What is the Difference Between Cellulase and Hemicellulase

Cellulase and hemicellulase are both important enzymes in plant cell wall degradation but serve different industrial purposes. Cellulase, targeting cellulose, is crucial in biofuels, paper, and textiles. Hemicellulase, acting on hemicellulose, is used in animal feed, fruit juice extraction, and brewing. Here is a detailed comparison between cellulase and hemicellulase.

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1. Substrate Targets

Cellulase:

Target: Cellulase primarily acts on cellulose, a polysaccharide found abundantly in plant cell walls. Cellulose is made up of glucose molecules connected by β-1,4-glycosidic bonds, making it strong and crystalline.

Function: Cellulase breaks the β-1,4-glycosidic bonds, converting cellulose into smaller sugar molecules, such as cellobiose and glucose.

Hemicellulase:

Target: Hemicellulase acts on hemicellulose, a polysaccharide made of various sugar molecules (such as xylose, arabinose, mannose, and galactose). Unlike cellulose, hemicellulose is amorphous and has a branched structure.

Function: Hemicellulase breaks the glycosidic bonds in hemicellulose, converting it into simple sugars like xylose, arabinose, and mannose.

hemicellulose in food

2. Structural Differences in Polysaccharides

Cellulose has a highly ordered crystalline structure, which is strong and difficult to degrade. It consists solely of glucose molecules connected by β-1,4-glycosidic bonds, giving it rigidity and resistance to degradation.

Hemicellulose has a more diverse and complex structure with many-branched chains and is amorphous. It includes a variety of sugar molecules, making it easier to degrade than cellulose.

3. Enzyme Types and Subtypes

Cellulase is typically composed of three types of enzymes:

Endocellulases: These enzymes randomly hydrolyze cellulose chains, producing cellobiose.

Exocellulases: These enzymes remove monosaccharides or oligosaccharides from the ends of the cellulose chains.

β-Glucosidases: These enzymes further hydrolyze cellobiose, converting it into glucose.

Hemicellulase can be divided into various types based on the type of sugar chain it acts upon, such as:

Xylanases: These enzymes hydrolyze xylan components.

Mannanases: These enzymes hydrolyze mannan components.

Arabinases: These enzymes hydrolyze arabinose components.

4. Role in Plant Cell Wall Degradation

Cellulase specifically targets cellulose, the main structural component of plant cell walls. It plays a crucial role in biofuel production, digestion in ruminant animals, and food processing by converting tough cellulose into fermentable sugars.

Hemicellulase targets hemicellulose, another key polysaccharide in plant cell walls, which often supports cellulose. Due to its less structured nature, hemicellulose is more easily degraded by hemicellulase, releasing simple sugars. This is essential for improving fiber digestibility in animal feed and increasing biofuel yields.

5. Industrial Applications

Cellulase Applications:

• Biofuel Production: Cellulase is vital for converting plant biomass (like wood, crop residues, or grass) into fermentable sugars, widely used in bioethanol production.

• Food & Beverage: It improves juice extraction, enhances the texture of baked goods, and reduces viscosity in fruit and vegetable products.

• Animal Feed: In animal feed, cellulase helps improve fiber digestibility, especially in non-ruminant animals.

• Textile & Pulp & Paper: In textiles, cellulase softens fabrics; in the paper industry, it helps break down cellulose fibers and improve paper quality.

Cellulase in food

Hemicellulase Applications:

• Animal Feed: Hemicellulase is used in animal feed to improve fiber digestibility, especially in non-ruminant animals.

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• Biofuel Production: Hemicellulase works alongside cellulase to break down hemicellulose into fermentable sugars, enhancing biofuel production.

• Food Processing: Hemicellulase improves the texture of food products (e.g., baked goods, grains, and beverages) by breaking down complex plant polysaccharides.

• Brewing: In brewing, the hemicellulase enzyme helps degrade complex sugars in barley, improving fermentation efficiency.

Conclusion

Cellulase and hemicellulase are distinct enzymes with different substrate specificities, enzyme compositions, roles in biodegradation, and application areas. Understanding the differences between these enzymes is essential for optimizing their use in various industrial processes. If you want to buy Hemicellulase enzyme, contact us for more details and free samples. We are confident our products will meet your needs.

