Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina.

Xyr1 (xylanase regulator 1) of the ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) was recently demonstrated to play an essential role in the transcriptional regulation of the xyn1 (xylanase 1-encoding) gene expression. Consequently, this study reports on the deletion of the xyr1 gene from the H. jecorina genome. Comparative studies of the growth behavior of the different mutant strains (deleted and retransformed xyr1) grown on various carbon sources pointed to the strongly reduced ability of the xyr1 deletion strain to utilize D-xylose and xylan. Transcriptional analysis of the xyl1 (D-xylose reductase 1-encoding) gene as well as measurements of corresponding enzymatic activities gave evidence that Xyr1 takes part in the control of the fungal D-xylose pathway, in particular in the regulation of D-xylose reductase. It could be demonstrated that the uptake of D-xylose into the fungal cell is uninfluenced in the Deltaxyr1 strain. Furthermore, transcriptional regulation of the major hydrolytic enzyme-encoding genes xyn1 and xyn2 (xylanases 1 and 2), cbh1 and cbh2 (cellobiohydrolases 1 and 2), and egl1 (endoglucanase 1) is strictly dependent on Xyr1. Regulation of the respective genes via Xyr1 is not affected by the substances mediating induction (xylose, xylobiose, and sophorose) and is indispensable for all modes of gene expression (basal, derepressed, and induced). Moreover, Xyr1, it was revealed, activated transcriptional regulation of inducer-providing enzymes such as beta-xylosidase BXLI and beta-glucosidase BGLI but was not shown to be involved in the regulation of BGLII.

Transcriptional regulation of xyn1, encoding xylanase I, in Hypocrea jecorina.

Two major xylanases (XYN I and XYN II) of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) are simultaneously expressed during growth on xylan but respond differently to low-molecular-weight inducers. In vivo footprinting analysis of the xylanase1 (xyn1) promoter revealed three different nucleotide sequences (5'-GGCTAAATGCGACATCTTAGCC-3' [an inverted repeat of GGCTAA spaced by 10 bp], 5'-CCAAT-3', and 5'-GGGGTCTAGACCCC-3' [equivalent to a double Cre1 site]) used to bind proteins. Binding to the Cre1 site is only observed under repressed conditions, whereas binding to the two other motifs is constitutive. Applying heterologously expressed components of the H. jecorina cellulase regulators Ace1 and Ace2 and the xylanase regulator Xyr1 suggests that Ace1 and Xyr1 but not Ace2 contact both GGCTAA motifs. H. jecorina transformants containing mutated versions of the xyn1 promoter, leading to elimination of protein binding to the left or the right GGCTAA box revealed either strongly reduced or completely eliminated induction of transcription. Elimination of Cre1 binding to its target released the basal transcriptional level from glucose repression but did not influence the inducibility of xyn1 expression. Mutation of the CCAAT box prevents binding of the Hap2/3/5 complex in vitro and is partially compensating for the loss of transcription caused by the mutation of the right GGCTAA box. Finally, evidence for a competition of Ace1 and Xyr1 for the right GGCTAA box is given. These data prompted us to hypothesize that xyn1 regulation is based on the interplay of Cre1 and Ace1 as a general and specific repressor with Xyr1 as transactivator.

Transcriptional regulation of xyn2 in Hypocrea jecorina.

The xylanase system of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) consists of two specific xylanases, Xyn1 and Xyn2, which are simultaneously expressed during growth on xylan but respond differentially to low-molecular-weight inducers. Using in vivo footprinting analysis of xylan-induced and noninduced mycelia, we detected two adjacent nucleotide sequences (5'-AGAA-3' on the noncoding strand and 5'-GGGTAAATTGG-3', referred to as the xylanase-activating element [XAE], on the coding strand, respectively) to bind proteins. Among these, binding to the AGAA-box is only observed under noninduced conditions, whereas binding to XAE is constitutive. Electrophoretic mobility shift assay with heterologously expressed components of the H. jecorina Hap2/3/5 protein complex and the cellulase regulator Ace2 suggests that these two transactivators form the protein complex binding to XAE. H. jecorina transformants, containing correspondingly mutated versions of the xyn2 promoter fused to the Aspergillus niger goxA gene as a reporter, revealed that the elimination of protein binding to the AGAA-box resulted in a threefold increase in both basal and induced transcription, whereas elimination of Ace2 binding to its target in XAE completely eliminated transcription under both conditions. Destruction of the CCAAT-box by insertion of a point mutation prevents binding of the Hap2/3/5 complex in vitro and results in a slight increase in both basal and induced transcription. These data support a model of xyn2 regulation based on the interplay of Hap2/3/5, Ace2 and the AGAA-box binding repressor.