Characterization of novel transcription factors ACEI and ACEII involved in regulation of cellulase and xylanase genes in Trichoderma reesei: Dissertation

Cellulose and hemicellulose are the most abundant renewable carbon sources on earth. Filamentous fungi produce a wide variety of extracellular enzymes that degrade these complex polymeric compounds and play an important role in carbon turnover in nature. The filamentous fungus Trichoderma reesei (Hypocrea jecorina) is the best studied cellulolytic fungus and it is widely used by the biotechnical industry for production of hydrolytic enzymes such as cellulases and xylanases. The production of these enzymes is regulated at the transcriptional level. The cellulase genes, especially the main cellulase cbh1, are very highly expressed and thus their promoters are among the strongest known in nature. Despite the importance of cellulases and xylanases very little is known of the regulatory mechanism involved in the high level of expression of cellulase and xylanase genes In this work a novel method was developed to isolate transcription factors without previous knowledge of the important DNA sequence elements in promoters or of the nature of the activator genes and proteins. This method selects for simultaneous binding and activation of the desired promoter in S. cerevisiae and is in general applicable for any organism and promoter. Using this method two new genes, ace1 and ace2, encoding transcription factors binding to the promoter of the main cellulase gene cbh1 of T. reesei were isolated. ace1 encodes a protein that contains three zinc finger motifs of Cys2-His2 type. Amino acid sequence similarity is seen towards A. nidulans protein StzA and deduced N. crassa protein, but not to yeast proteins suggesting that ace1 is a regulator specific for filamentous fungi. Gel mobility shift assays revealed at least eight putative binding sites for ACEI scattered in the cbh1 promoter all containing the core sequence 5'AGGCA. Deletion of ace1 gene in T. reesei resulted in increased expression of all the main cellulase genes cbh1, cbh2, egl1 and egl2 and the xylanase genes xyn1 and xyn2 indicating that ACEI down regulates cellulase and xylanase expression. The second factor ACEII belongs to the family of zinc binuclear cluster proteins found exclusively in fungi. ACEII binds to at least one sequence in vitro in the cbh1 promoter. Disruption of ace2 results in reduced expression levels of all the main cellulase genes and the fungus showed reduced cellulase activities when grown on cellulose containing media. Also the expression of gene encoding one of the main hemicellulases, b-xylanase XYNII was reduced on cellulose medium.