Research on regulation of cellulases and hemicellulases gene expression may be very useful for increasing the production of these enzymes in their native producers. Mechanisms of gene regulation of cellulase and hemicellulase expression in filamentous fungi have been studied, mainly in Aspergillus and Trichoderma. The production of these extracellular enzymes is an energy-consuming process, so the enzymes are produced only under conditions in which the fungus needs to use plant polymers as an energy and carbon source. Moreover, production of many of these enzymes is coordinately regulated, and induced in the presence of the substrate polymers. In addition to induction by mono- and oligo-saccharides, genes encoding hydrolytic enzymes involved in plant cell wall deconstruction in filamentous fungi can be repressed during growth in the presence of easily metabolizable carbon sources, such as glucose. Carbon catabolite repression is an important mechanism to repress the production of plant cell wall degrading enzymes during growth on preferred carbon sources. This manuscript reviews the recent advancements in elucidation of molecular mechanisms responsible for regulation of expression of cellulase and hemicellulase genes in fungi.

In order to enhance energy security and mitigate climate change, interest in finding renewable fuels to replace petroleum-based ones is enormously increasing. The biofuels ethanol and biodiesel represent potential options for meeting these needs in the transportation sector. The uniqueness of cellulosic ethanol as a sustainable liquid transportation fuel, which can be produced in high volumes and at low cost, and its many powerful benefits have been recognized for decades [ 1 - 5 ]. A recent awareness of the urgent need to advance cellulosic ethanol production is evidenced by the number of reviews reported on the theme of ethanol fuel production from lignocellulosic biomass, with great attention to ethanol production from lignocellulosic residues, such as crop and wood residues and municipal solid waste [ 6 - 15 ]. The key step for conversion of lignocellulosic biomass into fermentable sugars for fuel production is represented by the hydrolysis of polysaccharides, resulting from biomass pretreatment, by cellulases and hemicellulases. Filamentous fungi are the major source of cellulases and hemicellulases. As far as cellulases are concerned, three main enzymatic activities are involved in cellulose hydrolysis: 1) endoglucanases (EC 3.2.1.4); 2) exoglucanases, including d-cellodextrinases (EC 3.2.1.74) and cellobiohydrolases (EC 3.2.1.91); and 3) β-glucosidases (EC 3.2.1.21). As far as hemicellulases are concerned, endo-β-1,4-xylanases (EC 3.2.1.8) and β-xylosidases (EC 3.2.1.37) are required for degradation of the xylan backbone, while auxiliary enzymes such as α-glucuronidases (EC 3.2.1), α-arabinofuranosidases (EC 3.2.1.55), acetylesterases or acetyl xylan esterases (EC 3.1.1.6) are required to achieve the complete degradation of complex substituted xylans. Research on regulation of cellulase and hemicellulase genes' expression may be very useful for increasing production of these enzymes in their native producers. Mechanisms of cellulase and hemicellulase genes regulation have been studied in filamentous fungi, mainly in Aspergillus [ 16 , 17 ] and Trichoderma [ 18 ]. The production of these extracellular enzymes is an energy-consuming process, so the enzymes are produced only under conditions in which the fungus needs to use plant polymers as an energy and carbon source. Moreover, production of many of these enzymes is coordinately regulated, and induced in the presence of the substrate polymers. Induction mechanisms of cellulase and hemicellulase genes expression involve activation of gene expression by the respective hydrolysis and/or transglycosylation products of cellulose and/or xylan, such as gentiobiose for Penicillium [ 19 ], and sophorose for A. terreus and T. reesei [ 20 , 21 ]. In addition to induction by mono- and oligo-saccharides, genes encoding hydrolytic enzymes involved in plant cell wall deconstruction in filamentous fungi can be repressed during growth in the presence of easily metabolizable carbon sources, such as glucose. Carbon catabolite repression (CCR) is an important mechanism to repress the production of plant cell wall degrading enzymes during growth on preferred carbon sources [ 22 - 25 ].

