**15. The transcriptional activator XlnR**

Complementation by transformation of an *A. niger* mutant lacking xylanolytic activity led to the isolation of *xlnR* gene, a gene encoding the first known transcriptional activator controlling the expression of genes for xylanolytic and cellulolytic enzymes in filamentous fungi [108]. Ini‐ tially, it was thought that XlnR was able to regulate the expression of the xylanolytic genes en‐ coding two endoxylanases (*xlnB* and *xlnC*) and a β-xylosidase (*xlnD*), and the transcription of genes encoding some accessory enzymes involved in xylan degradation including α-glucuro‐ nidase A (*aguA*), acetylxylan esterase A (*axeA*), arabinoxylan arabinofuranohydrolase A (*ax‐ hA*), and feruloyl esterase A (*faeA*). Furthermore, XlnR has been found to activate the transcription of two endoglucanase-encoding genes, *eglA* and *eglB*, indicating that transcrip‐ tional regulation by XlnR includes cellulase-encoding genes [109]. Currently, it is known that XlnR actually controls the transcription of about 20-30 genes encoding hemicellulases and cel‐ lulases, and a gene encoding a D-xylose reductase (*xyrA*), involved in intracellular D-xylose metabolism [110]. These results demonstrate an interconnection of extracellular xylan degra‐ dation and intracellular D-xylose metabolism, coupled via transcriptional regulation of *xyrA* gene by XlnR. In this way, the fungus is able to adapt intracellular D-xylose metabolism to ex‐ tracellular xylan degradation, indicating a high level of metabolic regulation. Based on these findings, a model was proposed to explain the activation of XlnR regulon [111]. Basically, car‐ bon limitation minimize carbon catabolite repression, and this de-repressed condition favours monomeric sugars or their derivatives acting as inducers of cellulolytic/hemicellulolytic sys‐ tem. The nature of the monomeric sugar drives the polysaccharide enzyme system to be in‐ duced [110]. Figure 5 shows a schematic model for the regulation through XlnR of genes encoding CWDEs in *A. niger*. Besides *A. niger*, the gene encoding XlnR has also been isolated from *A. oryzae*, where the corresponding protein AoXlnR demonstrated to control the expres‐ sion of the xylanase-encoding genes *xynF1* and *xynF2* [112].

vated by this sugar. As described above, the most potent inducer for cellulolytic system in *T. re‐ esei* is sophorose, whereas hemicellulases appear to be induced during *T. reesei* growth in the presence of cellulose, xylan, sophorose, xylobiose and L-arabitol [6]. Despite of the diversity of inducers, it was demonstrated that the transcriptional regulation of the major hydrolytic en‐ zyme-encoding genes *cbh1*, *cbh2* and *egl1* (cellulases), *xyn1*, *xyn2* (xylanases) is dependent on Xyr1, the XlnR homologue in *T. reesei*. Xyr 1 appears to be an essential activator for all levels of xylanase genes *xyn1* and *xyn2* transcription, receiving or mediating different signals from the inducer molecules [114]. In *xyn1* and *xyn2* promoters, the Xyr1-binding elements resemble the consensus sequences of *T. reesei* transcriptional regulator Ace1 [117]. Ace1 apparently acts as an antagonist of the Xyr1-driven *xyn1* gene transcription. Deletion of *xyr1* gene in *T. reesei* led to increased expression of cellulase and xylanase genes studied in cellulose- and sophorose-in‐ duced cultures, suggesting a negative effect of Ace1 on the induced expression of these genes [118]. In contrast, *ace1* activated the *cbh1* promoter in yeast, suggesting that Ace1 could be able to act as an inducer or a repressor, depending on the context, but the mechanisms involved in

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**Figure 5. A proposed model for regulation of genes encoding polysaccharide-degrading enzymes in** *A. niger***.** Sugar monomers or polysaccharides provoke the binding of the transcription factor XlnR to the upstream activating system (UAS) in the promoter region of the genes, inducing the respective enzymatic system. Genes encoding xylanas‐ es (*xlnB*, *xlnC*), xylosidase (*xlnD*), glucuronidase (*aguA*) and feruloyl esterase (*faeA*) induce the xylanolytic enzymatic system. Genes encoding cellobiohydrolases (*cbhA*, *cbhB*) and endoglucanases (*eglA*, *eglB*, *eglC*) induce the cellulolytic system. Genes encoding galactosidases (*aglB*, *lacA*) induce the galactolytic system and the gene encoding arabinoxy‐

such regulation still remain to be investigated [6].

lan arabinofuranohydrolase induces the arabinolytic system.

The XlnR transcriptional activator belongs to the class of zinc binuclear cluster domain pro‐ teins (PF00172) [113]. The DNA-binding domain is found in the XlnR at the N-terminal of the protein and, in addition to this domain, a fungal specific transcription factor domain is also present (PF04082) [110]. Functional studies have been demonstrated that a putative coiled-coil domain is important for the XlnR function, as the disruption of the α-helix structure (Leu650Pro mutation) lead to cytoplasmic localization and loss of function of XlnR, due to a loss of transcription of the structural genes of the regulon [114]. As demonstrated by the same study, a C-terminal portion of XlnR appeared to be involved in transcriptional regulation, as a deletion of some amino acids of the C-terminus increased the expression of XlnR target genes, even under D-glucose repression conditions [114]. Efforts have been done in order to evaluate the behavior of XlnR regulon to optimize the expression of target genes. For instance, a model‐ ing study for the observation of XlnR regulon dynamics under D-xylose induction was per‐ formed. In this study, it was demonstrated that regulation of the *A. niger* XlnR network system was dictated mainly by transcription and translation degradation rate parameters, and by Dxylose consumption profile [115, 116]. Structural and functional studies of XlnR are pivotal for the development of new strains with improved cellulase/hemicellulase production, given its importance for the transcription of hydrolase-encoding genes.
