Disruption of the Trichoderma reesei cbh2 gene coding for cellobiohydrolase II leads to a delay in the triggering of cellulase formation by cellulose

Abstract

The role of the major conidial-bound cellulase — cellobiohydrolase II (CBH II) — in the triggering of cellulase formation in the fungus Trichoderma reesei was investigated by comparing the mutant strain QM 9414 with a recombinant strain unable to produce CBH II. For this purpose, the cbh2 gene was isolated from a chromosomal gene bank of T. reesei, cloned into pGEM-7Zf(+), and disrupted by insertion of the homologous pyr4 gene in its coding region to yield the plasmid vector pSB3. Transformation of the auxotrophic, pyr4-negative strain T. reesei TU-6 with pSB3 yielded 23 stable prototrophs, of which three were unable to produce CBH II — assessed by means of a monoclonal antibody — during growth on lactose or in the presence of sophorose. However, they formed cellobiohydrolase I (CBH I) at a rate comparable to strain QM 9414 under these conditions. Southern analysis of DNA of some CBH II− and CBH II+ transformants confirmed that pSB3 had integrated at the cbh2 locus in the CBH II− strains. The latter displayed normal growth on glucose or maltose as carbon source. They showed retarded growth on cellulose as sole carbon source, however, and exhibited a lag in the time course of CBH I and EG I formation, although producing roughly the same final cellulase activities. It is concluded from these results that CBH II is not essential for induction of cellulase formation by cellulose, but that it contributes significantly to the formation of lower molecular mass inducers in the early phase of growth of the fungus on cellulose.