FUNCTIONAL-ANALYSIS OF THE CELLOBIOHYDROLASE-I PROMOTER OF THE FILAMENTOUS FUNGUS TRICHODERMA-REESEI

Functional analysis of the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei was carried out using the Escherichia coli l acZ gene as a reporter. An assay based on cultivation on solid medium in microtiter plates was developed that allows rapid and reliable semi quantitative analysis of beta-galactosidase expression of a large numb er of transformants. A series of deletions and specifically designed a lterations were made covering 2.2 kb of the cbh1 promoter. Removal of sequences upstream of nucleotide - 500 in relation to the initiator AT G abolished glucose repression. Mutation of a single hexanucleotide se quence 5'GTGGGG at nucleotide - 720 was sufficient for derepression. T his site is similar to the binding sites of the glucose repressors MIG 1 of Saccharomyces cerevisiae and CREA/CREI of filamentous fungi. Remo val of the glucose repressor site did not affect sophorose induction. Sophorose induction of the promoter was retained even in deletion deri vatives lacking sequences upstream of position - 161, which retained a bout 70 bp upstream of the transcription start point and only 30 bp up stream of the TATA box.