TRANSFORMATION OF TRICHODERMA-REESEI WITH THE CELLOBIOHYDROLASE-II GENE AS A MEANS FOR OBTAINING STRAINS WITH INCREASED CELLULASE PRODUCTION AND SPECIFIC ACTIVITY

Trichoderma reesei QM 9414 was shown to contain a single copy of both the cbh1 and cbh2 gene, coding for cellobiohydrolase I (CBH I) and II (CBH II) synthesis. In the attempt to increase CBH II formation, multiple copies of the cbh2 gene were introduced by cotransformation with the Neurospora crassa pyr4 gene using a pyrG auxotrophic mutant of T. reesei QM 9414 (T. reesei TU-6). Transformants with mitotically stable pyrG prototrophy, were shown to display both homologous as well as ectopic integration of the cbh2 gene. The cbh2 copy number varied between 10- and 20-fold. Secretion of CBH I and II by the transformants during growth on lactose as a carbon source leading to cellulase formation was quantified by means of monoclonal antibodies. Three out of 14 transformants exhibited a 2- to 4-fold increased CBH II titer. Hence, no correlation between the cbh2 copy number and the amount of secreted CBH II was observed. The same three transformants exhibited a roughly 1.5-fold increase in specific cellulase activity (FPU). The crude cellulase mixture of transformant 3, which displayed a 3- to 4-fold elevated CBH II formation, exhibited an increased specific activity against crystalline cellulose in vitro. It is therefore concluded that transformation with individual cellulase genes can be a useful and simple tool to alter the quantitative pattern of cellulolytic enzymes produced by T. reesei.