditions. RUTM-BM- C30 exhibited both earlier onset of cellulase production and higher steady-state productivity. A rather lowM-BM- number of genes were regulated, most of them being specific to the NG14 strains. Clustering of these genes highlighted similar behavior for some functional categories, and allowed to distinguish between induction-related genes and productivity-related genes. The lower number of genes regulated in RUTM-BM- C30 could not formally be linked to the relief in catabolic repression that is characteristic of this strain. Cross-comparison of our transcriptome data with mutations previously identified revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting these clusters contain genes playing a role in strain performance. This is the first kinetic transcriptome study carried out in industry-relevant conditions with two related strains of T. reesei showing distinctive performances. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggest that the induction mechanism is essentially intact and that there is room for further improvement of T. reesei. We also provide some potential target for further genetic improvement of these strains. Two biological pool by condition in dye switch. For the two biological replicates on each four experiments we apply on the pretreated results the linear modeling approach implemented by lmFit and the empirical Bayes statistics implemented by eBayes from the limma R package (Smyth 2004). We select the list of statistically regulated genes using a 5% significance threshold.
Project description:Trichoderma reesei is known for its ability to produce large amounts of extracellular proteins and is one of the most important industrially used filamentous fungus. Xylanase regulator 1 (XYR1) as the master regulator is responsible for the activation of cellulase and hemicellulase gene expression, normally under inducing conditions. It has been reported that strains with point mutations in certain areas of xyr1 bypass the carbon catabolite repression, allowing cellulase and hemicellulase production even in the presence of glucose. These mutations also change the profile of produced proteins, shifting it more towards xylanase production, and increase the overall protein production in inducing conditions. However, how these mutations alter the metabolism and other cellular processes to cause these changes remains unclear. In the present study, our aim was to explore changes caused by a point mutation in xyr1 on transcriptomic and metabolic level to better understand the reasons behind the increased protein production in both repressing glucose and inducing lactose conditions. We observed that the xyr1 mutant strain built more biomass and produced more extracellular proteins during growth on lactose compared to the wild type xyr1 strain. Genes involved in oxidoreductive D-galactose catabolism pathway were upregulated in the xyr1 mutant strain, potentially contributing to the more efficient utilization of lactose. The shift from utilizing lactose to using glucose as carbon source seemed faster in the xyr1 mutant strain. Clustering and enrichment analysis showed over-representation of mitochondria-related Gene Ontology terms in clusters where gene expression was higher in the xyr1 mutant, indicating that mitochondria play a role in the altered metabolic state associated with the xyr1 mutation. Metabolomics revealed that free tyrosine was more abundant in the xyr1 mutant strain in all measured timepoints, whereas multiple fatty acids were less abundant in the mutant strain on glucose. The results contribute to more in-depth knowledge on T. reesei physiology and aid in finding new targets for improved protein production.
Project description:The mitosporic fungus Trichoderma reesei is an industrial producer of enzymes for degradation of lignocellulosic polysaccharides to soluble monomers that can be fermented to biofuels. The genes encoding these enzymes in T. reesei have recently been shown to be clustered in the genome. Here we will show that the expression of these genes is epigenetically controlled at the heterochromatin level by the protein methyltransferase LAE1. Deletion of lae1 led to a loss of expression of the major cellulase and hemicellulase encoding genes, and resulted in an inability to grow on cellulose. The cellulase null phenotype was also seen with known soluble inducers of enzymes active on cellulose. In contrast, introduction of a second copy of lae1 or its enhanced expression under a strong constitutive promoter resulted in increased levels of cellulases. Thus, our data provides an experiment-based explanation for the advantage for clustering of cellulases in the genome of T. reesei, and imply that the heterochromatin structure is a major determinant of cellulase gene expression and hence an attractive target for strain improvement. We used two biological replicas of four T. reesei strains growing on lactose, the parent strain (QM9414), a delta-lae1, and two overexpressing strains (tef1:lae1 mutant 1 and mutant 2).