Abstract
Cellobiohydrolase 1 (CBH1) is the major component of the Trichoderma cellulase system, amounting to more than 60 % of the total excreted proteins. As the production of CBH1 is inducible by various compounds and repressible by easily metabolizable carbon source like glucose, CBH1 synthesis thus appears to be controlled by its promoter in a complicated and variable mechanism. In order to investigate the mechanism of carbon catabolite repression in the filamentous fungus T. koningii G-39, the regulatory protein GRPT was obtained from the glucose-grown mycelia and purified from a DNA-ligand affinity column. The estimated molecular weight is comparable to the previous published Cre1, which is equivalent to the Aspergillus nidulans CREA and identified to be involved in the glucose repression. From the data of gel retardation, GRPT possess binding activity to the DNA fragments carrying putative consensus sequences. In vitro DNase I footprinting protection experiments reveal that binding of GRPT to the sequence 5-(G/C)TGGGG-3’, which matches well with the consensus sequences for MIG1/CREA repressors. We propose that the mediations by carbon catabolite repression in A. nidulans and Saccharomyces cerevisiae are operative in the cbh1 gene expression of T. koningii G-39. Moreover, reporter gene lacZ was expressed in S. cerevisiae to study the function of cbh1 promoter in vivo. Removal of the GRPT binding sites led the reporter □-galactosidase activity of yeast in 2 % glucose to a level similar to that observed in a derepressing carbon source (5 % glycerol). The results indicated that glucose inhibition affects the □–galactosidase activity more than galactose induction does. The data presented suggest that the five identified cis-acting elements, in cooperation with the repressor, GRPT, mediate the regulation of cbh1 transcription.