Regulation of transcription of cellulases- and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei).

The filamentous fungi Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) have been the subject of many studies investigating the mechanism of transcriptional regulation of hemicellulase- and cellulase-encoding genes. The transcriptional regulator XlnR that was initially identified in A. niger as the transcriptional regulator of xylanase-encoding genes controls the transcription of about 20-30 genes encoding hemicellulases and cellulases. The orthologous xyr1 (xylanase regulator 1-encoding) gene product of H. jecorina has a similar function as XlnR, although at points, the mechanisms seems to be different. Specifically in H. jecorina, the interaction of Xyr1 and the co-regulators Ace1 and Ace2 in the regulation of transcription of xylanases and cellulases has been studied. This paper describes the similarities and differences in the transcriptional regulation of expression of hemicellulases and cellulases in A. niger and H. jecorina.

Role of Ace2 (Activator of Cellulases 2) within the xyn2 transcriptosome of Hypocrea jecorina.

Ace2 (Activator of Cellulases 2)-encoding gene was deleted from and retransformed in the H. jecorinaQM9414 genome. Comparison of xylanase activity and xyn2 transcription of the corresponding strains after cultivation on inducing compounds (xylan, xylobiose) revealed a faster initial inducibility in the Deltaace2-strain, but final levels of xyn2 transcript and xylanase activity of the parental strain could not be reached. This suggests a role for Ace2 in the regulation of xyn2 induction mechanisms, moreover Ace2 is responsible for the basal level of xyn2 transcription. Furthermore, a palindrome in the xyn2 promoter consisting of a GGGTAA- and a CCAGCC-element was identified. Both Xyr1 and Ace2 are able to bind the complete motif, the latter also only to one part of it. Phosphorylation as well as dimerization are prerequisites for binding of Ace2 to the xyn2 promoter. Finally, the impact of Ace2 on xyr1 transcription could be demonstrated under inducing conditions.

Xyr1 receives the lactose induction signal and regulates lactose metabolism in Hypocrea jecorina.

This study reports the vital regulatory influence of Xyr1 (xylanase regulator 1) on the transcription of hydrolytic enzyme-encoding genes and hydrolase formation on lactose in Hypocrea jecorina. While the transcription of the xyr1 gene itself is achieved by release of carbon catabolite repression, the transcript formation of xyn1 (xylanase 1) is regulated by an additional induction mechanism mediated by lactose. Xyr1 has an important impact on lactose metabolism by directly activating xyl1 (xylose reductase 1) transcription and indirectly influencing transcription of bga1 (beta-galactosidase 1). The latter is achieved by regulating the conversion of D-galactose to the inducing carbon source galactitol.

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.