Ethanol production from lignocellulosic biomass, comprised primarily of cellulose and hemicellulose, appears to evolve as one of the most important technologies for sustainable development. Given its renewable nature, biomass is a potential raw material not only for the production of biofuels, but also chemicals, energy and other materials of main industrial interest (Zhang et al., 2006). The monosaccharides contained in the cellulosic (glucose) and hemicellulosic fractions (xylose, arabinose, mannose, and galactose) represent substrates that can be used for ethanol production via fermentation. To initiate the degradation of these fractions, it is necessary to overcome the physical and chemical barriers presented by the cohesive combination of the main biomass components, which hinders the hydrolysis of cellulose and hemicellulose into fermentable sugars. The above include high substrate viscosity, poor mass transfer conditions and long reaction times, during which hydrolysis reactors are susceptible to contamination. Fungi are the main decomposers of lignocellulosic biomass in terrestrial ecosystems and the enzymes they secrete to break down lignocellulose may be useful in industrial processes. Thermophilic fungi provide a potential source of plant cell wall degrading enzymes with higher levels of specific activity and better stability at higher temperatures, thus making it feasible to minimize the hydrolysis time, reduce substrate viscosity and contamination levels (Margaritis and Merchant, 1986).

Myceliophthora thermophila (synonym Sporotrichum thermophile) is a thermophilic filamentous fungus, classified as an ascomycete, which was isolated from soil in eastern Russia and constitutes an exceptionally powerful cellulolytic organism, which synthesizes a complete set of enzymes necessary for the breakdown of cellulose. The growth rate and cell density of this microorganism appear to be similar in media containing cellulose or glucose (Bhat and Maheshwari, 1987). The 38.7 Mbp genome of M. thermophila, comprising about 9500 genes, organized in 7 chromosomes, has been sequenced and annotated (Joint Genome Institute, University of California, http://genome.jgi-psf.org; Berka et al., 2011). It revealed a large number of genes putatively encoding industrially important enzymes, such as carbohydrate-active enzymes (CAZy), proteases, oxido-reductases, and lipases, while more than 200 sequences have been identified exclusively for plant cell-wall-degrading enzymes. These sequences encode a large number of glycoside hydrolases (GH) and polysaccharide lyases, covering the most of the recognized families (Table 1). In addition, M. thermophila was developed into a proprietary mature enzyme production system with easy scaling (C1 strain; Visser et al., 2011). The main features of C1 are the high production levels (up to 100 g/L protein), as well as the maintenance of low viscosity levels of the culture medium, thus enabling fermentation process to reach very high densities.

M. thermophila exhibits an impressing number of accessory enzymes belonging to AA9 (previously described as GH61) and family 1 carbohydrate binding modules (CBM), which are the highest found in fungi (Berka et al., 2011). Family 1 CBM presents a cellulose-binding function and is almost exclusively found in enzymes of fungal origin (http://www.cazy.org; Guillén et al., 2009). In addition, M. thermophila distinguishes itself from other cellulolytic fungi, such as Aspergillus niger and Trichoderma reesei by the presence of a relatively high number of (glucurono) arabinoxylan degrading enzymes (Hinz et al., 2009). Eleven putative xylanases were found that belong into GH 10 and 11 families compared to five in both A. niger (Broad Institute of Harvard and MIT, http://www.broadinstitute.org) and T. reesei (Joint Genome Institute, University of California, http://genome.jgi-psf.org), while 14 arabinofuranosidases belonging to GH 43, 51, and 62 families were found compared to 13 in A. niger and three in T. reesei, rendering M. thermophila a promising source of hemicellulolytic enzymes. Studying the secretome of M. thermophila after 30 h of growth in barley and alfalfa straws, it was found to comprise of 683 predicted proteins, 230 of which are proteins with unknown function (Berka et al., 2011). Based on transcriptome analysis, many secreted enzymes including accessory enzymes, hypothetical proteins and proteins with unknown function were upregulated, when the fungus is grown in more complex substrates, such as agricultural straws, compared to glucose, indicating their crucial role in lignocellulose degradation (Berka et al., 2011).

