**19. Carbon catabolite repression in Aspergilli**

As briefly described above, microorganisms are known to adjust their carbon metabolism in order to minimize energy demands. One of these regulatory mechanisms is the carbon catabolite repression (CCR). Readily metabolizable carbon sources, such as glucose, are preferably catabolized and, in general, suppress the utilization of alternative carbon sour‐ ces, repressing mainly the enzymatic system required for the catabolism of less favorable carbohydrates. For general carbon catabolite repression in some Aspergilli species, the DNA-binding Cys2His2 zinc-finger repressor CreA is absolutely necessary [75]. In general, the negative effect of this regulatory system depends on the concentration of the prefera‐ ble carbon source (elicitor). For instance, higher concentrations of the elicitor usually in‐ duce stronger transcriptional repression [137]. The presence of the repressing elicitors initiates signal transduction pathways to result in transcriptional repression of the catabo‐ lism of poor carbon sources. In this context, the molecular mechanisms leading to CCR is well known for the ethanol utilization in *A. nidulans*, and therefore ethanol catabolism in this specie is commonly used as a model for studying CCR gene regulation [138]. In *A. ni‐ dulans*, ethanol, ethylamine and L-threonine can be used as sole carbon sources via their conversion into acetaldehyde and acetate [139, 140]. Furthermore, acetate enters the main metabolism in its activated form, acetyl-CoA. Alcohol dehydrogenase I (*alcA* gene) and al‐ dehyde dehydrogenase (*aldA* gene) are the two enzymes involved in the oxidation of etha‐ nol into acetate. The genes *alcA* and *aldA* are activated through the transcriptional regulator AlcR in the presence of a co-inducer compound [141]. AlcR is a positive regula‐ tory protein of the zinc binuclear class, is autoregulated and binds to specific sites on the *alcA* and *alcR* promoter regions.

In this context, CreA appears as a sole transcriptional repressor of the system, exerting its function in the presence of a co-repressor [139, 142 - 143]. It is well known that in the *alc* genes repression system, CreA exerts its repressing function in three main different levels: (i) direct repression of *alcR*; (ii) indirect repression, via *alcR* repression, of the structural genes (*aldA* and *alcM*); and (iii) combined direct and indirect repression of the structural *al‐ cA* and *alcS* genes, by the "double-lock" mechanism. At the molecular level, a competition between AlcR and CreA results in partial repression of the *alcR* gene and a complete repres‐ sion of *alcA*. This mechanism is important under growth conditions in which poor carbon sources such as ethanol are simultaneously present with high amounts of a preferable car‐ bon source such as glucose, and the fungus is able to fine-tune the regulation in order to adapt to new nutrient. A second mechanism involves complete repression of the *alcR* gene, operating at high glucose concentration. Under these conditions, expression of the *alc* genes does not occur, and the fungus metabolizes only the rich carbon source (reviewed in [141]).

Homologs of *clr-1* and *clr-2* are present in the genomes of many filamentous ascomycete species capable of degrading plant-cell wall material, including *A. nidulans*. The *N. crassa* TFs *clr-1* and *clr-2* were able to induce all major cellulase and some hemicellulase genes, and functional CLR-1 was necessary for the expression of *clr-2* and efficient cellobiose utilization by the fungus. Besides, in *A. nidulans*, a deleted strain of the *clr-2* homolog (*clrB*) failed to induce cellulase gene expression and lacked cellulolytic activity on Avicel [136]. These au‐ thors reinforced the idea that further manipulation of the transcriptional regulation of cellu‐ lase/hemicellulase system may improve yields of cellulases for industrial applications, e.g.,

