CHARACTERIZATION OF THE TRICHODERMA-REESEI CBH2 PROMOTER

A 613-bp fragment of the 5' upstream region of the Trichoderma reesei cbh2 gene (coding for the cellulolytic enzyme cellobiohydrolase II) has been isolated and sequenced. Fusion of this fragment to the E. coli uidA gene (coding for beta-glucuronidase) leads to - albeit low - expression of beta-glucuronidase activity in the presence of cellulose and upon the addition of low molecular weight inducers (sophorose, lactose) of cellobiohydrolase II. It also governed the formation of beta-glucuronidase activity during sporulation and its transport to the conidial surface. However, despite the presence of a signal peptide in the cbh2:uidA fusion, beta-glucuronidase was not secreted in T reesei. Defined fragments of the 613-bp promoter region were isolated and used to identify areas involved in the regulation of cbh2 expression by protein-DNA binding assays. At least two binding areas - between - 443/ - 363 and - 363/ - 173, respectively - were identified. In both areas, the DNA-protein complex observed was appreciably larger when cell-free extracts from sophorose-induced mycelia were used. This suggests that at least one of the proteins regulating cbh2 transcription is itself induced by cellulose.