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:Enzyme production by T. reesei Rut C-30 was studied in submerged cultures on five different cellulose-rich substrates, namely, commercial cellulose Avicel® and industrial-like cellulosic pulp substrates which consist mainly of cellulose, but also contain residual hemicellulose and lignin. In order to evaluate the hydrolysis of the substrates by the fungal enzymes, the spatial polymer distributions were characterised by cross-polarization magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS 13C-NMR) in combination with spectral fitting. Proteins in culture supernatants at early and late stages of enzyme production were labeled by Tandem Mass Tags (TMT) and protein profiles were analysed by liquid chromatography-tandem mass spectrometry.
Project description:Trichoderma reesei is the main industrial producer of cellulases and hemicellulases used to depolymerize biomass in many biotechnical applications. Many production strains in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in hyperproducing mutants of T. reesei by high-resolution comparative genomic hybridisation tiling array. We carried out aCGH analysis of four hyperproducing strains (QM9123, QM9414, NG14 and RutC-30) using QM6a genome as a reference. ArrayCGH analysis identified dozens of mutations in each strain analyzed.
Project description:Trichoderma reesei is the main industrial producer of cellulases and hemicellulases used to depolymerize biomass in many biotechnical applications. Many production strains in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in hyperproducing mutants of T. reesei by high-resolution comparative genomic hybridisation tiling array. We carried out aCGH analysis of four hyperproducing strains (QM9123, QM9414, NG14 and RutC-30) using QM6a genome as a reference. ArrayCGH analysis identified dozens of mutations in each strain analyzed. 2.1 million oligonucleotide probe custom aCGH (HD2 format, RocheNimblegen) was designed according to T. reesei strain QM6a genome v2.0 (http://genome.jgi-psf.org/Trire2/Trire2.home.html). 14 samples are included in this set; 3 replicates of each strain (except two replicates of QM9123) were analyzed (four mutant strains and QM6a control strain for self-hybridization)
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