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

As briefly described above, microorganisms are known to adjust their carbon metabolism in order to minimize energy demands. One of these regulatory mechanisms is the carbon catabolite repression (CCR). Readily metabolizable carbon sources, such as glucose, are preferably catabolized and, in general, suppress the utilization of alternative carbon sour‐ ces, repressing mainly the enzymatic system required for the catabolism of less favorable carbohydrates. For general carbon catabolite repression in some Aspergilli species, the DNA-binding Cys2His2 zinc-finger repressor CreA is absolutely necessary [75]. In general, the negative effect of this regulatory system depends on the concentration of the prefera‐ ble carbon source (elicitor). For instance, higher concentrations of the elicitor usually in‐ duce stronger transcriptional repression [137]. The presence of the repressing elicitors initiates signal transduction pathways to result in transcriptional repression of the catabo‐ lism of poor carbon sources. In this context, the molecular mechanisms leading to CCR is well known for the ethanol utilization in *A. nidulans*, and therefore ethanol catabolism in this specie is commonly used as a model for studying CCR gene regulation [138]. In *A. ni‐ dulans*, ethanol, ethylamine and L-threonine can be used as sole carbon sources via their conversion into acetaldehyde and acetate [139, 140]. Furthermore, acetate enters the main metabolism in its activated form, acetyl-CoA. Alcohol dehydrogenase I (*alcA* gene) and al‐ dehyde dehydrogenase (*aldA* gene) are the two enzymes involved in the oxidation of etha‐ nol into acetate. The genes *alcA* and *aldA* are activated through the transcriptional regulator AlcR in the presence of a co-inducer compound [141]. AlcR is a positive regula‐ tory protein of the zinc binuclear class, is autoregulated and binds to specific sites on the

In this context, CreA appears as a sole transcriptional repressor of the system, exerting its function in the presence of a co-repressor [139, 142 - 143]. It is well known that in the *alc* genes repression system, CreA exerts its repressing function in three main different levels: (i) direct repression of *alcR*; (ii) indirect repression, via *alcR* repression, of the structural genes (*aldA* and *alcM*); and (iii) combined direct and indirect repression of the structural *al‐ cA* and *alcS* genes, by the "double-lock" mechanism. At the molecular level, a competition between AlcR and CreA results in partial repression of the *alcR* gene and a complete repres‐ sion of *alcA*. This mechanism is important under growth conditions in which poor carbon

for biofuel production.

*alcA* and *alcR* promoter regions.

**19. Carbon catabolite repression in Aspergilli**

A variety of studies have been demonstrated the mechanisms through which CreA re‐ presses some polysaccharide-degrading enzymatic systems in fungi. It was shown that CreA appears to repress *xlnA* transcription by the "double-lock" mechanism in *A. nidulans*, repres‐ sing directly the gene through its binding to the consensus *xlnA*.C1 site of the promoter, as well as indirect repression [144]. Studies on *A. nidulans xlnB* gene repression demonstrated that the four CreA target sites located in *xlnB* gene promoter region lack physiological rele‐ vance, suggesting that the repression exerted by CreA on *xlnB* is by an indirect mechanism [145]. The latter results suggested that an additional level of CreA repression via the xylano‐ lytic activator is present in *A. nidulans*. The authors suggested that this mechanism of regula‐ tion would be analogous to that described above for the *alc* regulon, where certain genes are under a double-lock mechanism of repression by CreA while others are not subject to direct repression, being regulated via CreA repression of the *alcR* regulatory gene. In fact, studies have been shown that the *xlnR* (the xylanolytic transcriptional activator) promoter is re‐ pressed by glucose via CreA in *A. nidulans*, and when this repression is eliminated, by pro‐ moter exchange, transcription of xylanolytic genes such as *xlnA*, *xlnB* and *xlnD* is derepressed [146]. These results demonstrated that a transcription factor cascade involving CreA and XlnR regulates CCR of *A. nidulans* xylanolytic genes.

F-box proteins are proteins containing at least one F-box domain in their structures. The Fbox domain is a protein structural motif of about 50 amino acids that mediates protein-pro‐ tein interactions [147]. Usually, F-box proteins mediate ubiquitination of proteins targeted for degradation by the proteasome, but these proteins have also been associated with cellu‐ lar functions such as signal transduction and regulation of cell-cycle [148]. A study that per‐ formed a screening of 42 *A. nidulans* F-box deletion mutants grown either on xylose or xylan as a sole carbon source in the presence of 2-deoxy-D-glucose was able to identify mutants with de-regulated xylanase induction [149]. In this study, a null mutant in a gene (*fbxA*) with decreased xylanase activity and reduced *xlnA* and *xlnD* mRNA accumulation was identi‐ fied. This mutant interacted genetically with *creA* mutants, emphasizing the importance of the CCR and ubiquitination in the *A. nidulans* xylanase induction. In addition, the identifica‐ tion of FbxA protein provides evidence for another level of regulatory network concerning xylanase induction in filamentous fungi [149].

