Project description:The fungus Trichoderma reesei is one of the most used industrial cellulase producers due to its high capacity of protein secretion. The strain was shown to possess an expanded endoplasmic reticulum, but the genetic factors responsible for this phenotype remain still unidentified. Recently, three new transcription factors were described in Neurospora crassa which were demonstrated to be involved in protein secretion. The orthologous gene of one of them, Res-2, was deleted in the T. reesei hyperproducer strain Rut-C30 and its role on protein secretion analyzed. The mutant strain showed slower growth on all substrates tested, but the presence of the stress agent DTT had contrasting effects. Protein secretion tended to be lower in the deletion strain compared to the parental strain Rut-C30. Analysis of the transcriptomes of the Rut-C30 and the Δres-2 mutant strain in secretion stress conditions reveiled remarkably few differences, but induction of cellulase secretion and/or presence of DTT led to differential expression of genes coding for transporters, redox genes and lipid metabolism. These results suggest that in the T. reesei Rut-C30 strain, Res-2 does not act as a master regulator of the secretion pathway, but contributes to a high protein secretion by adjusting the expression of genes involved in membrane turnover and the redox status of the organism or specific organelles.
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:We perform a self hybridisation comprative genomic hybridization (CGH) in order to validate the probe tiling design we done on Trichoderma reesei. This hybridization was done using QM6a wild type strain.
Project description:We perform a self hybridisation comprative genomic hybridization (CGH) in order to validate the probe tiling design we done on Trichoderma reesei. This hybridization was done using QM6a wild type strain. One biological replicate