Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei

Abstract

 Functional analysis of the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei was carried out using the Escherichia coli lacZ gene as a reporter. An assay based on cultivation on solid medium in microtiter plates was developed that allows rapid and reliable semiquantitative analysis of β-galactosidase expression of a large number of transformants. A series of deletions and specifically designed alterations were made covering 2.2 kb of the cbh1 promoter. Removal of sequences upstream of nucleotide −500 in relation to the initiator ATG abolished glucose repression. Mutation of a single hexanucleotide sequence 5′GTGGGG at nucleotide −720 was sufficient for derepression. This site is similar to the binding sites of the glucose repressors MIG1 of Saccharomyces cerevisiae and CREA/CREI of filamentous fungi. Removal of the glucose repressor site did not affect sophorose induction. Sophorose induction of the promoter was retained even in deletion derivatives lacking sequences upstream of position −161, which retained about 70 bp upstream of the transcription start point and only 30 bp upstream of the TATA box.