Transcriptional regulation of xyn3 in the filamentous fungus Trichoderma reesei PC-3-7

The filamentous fungus Trichoderma reesei is a one of the most efficient producers of xylanolytic and cellulolytic enzymes. In T. reesei, the production of these enzymes is regulated at the transcriptional level depending on the carbon source available. Recently, isolation of transcription factors involved in cellulase and xylanase genes has been reported. However, the roles of these factors have only been investigated for the major cellulase genes (cbh1, cbh2, egl1 and egl2) and two major xylanase genes (xyn1 and xyn2). Furthermore, the physiological binding sequence of the regulators has not yet been characterised.

In order to elucidate the molecular mechanisms regulating xyn3 expression in T. reesei PC-3-7, here we defined cis-acting elements in the 1071-bp upstream region of xyn3 by using detailed deletion and mutation analysis. In addition to the typical Xyr1/ACEII-binding motif (5’-GGCTAA-3’), the analogous motifs 5’-GGCTAT-3’ and 5’-GGCAAA-3’ present as an inverted repeat spaced by 16-bp internal sequences were identified as essential elements for xyn3 expression. Electrophoretic mobility shift assay using heterologously expressed DNA binding domain of Xyr1 demonstrates that all the identified cis-acting elements are able to interact with Xyr1. Furthermore, no xyn3 transcripts were formed in the xyr1-knockout strain upon induction by sophorose and L-sorbose. These results indicate that xyn3 expression in T. reesei PC-3-7 is transcriptionally regulated by Xyr1, and suggest that the 5’-GGCTAT-3’ and 5’-GGCAAA-3’ motifs play roles in Xyr1-mediated cellulase and xylanaser gene expression in T. reesei.