2. REGULATION OF PRODUCTION OF CELLULASES AND HEMICELLULASES IN TRICHODERMA REESEI

The cellulolytic machinery of T. reesei is one of the most widely studied [26]. T. reesei genome (http://genome.jgi psf.org/Trire2/Trire2.home.html) contains ten cellulase and sixteen hemicellulase genes [27]. The enzymes so far identified and characterized as responsible for the cellulolytic activity of T. reesei include five endoglucanases -EGI/Cel7B, EGII/Cel5A, EGIII/Cel12A [28, 29], EGIV/Cel61A [30], and EGV/Cel45A [31] and two exoglucanases -the cellobiohydrolases CBHI/Cel7A and CBHII/Cel6A [32]. These enzymes act synergistically to convert cellulose into cellobiose [28-33], whose hydrolysis into glucose involves then two β-glucosidases -BGLI/Cel3A [34] and BGLII/Cel1A [35]. An additional protein, swollenin (encoded by the gene swo1), has been described, that disrupts crystalline cellulose structures, presumably making polysaccharides more accessible to hydrolysis [36]. The cellulases CBHI/Cel7A, CBHII/ Cel6A, EGI/Cel7B, and EGII/Cel5A are the most abundantly produced by T. reesei secreting them up to 40 g/liter [37]. Due to the enormous level of cellulase production, T. reesei revealed to be a potential candidate for advancing cellulosic ethanol by I Consolidated BioProcessing, engineering it to ferment monosaccharides into ethanol in high yields [38].

The T. reesei genome also contains sixteen hemicellulases including two GH43, one GH10, four GH11, one GH74, one GH62, two GH54, one GH67 and four GH95 [27]. Among these, two major endo-β-1,4-xylanases XYNI and XYNII (EC 3.2.1.8) [39]; and one β-xylosidase, BXLI (EC 3.2.1.37) [40] have been characterized.

The presence of cellulose, xylan or mixtures of plant polymers in the fungal culture medium causes abundant production of cellulolytic and xylanolytic activities by T. reesei, as already reported by the earlier studies [41-44]. Pure (oligo)saccharides, such as sophorose [20, 21], β-cellobiono-1,5-lactone, D-xylose, xylobiose, galactose, and lactose, have been also reported to induce cellulase and hemicellulase production in T. reesei (Table 1) [24, 45-49].

Inability of the fungal cells to incorporate insoluble polymeric compounds, such as cellulose and xylan, aroused the question on how these polymers can initiate production of hydrolytic enzymes. Several studies investigating this aspect postulated the inducer function of a low molecular weight and soluble compound derived from cellulose. One of the proposed mechanisms is that the fungus produces basal levels of cellulase (mainly CEL7A and CEL6A) and that the activity of these extracellular enzymes on cellulose produces a soluble inducer, which can enter the cell and affect induction [50, 51]. In support of this mechanism, it was shown that antibodies against CBHI, CBHII, EGI and EGII blocked the expression of cbh1/cel7a gene in the presence of cellulose but not the soluble inducer sophorose [50]. The constitutive levels of these cellulases and their role in cellulase induction were afterwards demonstrated by Carle-Urioste et al. [51]. These authors showed that the mRNAs cbh1 and egl1 are transcribed under uninduced conditions, and that induction with cellulose results in at least -fold increase of both transcripts, as demonstrated by Northern blots. The basal activity of the cbh1 promoter was also examined by using a chimeric vector in which the gene encoding hygromycin B phosphotransferase [52] was placed under the control of the 59-flanking DNA sequence of the cbh1 gene. Under uninduced conditions, resistance to the antibiotic hygromycin B was observed with T. reesei cells transformed with this vector and grown on medium lacking cellulose. An antisense RNA strategy was also adopted by the same authors to gain in vivo evidence for the requirement of the basal expression of the cellulase in induction of the cellulase transcripts by cellulose [51]. The results demonstrated that the expression of this antisense RNA produced marked effects on the induction of the cbh1 transcript using cellulose (reduction of the cbh1 transcript expression between 80 and 90%) but not sophorose as an inducer. The authors also showed that the initial hydrolysis of cellulose is the rate-limiting step in the induction, as suggested by the observation that the addition of the cellulase system or its purified enzyme members to a culture of T. reesei, in the presence of cellulose, resulted in earlier detection of the cbh1 and egl1 transcripts. The time required for induction of cbh1 and egl1 transcripts using cellulose, cellulose + cellulase, or sophorose is 14, 10, and 4 h, respectively. This result supports the hypothesis that oligosaccharide( s) is(are) formed in vivo from cellulose by the activity of a low, constitutive, and extracellular cellulase activity. The relatively slow induction by sophorose could be explained by the fact that the inductive process is protein synthesis-dependent. In addition, it has recently been shown that a sophorose-inducible β-diglucoside permease is involved in the induction of the cellulase system in T. reesei [53]. Subsequently, Foreman et al. [54] identified further genes whose regulatory behavior is consistent with their role in primary inducer formation for cellulase expression. Among them, the mRNA of cel5b was moderately expressed during growth on glycerol, glucose, sophorose and lactose, and only slightly induced over this level by cellulose. It is worth noting that CEL5B contains the consensus sequence for membrane-anchoring via a glycosylphosphatidylinositol residue. All these properties make it an interesting candidate for generating the inducer of cellulase formation. Similarly, the acetyl xylan esterase Axe2, which is also predicted to contain a glycosylphosphatidylinositol anchor, may be involved in primary induction of some hemicellulases [54].

