**16. The transcriptional regulators Xyr1, Ace1 and Ace2**

In *Aspergillus*, the xylanolytic and cellulolytic enzymatic system is strictly co-regulated via the inducer D-xylose, while enzymes involved in the same systems in *T. reesei* are not mainly acti‐ 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 such regulation still remain to be investigated [6].

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‐

224 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

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

In *Aspergillus*, the xylanolytic and cellulolytic enzymatic system is strictly co-regulated via the inducer D-xylose, while enzymes involved in the same systems in *T. reesei* are not mainly acti‐

sion of the xylanase-encoding genes *xynF1* and *xynF2* [112].

importance for the transcription of hydrolase-encoding genes.

**16. The transcriptional regulators Xyr1, Ace1 and Ace2**

**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‐ lan arabinofuranohydrolase induces the arabinolytic system.

Ace2 belongs to a zinc-binuclear cluster DNA-binding protein and appears to be an activa‐ tor of cellulase and hemicellulase genes in cellulose-induced cultures of *T. reesei*. Deletion of the *ace2* gene resulted in decreased expression of the cellulase genes *cbh1*, *cbh2*, *egl1*, *egl2*, and the xylanase gene *xyn2* upon *T. reesei* growth on cellulose as sole carbon source [119]. The same study has been demonstrated that the expression of these genes were not affected after *T. reesei* growing on sophorose, suggesting partially different mechanisms for hydro‐ lase-encoding genes expression upon different carbon sources [119]. Homologs of *ace2* in *A. nidulans*, *A. niger*, *N. crassa* and *Magnaporthe grisea* were not found to date, suggesting that different transcriptional regulators are used by these fungi to induce the expression of hy‐ drolytic-encoding genes.

Furthermore, it was found that in *Aspergillus aculeatus*, a new XlnR-independent pathway for the regulation of cellulase- and hemicellulase-encoding genes exists [127]. This study suggests that cellobiose from Avicel (crystalline cellulose) degradation stimulates only the XlnR-independent signaling pathway for cellulase/hemicellulase production in *A. aculeatus*. In addition, sequential promoter truncation studies on *cbh1* gene demonstrated that a con‐ served sequence in the promoter region of *cbh1*, namely CeRe (required for *eglA* induction in *A. nidulans*; [128], plays a pivotal role on the expression regulated by the XlnR-independent

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http://dx.doi.org/10.5772/54325

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AmyR is another transcriptional activator found in Aspergilli. AmyR was first described as a transcriptional regulator of genes encoding enzymes involved in starch and maltose hy‐ drolysis [129]. Nowadays, studies have been demonstrated a broader role for AmyR, which appears to regulate another gene expression systems. High levels of both α- and β-glucosi‐ dase as well as α- and β-galactosidase in the *amyR* multicopy strain of *A. niger* were found [130]. This study also demonstrated that AmyR-regulated genes in *A. niger* are induced dur‐ ing growth in low levels of D-glucose, as their expression increased during the cultivation. As D-glucose has been shown to act as a repressor through the carbon catabolite repressor protein CreA [75] (see next section), the authors suggested that repression levels of those genes are likely a balance between induction through AmyR and repression through CreA.

**18. Transcriptional regulators of plant polysaccharide degradation genes**

The filamentous ascomycete fungus *Neurospora crassa* has been commonly used as a model laboratory organism [132]. In nature, *N. crassa* can be found on burnt plant material, primar‐ ily grasses, including sugarcane and *Miscanthus* [133]. Previous studies have been demon‐ strated that *N. crassa* is able to express and secrete many plant cell wall degrading enzymes after grown on ground *Miscanthus* stems and crystalline cellulose [134]. Studies conducted with strains containing deletions of predicted transcription factors (TFs) in *N. crassa* demon‐ strated that a specific TF, named XLR1 (xylan degradation regulator-1), is essential for hemi‐ cellulose degradation in *N. crassa* [135]. The *xlr-1* gene is an ortholog to XlnR/Xyr1 found in *Aspergillus* and *Trichoderma* species, respectively. The results presented in this study have been shown that deletion of *xlr-1* in *N. crassa* abolished growth on xylan and xylose, but growth on cellulose and cellulolytic activities were not highly affected. The transcriptional profiling showed that *xlr-1* is required for induction of hemicellulase and xylose metabolism genes, and modulated the expression levels of few cellulase genes, but these genes do not require XLR-1 for induction [135]. These findings suggested that unknown TFs in *N. crassa* could be important for the induced expression of genes encoding cellulases in response to

In fact, studies assessing a near-full genome deletion strain set in *N. crassa*, have been shown two transcription factors, named *clr-1* and *clr-2*, required for degradation of cellulose [136].

signaling pathway triggered by cellulosic compounds in *A. aculeatus* [127].

Similar results were obtained for AmyR in *Aspergillus oryzae* [131].

**in Neurospora crassa**

the presence of cellulose.