In summary, an intricate and fine-tuned regulation network exists in order to control the ex‐ pression of plant cell-wall degradation genes in fungi. A variety of transcriptional regulators are able to respond to different nutritional requirements of the fungus, depending on its life‐ style. In general, readily metabolizable carbon sources such as glucose represses the tran‐ scription of genes responsible for the poor carbon source catabolism, via different mechanisms. The carbon catabolite repression in fungi is a common mechanism of regula‐ tion through which the organism adapts to nutritional availability in their environment. For instance, in *A. nidulans*, the xylanolytic transcriptional activator XlnR is repressed by glucose via CreA, the transcriptional factor responsible for CCR in this specie. The comprehension of such sophisticated regulatory network is essential for genetic engineering of new strains able to produce a wide range of lignocellulolytic enzymes.

It is worth to mention that synthetic bioengineering could be applied for any microorgan‐

Microbial Degradation of Lignocellulosic Biomass

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

231

While most biological routes being studied for the processing of lignocellulosic biomass fo‐ cused on the separate production of hydrolytic enzymes, in a process that usually comprises several steps, another approach is suggested to achieve this goal. This approach, termed consolidated bioprocessing (CBP) involves the production of cellulolytic enzymes, hydroly‐ sis of biomass, and fermentation of resulting sugars in a single stage via microorganisms or a consortium [157]. CBP appears to offer very large costs reduction if microorganisms can be developed that possess the required combination of substrate utilization and product forma‐ tion properties [158]. In a 2006 report in biomass conversion to biofuels, the U.S. Department of Energy endorsed the view that CBP technology is "the ultimate low-cost configuration for cellulose hydrolysis and fermentation" (DOE Joint Task Force, 2006; energy.gov). Currently, CBP technology is developing fast, especially due to partnerships with venture capital in‐ vestors and researchers. The main challenge of CBP is to generate engineered microorgan‐ isms able to produce the saccharolytic enzymes and converting the sugars released by those enzymes into the desired end-products. In addition, CBP microorganisms need to be able to perform these tasks rapidly and efficiently under challenging, industrial processes. A suc‐ cessful microbial platform for production of bioethanol from microalgae is currently availa‐ ble, and demonstrates an application of the CBP [159]. A DNA fragment encoding enzymes for alginate transport and metabolism from *Vibrio splendidus*, abundant and ubiquitous ma‐ rine bacteria, was introduced in the genome of *Escherichia coli*, a well-characterized microor‐ ganism. This microbial platform was able to simultaneously degrade, uptake and metabolize alginate, an abundant polysaccharide present in microalgae. When further engi‐ neered for ethanol synthesis, this platform enabled bioethanol production with satisfactory

ism, since it is a rational design of metabolic pathways.

yield directly from microalgae via a consolidated bioprocess [159].

pletely the lignocellulosic biomass.

**21. Conclusions and future perspectives**

The approach required for generation of CBP microorganisms involves the knowledge of many topics discussed in this chapter, concerning to fundamental principles of microbial cellulose utilization and its regulation. Moreover, the principles of synthetic bioengineering discussed above can be applied to the development of new strains for CBP technology, and therefore the generation of new microbial platforms able to uptake and metabolize com‐

A large quantity of lignocellulosic residues is accumulating over the world, mainly due to the expansion of industrial processes, but other sources such as wood, grass, agricultural, forestry and urban solid wastes contribute to accumulation of lignocellulosic material. These residues constitute a renewable resource from which many useful biological and chemical products can be derived. The natural ability of fungi and other microorganisms to degrade lignocellulosic biomass, due to highly efficient enzymatic systems, is very attractive for the development of new strategies concerning industrial processes. Paper manufacture, com‐