The surface-bound cellulolytic activity displayed by conidia of T. reesei, mainly due to CEL6A/CBHII [55, 56] is also considered important for cellulase induction since its elimination by detergents hinders germination of the conidia on cellulose. These conclusions were deduced by the observation that introduction of multiple copies of the cel6a gene into T. reesei caused an enhanced secretion of CEL7A and CEL6A on cellulose and an increased cellulase activity on cellulose corresponding to enhanced level of conidial-bound CEL6A [56, 57]. Consistently, a cel6a knocked out strain showed a delay in growth and cellulase formation on cellulose [58]. In more details, comparing strains in which the corresponding genes of the main cellulases (cel6a, cel7a, cel7b, cel5a) had been deleted, Seiboth et al. [58] showed that strains knocked out for cel6 and cel5a, respectively, exhibited a significantly reduced expression of the remaining cellulase genes, while strains carrying the cel7a or cel7b deletion showed these transcripts. A strain showing both the cellobiohydrolases cel6a and cel7a deletion, was unable to initiate growth on cellulose. During growth on lactose, these strains showed no significant alteration in their ability to express the respective other cellulase genes. These data support the role of CEL6A and other conidial-bound cellulases (such as CEL5A, for which a conidial location is not yet known) in the induction of cellulases and germination on cellulose.

Ilmèn et al. [59] investigated basic features of expression regulation of the T. reesei cellobiohydrolases cbh1 and cbh2 and endoglucanases egl1, egl2 and egl5 encoding genes, at the mRNA level, showing that these cellulase genes are coordinately expressed and the steady-state mRNA levels of cbh1/Cel7A is the highest. The highest induction level was achieved with cellulose and sophorose and moderate expression was observed when cellobiose or lactose were used as the carbon source. No expression could be observed on glucose-containing medium and high glucose levels abolish the inducing effect of sophorose. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression. It was also found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels.

The best inducer of cellulase expression so far known is sophorose ((2-O-β-glucopyranosyl-D-glucos) [60, 21, 61], whose synthesis from cellobiose involves the transglycosylation activity of β-glucosidase [62]. Induction by sophorose is affected by various parameters such as its concentration and rate of uptake [61, 63]. Two pathways of sophorose utilisation were for the first time hypothesised by Loewenberg and Chapman [64]: a catabolic pathway characterized by a high capacity but low affinity for sophorose, and a cellulase inducing pathway endowed with a lower capacity but higher affinity for sophorose. As a matter of fact, Kubicek et al. [53] showed that sophorose is transported by a cellobiose permease, characterized by low KM and Vmax for sophorose, and thus competing with the extracellular β-glucosidase, which has a much higher KM but also Vmax for it.

Most authors implied a β-glucosidase in the process of sophorose production. T. reesei produces β-glucosidases having different cellular localizations [65-69]. The gene cel3a [65, 70] encodes the major extracellular β-glucosidase identified as one of the β-glucosidases involved in inducer formation. Knock-out of the cel3a gene causes a delay in induction of the other cellulase genes by cellulose, but not by sophorose, whilst a cel3a-multicopy strain is able to produce higher levels of cellulases than the wild-type strain under nonsaturating concentrations of sophorose, but both strains were comparably efficient at saturating concentrations [48]. The observation that the β-glucosidase inhibitor nojirimycin inhibits cellulase induction also in the cel3a disrupted strain suggests that the CEL3A is not the only β-glucosidase involved in inducer formation [48]. An additional β-glucosidase-encoding gene has been cloned [35] and properties and intracellular localisation of the corresponding enzyme have been characterised [69]. However, as no multicopy or gene deletion studies have yet been carried out, ascertainment of its involvement in cellulase induction requires further investigation.

