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To construct a yeast one-hybrid system and isolate transcriptional activators.A 1·1-kb promoter region of xylanase2 from Trichoderma reesei was cloned by PCR and sequenced (GenBank accession number: AY263380). Sequence analysis revealed that typical binding sites for several transcription factors in filamentous fungi, such as CREI, XLNR, ALCR, AREA and CCAAT enhancer, are located in the promoter. To isolate xyn2 transcription factors, the reporter plasmid of a yeast one-hybrid system was constructed on the backbone of the plasmid pRS415 containing the leu2 selective marker, with the xyn2 promoter region and Saccharomyces cerevisiae his4 as a reporter gene. The reporter gene contained 123-bp minimal promoter region. The S. cerevisiae H158 strain containing the reporter plasmid was transformed with a T. reesei expression cDNA library, and 34 transformants were collected from SC-Leu-His-Ura plates. The isolation of the gene ace2 from several transformants showed that the one-hybrid system approach was successful. Then, approx. 59 mg l−1 of ace2 was overexpressed in Escherichia coli BL21.The yeast one-hybrid system is suitable for isolating transcription factors of filamentous fungi. ACE II is a main and universal transcriptional activator that controls cellulase and hemicellulase transcription regulation in T. reesei.