Trichoderma reesei

About: Trichoderma reesei is a research topic. Over the lifetime, 3832 publications have been published within this topic receiving 152877 citations. The topic is also known as: Trichoderma reesi.

Three-dimensional structures of three engineered cellulose-binding domains of cellobiohydrolase I from Trichoderma reesei.

Abstract: Three-dimensional solution structures for three engineered, synthetic CBDs (Y5A, Y31A, and Y32A) of cellobiohydrolase I (CBHI) from Trichoderma reesei were studied with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. According to CD measurements the antiparallel beta-sheet structure of the CBD fold was preserved in all engineered peptides. The three-dimensional NMR-based structures of Y31A and Y32A revealed only small local changes due to mutations in the flat face of CBD, which is expected to bind to crystalline cellulose. Therefore, the structural roles of Y31 and Y32 are minor, but their functional importance is obvious because these mutants do not bind strongly to cellulose. In the case of Y5A, the disruption of the structural framework at the N-terminus and the complete loss of binding affinity implies that Y5 has both structural and functional significance. The number of aromatic residues and their precise spatial arrangement in the flat face of the type I CBD fold appears to be critical for specific binding. A model for the CBD binding in which the three aligned aromatic rings stack onto every other glucose ring of the cellulose polymer is discussed.

The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on d-galactose.

Abstract: Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei). Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H. jecorina, which catalyses the first step in d-galactose catabolism. It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis. Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding. A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d-galactose, l-arabinose and their corresponding polyols. Deletion of gal1 reduces growth on d-galactose but does only slightly affect growth on lactose. This is the result of the operation of a second pathway for d-galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta-gal1 strain. In this pathway, galactitol is catabolised by the lad1-encoded l-arabinitol-4-dehydrogenase, because a gal1/lad1 double delta-mutant failed to grow on d-galactose. In the delta-gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose-1-phosphate uridylyltransferase) by d-galactose, but not by l-arabinose, is impaired. Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose-derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.

Characterization of a Thermomonospora fusca exocellulase

Abstract: The exocellulase E3 gene was cloned on a 7.1 kb NotI fragment from Thermomonospora fusca genomic DNA into Escherichia coli and expressed in Streptomyces lividans. The E3 gene was sequenced and encoded a 596 residue peptide. The molecular masses of the native and cloned E3s were determined by mass spectrometry, and the value for E. coli E3, 59,797 Da, agreed well with that predicted from the DNA sequence, 59,646 Da. The value of 61,200 Da for T. fusca E3 is consistent with E3 being a glycoprotein. E3 is thermostable, retaining full activity after 16 h at 55 degrees C. It also has a broad pH optimum around 7-8, retaining 90% of its maximal activity between pH 6 and 10. The cloned E3s were identical to the native enzyme in their activity, cellulose binding, and thermostability. Papain digestion produced a 45.7 kDa catalytic domain with 77% of the native activity on amorphous cellulose and 33% on crystalline cellulose. E3 belongs to cellulase family B and retains the residues that have been identified to be crucial for catalytic activity in Trichoderma reesei cellobiohydrolase II and T. fusca E2. The E3 gene contains a 14 bp inverted repeat regulatory sequence 212 bp before the translational start codon instead of the 30-70 bp found for the other T. fusca cellulase genes. An additional copy of this sequence with one base changed is 314 bp before the translational start codon. The transcriptional start site of the E3 gene was shown to be between these two inverted repeats.