2.1. Transcriptional Factors Involved in Regulation of Cellulase and Hemicellulase Genes' Expression in T. reesei

Foreman et al. [54] performed investigations on regulation of cellulase and hemicellulase genes' expression in T. reesei by microarrays showing that most of the genes encoding known and putative biomass-degrading enzymes are transcriptionally co-regulated. This co-regulation indicates a tightly coordinated cooperation of the corresponding transcription factors, five of which have been so far identified (Fig. 1): the positive regulators XYR1, ACE2 and the HAP2/3/5 complex, the repressor ACE1 and the carbon catabolite repressor CRE1 (Table 2) [25].

Fig. (1).

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Schematic representation of transcriptional factors affecting cellulases and xylanases expression in T. reseei (black box), N. crassa (grey box) and Aspergillus spp. (white box). The carbon catabolite repressor CRE, the activators clbR, Xyr/xlR/XlnR,Clr,ACE2, the repressor ACE1, the CCAAT binding Hap2/3/5 complex, the pH regulator PacC, and the nitrogen regulators AreA and Nit2 are shown. The repression activity (), the induction activity () and also promoter () and coding region () are indicated.

Table 2.

Positive and Negative Regulators of Expression of Genes Coding for (hemi)cellulolytic Enzymes and Their Binding consensus Sequences in the Target Promoters

Trichoderma reesei Positive Regulators Name Structure Consensus Region References XYR1 Zinc binuclear cluster protein 5'-GGCTAA [194]
[25] ACE2 Zinc binuclear cluster proteins 5ʹGGCTAATAA HAP2 Multimeric protein complex 5'- CCAAT HAP3 HAP5 Negative regulators Name Structure Consensus region References ACE1 Three Cys2His2-type zinc fingers 5'-AGGCA [25]
[195] CRE1 Cys2His2 type transcription factor 5'-SYGGRG Neurospora crassa Positive regulators Name Structure Consensus region References CLR-1/-2 Two zinc binuclear cluster - [145] PacC Three Cys2His2 zinc fingers 5'-GCCARG [147] Neurospora crassa Positive regulators Name Structure Consensus region References XLR1 Zinc binuclear cluster protein [148] NIT2 Single zinc finger protein 5'- TATCTA [149] Negative regulators Name Structure Consensus region References CRE1 Cys2His2 type transcription factor 5'-SYGGRG- [152] Aspergillus spp. Positive regulators Name Structure Consensus region References AmyR Zn(II)2Cys6-binuclear cluster DNA-binding motif 5'-CGGN8CGG-3' [194,-196] AraR Zn(2)Cys(6) binuclear cluster domain [197,198] PacC Three Cys2His2 zinc fingers 5'- GCCARG [22] XlnR Zinc binuclear cluster protein 5'-GGCTAAA [48] ClbR Zn(II)2Cys6-binuclear cluster DNA-binding motif CGG or CCG triplets [174] AreA Highly conserved DNA binding motif comprising a Cys(4) zinc finger followed by a basic domain 5'-GATA (core sequence) [182] Negative regulators Name Structure Consensus region References CREA Cys2His2 type transcription factor 5'-SYGGRG [183-187] CREB CREC Open in a new tab

The main positive regulator of cellulase and hemicellulase gene expression is represented by XYR1 (xylanase regulator 1) [48, 18], a zinc binuclear cluster protein binding to a GGCTAA-motif arranged as an inverted repeat [48]. xyr1 deletion abolishes cellulase induction on cellulose and sophorose and impairs the induction of hemicellulase genes involved in xylan and arabinan degradation [71, 18], thus proving its essential role in the induction process. xyr1 transcription seems not to be induced during growth on cellulose [72]. Most of eukaryotic transcriptional activators are present in cells only in small amounts required to start gene expression [73], and, in many cases, they are further induced by the conditions for which they are needed and are degraded once they are no longer required [74]. On the contrary, xyr1 expression is regulated solely by CRE1-dependent CCR and by repression by the specific transcription factor ACE1, not by induction [72, 49]. Whether an increase in constitutive expression of xyr1 would increase enzyme formation is not sufficiently understood. Aigner-Mach et al. [72] fused the xyr1 gene under the regulatory signals of the nag1 (N-acetyl-β-D-glucosaminidase) promoter, which resulted in a slightly earlier beginning of xylanase formation but did not significantly enhance the final enzyme titre. However, these studies used the uninduced, basal expression level of nag1, which is not much higher than that of xyr1 itself, and studies using stronger expressed promoters (such as those for glycolytic or hydrophobin genes) must be used to clarify whether the constitutive expression of xyr1 would enhance cellulase and/or xylanase formation.

