The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30
In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated PCR-primers were designed to amplify a 0.7-bp fragment of the cre1 gene, which was used to clone the entire gene. It encodes a 402-amino acid protein with a calculated M(r) of 43.6 kDa. Its aa-sequence shows 55.6% and 54.7% overall similarity to the corresponding genes of Aspergillus nidulans and A. niger, respectively. Similarity was restricted to the aa-region containing the C2H2 zinc finger and several aa-regions rich in proline and basic amino acids, which may be involved in the interaction with other proteins. Another aa-region rich in the SPXX-motif that has been considered analogous to a region of yeast RGR1p, was instead identified as a domain occurring in several eucaryotic transcription factors. The presence of the cre1 translation product was demonstrated with polyclonal antibodies against Cre1, which identified a protein of 43 (+/- 2) kDa in cell-free extracts from T. reesei. A Cre1 protein fragment from the two zinc fingers to the region similar to the aa-sequence of eucaryotic transcription factors, was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. EMSA and in vitro footprinting revealed binding of the fusion protein to the sequence 5'-GCGGAG-3', which matches well with the A. nidulans consensus sequence for CreA binding (5'-SYGGRG-3'). Cell-free extracts of T. reesei formed different complexes with DNA-fragments carrying this binding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated. We conclude that T. reesei Cre1 is the functional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.