M. thermophila grows in temperatures between 25 and 55°C, while a relative growth performance study on mycobroth agar plates indicated that the optimum condition is at 45°C (Morgenstern et al., 2012). The temperature optima for several enzymes with the same specific activity, characterized from M. thermophila, range from 50 to 70°C. For example, StEG5 endoglucanase, expressed in A. niger, exhibits a T_opt of 70°C (Tambor et al., 2012), while recombinant MtEG7 expressed in Pichia pastoris exhibited an optimal temperature of 60°C (Karnaouri et al., 2014). The same characteristic is also observed for M. thermophila xylanases expressed in A. niger, showing optimal activity at temperatures between 50 and 70°C (Berka et al., 2011), underpinning the enzymatic potential that is not only diverse in catalytic activities, but also in properties increasing its efficiency in various temperatures.

Individual cellulolytic enzymes exhibit comparable activities on cellulose; however, synthetically composed multienzyme mixtures display a much higher performance than those from other lignocellulolytic thermostable fungi (Szijártó et al., 2011; Zhang et al., 2013). This can be attributed to synergistic mode of action between the enzymes. For example, synergism between GH 11 xylanase and type C feruloyl esterase has been proved (Moukouli et al., 2010), as well as between cellobiohydrolases acting on the reducing and the non-reducing end of cellulose molecules (Gusakov et al., 2007).

In this review, an overview will be given of the cellulolytic and hemicellulolytic potential of M. thermophila regarding the degradation of plant cell wall material. The genomic potential of this thermophilic fungus demonstrates a strong enzymatic toolbox including hydrolytic, oxidative and accessory activities that may enhance its ability to decompose plant biomass. Many of these enzymes have been isolated from culture supernatant or selectively overexpressed in M. thermophila (C1 strain) or in other heterologous hosts and have been characterized. All sequences used in this study were extracted from Genome Portal database (http://genome.jgi-psf.org) and the continually updated CAZy database (http://www.cazy.org/; Lombard et al., 2014). The conserved domains were found with Pfam/InterProscan (http://pfam.sanger.ac.uk/; Punta et al., 2012), while the theoretical molecular mass and isoelectric point for each protein were calculated using the ProtParam tool of ExPASY (http://web.expasy.org/protparam/). Post-translational glycosylation sites were predicted with NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/) and NetOGlyc 3.1 server (http://www.cbs.dtu.dk/services/NetOGlyc/). Predicted secretome was extracted using SignalP v4.0 (http://www.cbs.dtu.dk/services/SignalP/).

Cellulose is composed of β-D-anhydroglucopyranose units linked by (1,4)-glycosidic bonds. Polymorphism or allotropy refers to the existence of more than one crystalline forms differing in physical and chemical properties. Cellulose degradation is attributed to the synergistic action of three complementary enzyme activities: (1) endoglucanases (EGs, EC 3.2.1.4); (2) exoglucanases, including cellodextrinases (EC 3.2.1.74) and cellobiohydrolases (CBHs, EC 3.2.1.91 for the non-reducing end acting CBHs and EC 3.2.1.176 for the reducing end acting ones) and (3) β-glucosidases (BGs, EC 3.2.1.21) (Lynd et al., 2002). Amorphous regions of the polysaccharide chain are cleaved randomly by EGs, while CBHs remove processively cellooligosaccharides from chain ends. The latter are the most abundant enzymes in the secretome of cellulolytic fungi (Jun et al., 2011; Ribeiro et al., 2012). Their main representatives are GH family 7 (CBH I) that attack the reducing end of a cellulose chain and GH family 6 (CBH II) that are specific toward the non-reducing end of the chain. Until very recently, CBHs were considered as the main degraders of the crystalline part of cellulose (Sweeney and Xu, 2012).