The cellulase activator ACE2 also belongs to the class of zinc binuclear cluster proteins [75]. It has so far been shown to occur only in Trichoderma spp. Deletion of ace2 lowers the transcript levels of the major cellulases and causes a decrease of cellulase activity during growth on cellulose [75, 76], whilst it does not affect cellulase induction by sophorose [75]. It is worth noting that the DNA-binding domain of ACE2 is able to bind to the promoter motif [GGC(T/A)4] present in the cbh1 promoter also recognized by XYR1 [77]. Stricker et al. [76] suggested that phosphorylation and dimerization are needed for the binding of ACE2 to the corresponding promoter element.

The CCAAT motif is a common cis-acting element found in either orientation in the promoter and enhancer region of a large number of eukaryotic genes. Particularly, in yeasts, as well in filamentous fungi, the CCAAT box-binding proteins identified so far all belong to the group of HAP-like factors. Site-directed mutagenesis of the promoter of one of the most abundant cellulase produced by T. reesei, cbh2, revealed the existence of an undecameric nucleotide motif which is essential for gene expression in vivo. Moreover, experiments of promoter mutation and in vivo footprinting analysis allowed to show that expression from the cel6a promoter is dependent on a CCAAT box bound by the HAP2/3/5 protein complex [78]. The CCAAT motif is found in approximately 30% of the 5'- non-coding regions of eukaryotic genes [79]. In analogy to the mammalian NF-Y complex containing NF-YA, NFYB and NF-YC orthologues of HAP2, HAP3 and HAP5, respectively, they contain a histone fold motif, a structural feature of histones suggesting that NF-Y might be involved in the organisation of the chromatin structure [80]. Thereby the action of acetyltransferases may play a role in the local disruption of nucleosomes since an association of GATA-1 and NF-Y with acetyltransferases p300/CBP has been shown [81, 82]. The corresponding hap2, hap3 and hap5 genes from T. reesei were cloned by Zeilinger et al. [83] showing that they encode proteins similar to Hap homologues from other organisms and essential for binding to the CAE (cbh2-activating element) in the T. reesei cel6a promoter. The HAP2/3/5 complex is considered needed for generating an open chromatin structure required for full transcriptional activation [84]. The hypothesis that the CCAAT sequences in the cellulase promoters could play a conserved role in the generation of an open chromatin structure necessary for full transcriptional activation is supported by the detection of a nucleosome-free region around the XYR1/ACE2/HAP2/3/5- binding area in the cel6a promoter, which is flanked by strictly positioned nucleosomes [84]. Induction by sophorose results in a loss of positioning of nucleosomes -1 and -2 downstream of the binding area, thus making the TATA box accessible. A mutation in the CCAAT box shifted this positioning, thus proving the role of the HAP2/3/5 complex in this process [84]. These data provide an experiment based explanation of the advantage for clustering of cellulases in the genome of T. reesei and illustrate that chromatin regulation is a suitable target for strain improvement. For instance, it is worth noting that Zou et al. [85] have recently demonstrated that replacement of the CREI binding sites within the cbh1 promoter of T. reesei with the binding sites of transcription activator, namely the HAP2/3/5, besides the ACEII, led to improvement of promoter efficiency. The new developped promoter was shown able to induce expression of the green fluorescent protein reporter by 5.5-fold in inducing culture medium and 7.4-fold in repressing culture medium.

ACE1 contains three Cys2His2-type zinc fingers and it was shown to bind in vitro to eight sites containing the core sequence 5'-AGGCA scattered along the 1.15-kb cel7a promoter [86]. Deletion of ace1 resulted in an increase in the expression of all the main cellulase and hemicellulase genes in sophorose- and cellulose-induced cultures, indicating that ACE1 acts as a repressor of cellulase and xylanase expression [87] and of xyr1 during growth on D-xylose [72]. A strain bearing a deletion of both the ace1 gene and ace2 gene expressed cellulases and xylanases similar to the Δace1 strain, probably due to the remaining activity of XYR1 [87].

