Project description:BACKGROUND:The presence of low complexity and repeated regions in genomes often results in difficulties to assemble sequencing data into full chromosomes. However, the availability of full genome scaffolds is essential to several investigations, regarding for instance the evolution of entire clades, the analysis of chromosome rearrangements, and is pivotal to sexual crossing studies. In non-conventional but industrially relevant model organisms, such as the ascomycete Trichoderma reesei, a complete genome assembly is seldom available. RESULTS:The chromosome scaffolds of T. reesei QM6a and Rut-C30 strains have been generated using a contact genomic/proximity ligation genomic approach. The original reference assembly, encompassing dozens of scaffolds, was reorganized into two sets of seven chromosomes. Chromosomal contact data also allowed to characterize 10-40 kb, gene-free, AT-rich (76%) regions corresponding to the T. reesei centromeres. Large chromosomal rearrangements (LCR) in Rut-C30 were then characterized, in agreement with former studies, and the position of LCR breakpoints used to assess the likely chromosome structure of other T. reesei strains [QM9414, CBS999.97 (1-1, re), and QM9978]. In agreement with published results, we predict that the numerous chromosome rearrangements found in highly mutated industrial strains may limit the efficiency of sexual reproduction for their improvement. CONCLUSIONS:The GRAAL program allowed us to generate the karyotype of the Rut-C30 strain, and from there to predict chromosome structure for most T. reesei strains for which sequence is available. This method that exploits proximity ligation sequencing approach is a fast, cheap, and straightforward way to characterize both chromosome structure and centromere sequences and is likely to represent a popular convenient alternative to expensive and work-intensive resequencing projects.
Project description:A 1,4-beta-D-glucan cellobiohydrolase (EC 188.8.131.52) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography. A single protein band in polyacrylamide-gel electrophoresis and one arc in immunoelectrophoresis corresponded to the enzyme activity. The Mr was 65 000. The pI was 4.2-3.6. The purified enzyme contained about 10% hexose. The enzyme differs from previously described cellobiohydrolases in being more effective in the hydrolysis of cellulose.
Project description:Filamentous fungi are indispensable biotechnological tools for the production of organic chemicals, enzymes, and antibiotics. Most of the strains used for industrial applications have been--and still are--screened and improved by classical mutagenesis. Sexual crossing approaches would yield considerable advantages for research and industrial strain improvement, but interestingly, industrially applied filamentous fungal species have so far been considered to be largely asexual. This is also true for the ascomycete Trichoderma reesei (anamorph of Hypocrea jecorina), which is used for production of cellulolytic and hemicellulolytic enzymes. In this study, we report that T. reesei QM6a has a MAT1-2 mating type locus, and the identification of its respective mating type counterpart, MAT1-1, in natural isolates of H. jecorina, thus proving that this is a heterothallic species. After being considered asexual since its discovery more than 50 years ago, we were now able to induce sexual reproduction of T. reesei QM6a and obtained fertilized stromata and mature ascospores. This sexual crossing approach therefore opens up perspectives for biotechnologically important fungi. Our findings provide a tool for fast and efficient industrial strain improvement in T. reesei, thus boosting research toward economically feasible biofuel production. In addition, knowledge of MAT-loci and sexual crossing techniques will facilitate research with other Trichoderma spp. relevant for agriculture and human health.
Project description:The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.
Project description:Trichoderma reesei QM9414, Trichoderma viride 3.316, Aspergillus niger M85, and Aspergillus niger M92 were screened for hydrolyzing geniposide into genipin. T. reesei was selected according to the ?-glucosidase activity of the fermentation broths using geniposide as a substrate. T. reesei was immobilized by embedding method using sodium alginate as the carrier. Geniposide was hydrolyzed by immobilized T. reesei at 28°C (200?rpm) for 34?h, and the yield of genipin was 89%. The product was purified and identified by UV, IR, EIMS, and 1H-NMR. Since there were two sets of signals in 1H-NMR spectra, a series of experiments were performed and verified that the existence of two conformations was the main reason. Generally, biotransformation of geniposide into genipin by immobilized T. reesei provides a promising solution to the genipin production.
Project description:Species of the fungal genus Trichoderma (Hypocreales, Ascomycota) are well-known for their production of various secondary metabolites. Nonribosomal peptides and polyketides represent a major portion of these products. In a recent phylogenomic investigation of Trichoderma polyketide synthase (PKS)-encoding genes, the pks4 from T. reesei was shown to be an orthologue of pigment-forming PKSs involved in synthesis of aurofusarin and bikaverin in Fusarium spp. In this study, we show that deletion of this gene in T. reesei results in loss of green conidial pigmentation and in pigmentation alteration of teleomorph structures. It also has an impact on conidial cell wall stability and the antagonistic abilities of T. reesei against other fungi, including formation of inhibitory metabolites. In addition, deletion of pks4 significantly influences the expression of other PKS-encoding genes of T. reesei. To our knowledge, this is the first indication that a low-molecular-weight pigment-forming PKS is involved in defense, mechanical stability, and stress resistance in fungi.
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
Project description:Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50 °C, and V max = 2646 nkat/mg with a K m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes.
Project description:Background:The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) displays increased cellulase expression while growing on inducers such as lactose or cellulose. However, the mechanism of cellulase induction in T. reesei is not yet completely characterized. Here, a protein annotated as β-glucosidase (BGL3I) was found to be involved in cellulase induction in T. reesei. The effects of BGL3I on cellulase production have not yet been fully understood. Results:Deletion of the bgl3i gene had no influence on the growth of T. reesei, but significantly increased its cellulase activities. Deletion of bgl3i also resulted in decreased extracellular galactosidase activity, but significantly increased transcription of lactose permeases, which might be involved in lactose transport. Furthermore, deletion of bgl3i enhanced the transcription levels of intracellular β-glucosidases cel1a, cel1b and the regulator xyr1, which are all essential for lactose induction in T. reesei. BGL3I was found to have a relatively high ability to hydrolyze sophorose, which is proposed to be the strongest natural inducer of cellulase synthesis in T. reesei. Conclusions:BGL3I may take part in the complex regulating system of cellulase induction. The deletion of bgl3i offers a new strategy to improve T. reesei strain performance.
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. Overall design: One biological replicate