All together the above data suggest that the substrate-unspecific activator XYR1 is fine-tuned by more specific transcriptional regulators such as ACE1 and ACE2 (Fig. 1). This working model concurs with the findings that XYR1 binds to an inverted repeat either as a homo- or a heterodimer, respectively, thereby providing the opportunity for specific regulatory proteins to interact with the accordant promoter and/or XYR1. The role of the HAP2/ 3/5 complex in this regulation may be that of a general transcriptional enhancer raising the accessibility of the other factors to the cellulase promoters.

The putative methyltransferase LaeA is a global regulator that affects the expression of multiple secondary metabolite gene clusters in several fungi, and it can modify heterochromatin structure in Aspergillus nidulans. Seiboth et al. [88] showed that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei LAE1: the protein methyltransferase LAE1. In a lae1 deletion mutant a complete loss of expression of all seven cellulases was observed, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1- modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. Chromatin immunoprecipitation followed by highthroughput sequencing ('ChIP-seq') showed that lae1 expression was not obviously correlated with H3K4 dior trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZY (Carbohydrate-Active enZYmes) coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. These data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified.

To learn more about the function of LAE1 in T. reesei, Karimi-Aghcheh et al. [89] further assessed the effect of deletion and overexpression of lae1 on genome-wide gene expression. They found that in addition to positively regulating 7 of 17 polyketide or nonribosomal peptide synthases, genes encoding ankyrinproteins, iron uptake, heterokaryon incompatibility proteins, PTH11-receptors, and oxidases/monoxygenases are major gene categories also regulated by LAE1. Chromatin immunoprecipitation sequencing with antibodies against histone modifications known to be associated with transcriptionally active (H3K4me2 and -me3) or silent (H3K9me3) chromatin detected genes bearing one or more of these methylation marks, of which 75 exhibited a correlation between either H3K4me2 or H3K4 me3 and regulation by LAE1.

CRE1 is the main transcription factor mediating CCR [90, 91], a mechanism promoting the assimilation of high-energy yielding carbon sources over that of sources yielding less energy, described in more details below.

2.2. Carbon Catabolite Repression of Cellulase and Hemicellulase Genes' Expression in T. reesei

Expression of most of T. reesei cellulase and hemicellulases genes does not occur in the presence of glucose in culture medium. Two mechanisms are responsible for this phenomenon: inducer exclusion (that is, inhibition of inducer [= sophorose] uptake) by D-glucose [53] and glucose repression [59, 84, 92]. The latter specifies a transcriptional regulation controlling the preferential use of substrates such as D-glucose or other monosaccharides whose catabolism provides a high yield of ATP namely CCR.

Consequently, one of the earliest attempts for engineering cellulase production was removal of CCR. Classical mutagenesis combined with selection for 2-desoxyglucose resistance (an agent believed primarily to enrich carbon catabolite-resistant mutants) [93] has led to increased cellulase producers such as T. reesei RUT C30 [94], RL-P37 [95] and CL847 [96], thus supporting the possible importance of CCR in cellulase formation.

In Trichoderma spp., the key player in this glucose repression is the Cys2His2 type transcription factor CREI [90, 97]. cre1 is missing in the cellulase hyperproducer strain RUT C30 [90] and importance of its deletion for the increase of cellulase production has been highlighted recently [98]. The cre1 gene is located on scaffold 2: - (ID ), and the mutant is characterized by a loss of a -base pair fragment, which starts downstream of the region encoding the CRE1 zinc finger and reaches into the 3'-non-coding region [99]. Le Crom et al. [100] discovered that in Rut-C30, in addition to the 29 genes deleted during the generation of NG14, the truncation of cre1 gene and the frameshift in glucosidase II, nearly 45% of the genes mutated encode transcription factors, components of nuclear import, mRNA metabolism, protein secretion, and vacuolar sorting.

The knowledge of mutations in the hyperproducer T. reesei strains was widened by Vitikainen et al. [101], reporting an aCGH (Array-Comparative Genomic Hybridization) analysis of the high-producing strains QM, QM, NG14 and Rut-C30. These authors showed that the 85 kb deletion is not responsible for the high ability of cellulase producing in Rut-C30.

In vivo functionality of the CRE1 binding sites has been shown for the cbh1 and xyn1 promoters of T. reesei where mutations in the binding sequences led to constitutive expression of these genes in the presence of D-glucose [92, 102]. Functional CREI binding sites have been shown to consist of two closely spaced 5'-SYGGRG motifs, and it has been suggested that direct CREI repression would occur only through such double binding sites. Phosphorylation of a serine in a conserved short stretch within an acidic region of T. reesei CREI has been demonstrated to regulate its DNA binding [103]. Phosphorylation of this serine may involve a casein kinase 2. Casein kinases of this class are known from various other organisms to play a role in the regulation of a large number of transcription factors [104]. However, the SNF1 kinase, which plays a central role in the regulation of CCR in yeasts [105], appears not to be involved in the phosphorylation of CRE1 in T. reesei [106].

Another gene whose product is involved in CCR in T. reesei is represented by cre2 whose disruption led to deregulation of genes normally subjected to CCR [107]. Interestingly, the E3 ubiquitin ligase LIM1 also responds to cellulase inducing conditions and binds to the cbh2-promoter [108].

The way in which the presence of glucose triggers CCR is still only poorly understood in filamentous fungi. In S. cerevisiae, the D-glucose and D-fructose phosphorylating enzymes are also involved in D-glucose and carbon catabolite sensing, due to the presence of three hexose-6-phosphorylating enzymes including two hexokinases and one glucokinase. Each of them enables S. cerevisiae to grow on D-glucose, but the hexokinase Hxk2p is responsible for the main enzymatic activity and glucose repression mediated by the carbon catabolite repressor Mig1p (whose DNA-binding domain is highly similar to that of CRE1) [109-111]. The mechanism by which Hxk2p contributes to glucose repression has not yet been fully elucidated, but its catalytic activity seems to be dispensable and thus signal transmission may rather depend on substrate binding-induced conformational changes in the Hxk2p protein or a direct regulatory role of the Hxk2p in the nucleus (discussed, for example, in Linhoff et al. [80]).

Portnoy et al. [112] investigated how xyr1, ace1 and ace2 are regulated in cellulase induction conditions and how this regulation relates to carbon catabolite repression in the low cellulase producer strain T. reesei strain QM , the high-producer strain RUT C30 [94, 113] and the hyperproducer strain T. reesei CL847 [96]. They demonstrated that in QM all three genes are induced by lactose and xyr1 is also induced by D-galactose. Moreover, ace1 is carbon catabolite repressed, whereas full induction of xyr1 and ace2 requires CRE1. These regulatory patterns showed significant differences in RUT C30 and CL847 strains. Rate of cellulase production by strain CL847 on lactose was around 15-fold higher than that for strain QM , consistently with the 15-fold-increase of the cbh1 transcript level. These data indicate that gene expression is a major limiting step for cellulase biosynthesis. Consistent with its role as the major transcriptional regulator of cellulase gene expression, a strongly increased basal expression of xyr1 was observed in strain CL847, which was further induced by lactose. This increase indicates an improved function of the transcriptional machinery required for xyr1 expression in strain CL847. The basal expression of ace2 was not significantly altered in strain CL847, and the inducible level was the same as that in strain QM . This indicates that the lack of CRE1 function, which seems to be required for ace2 gene expression, as indicated by the lower expression levels in the Δcre1 mutant, has been overcome during the breeding of CL847. While these data suggest that ace2 expression is not limiting for cellulase induction on lactose, they nevertheless show that wild-type expression levels appear to be necessary for the formation of high levels of cellulase. Expression of ace1'even though it is a repressor of cellulase formation'was also increased in the mutant strain CL847. However, ace1 is subject to CRE1-dependent CCR. The comparison reveals that the basal expression level of ace1 in CL847 is lower than that in the Δcre1 strain and decreases during the glucose feed. The approximate doubling of this level during the lactose feed is conserved, however. It has therefore been concluded that carbon catabolite derepression of ace1 has partially reverted in CL847, leading to a lower concentration of this repressor under cellulase-producing conditions. The present findings of reduced xyr1 but increased cbh1 transcription in the Δcre1 strain would be consistent with the operation of post-translational modification of XYR1. Nevertheless, these data show clearly that the expression of xyr1, ace1, and ace2 has been significantly altered in the hyperproducer CL847, suggesting that their wild-type expression was insufficient for hyperproduction. Identification of the proteins and genes responsible for the mechanisms observed may result in a major breakthrough in the understanding of cellulase formation and may offer a straightforward means for its improvement. These observations suggest that a strongly elevated basal transcription level of xyr1 and reduced upregulation of ace1 by lactose may have been important for generating the hyperproducer strain and that thus, these genes are major control elements of cellulose production.

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