EGs are widespread among GH families, with examples described for families 5–9, 12, 44, 45, 48, 51, and 74 on the continually updated CAZy database (http://www.cazy.org/; Lombard et al., 2014). Most of them show optimal activity at neutral or acidic pH and at temperatures below 50°C (Maheshwari et al., 2000). Exo-glucanases (or CBHs) act in a processive manner (Davies and Henrissat, 1995) and are classified only to two families, as referred previously. One of the important features of all CBHs is that they can act on microcrystalline cellulose (Terri, 1997). BGs include enzymes of GH1 and GH3 families that hydrolyze cellobiose and short (soluble) cellooligosaccharides to glucose that could subsequently fermented to ethanol; e.g., the hydrolysis reaction is performed in the liquid phase, rather than on the surface of the insoluble cellulose particles, such as EGs and CBHs. The removal of cellobiose is an important step of the enzymatic hydrolysis process, as it assists in reduction of the inhibitory effect of cellobiose on EG and CBH. BG activity has often been found to be rate-limiting during enzymatic hydrolysis of cellulose (Duff and Murray, 1996; Tolan and Foody, 1999), and due to that the commercial cellulase enzyme preparations are often supplemented with BG activity.

Until recently, only hydrolytic enzymes were thought to play a role in the degradation of recalcitrant cellulose and hemicelluloses to fermentable sugars. Recent studies demonstrate that enzymes from the GH family 61 show lytic polysaccharide monooxygenase activity (LPMO) and have an enhancing cellulolytic effect when combined with common cellulases (Horn et al., 2012). Together with cellobiose dehydrogenase (CDH; EC 1.1.99.18), an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and boosts the enzymatic conversion of lignocellulose. It has long been thought that the proteins of GH family 61 are accessory proteins enhancing cellulose decomposition. They were thus frequently referred to as the “cellulose enhancing factors” (Harris et al., 2010) and previously thought to have no or only weak endoglucanase activity (Karlsson et al., 2001). Now, these enzymes are now reclassified to AA9 family of CAZy database and their mode of action provide a new dimension to the classical concept of cellulose degradation, as recently reviewed by Dimarogona et al. (2013). These copper-dependent enzymes were shown to cleave cellulose by an oxidative mechanism provided that reduction equivalents from CDH or low molecular weight reducing agents (e.g., ascorbate) are available (Langston et al., 2011). In some genomes, AA9 genes even outnumber cellulose genes. It remains to be elucidated whether all of these encoded enzymes have PMO activity, but their large number emphasizes the importance of oxidative cellulose cleavage. M. thermophila's genome has 25 AA9 genes, encoding putative proteins acting as accessory LPMOs enzymes (Berka et al., 2011). This number is outstanding in comparison to common lignocellulolytic organisms, as A. niger (seven sequences) and T. reesei (nine sequences). This difference can explain the high efficiency of hydrolysis of Myceliopthora in nature substrates and reveals the crucial role of these enzymes in the whole procedure.

Throughout the genome of M. thermophila, there are eight sequences encoding EG activity, seven sequences of CBH activity and nine sequences of BG activity (Figure 1; Table 2). The theoretical average molecular weight of the translated proteins is calculated at 51.05 ± 16.2 kDa and the theoretical pI at 5.58 ± 0.3. EGs are distributed to families GH5, 7, 12, and 45, all predicted to possess a secretion signal and several N- and O-glycosylation sites. Only two of them exhibit a CBM that belong in family 1. CBHs represent three non-reducing acting enzymes of GH6 family and four reducing-end acting enzymes of GH7 family. All of these enzymes seem to be targeted to secretion pathway and modified with glycans during post-translational modifications. BGs are classified to GH3 family, except one GH1 sequence, while none of them exhibit a CBM, as expected. Four are secreted and have potential N- and O-glycosylation sites, showing the highest molecular weight compared to the other cellulases, with a theoretical average value of 85.18 ± 3.2 kDa.

Totally, 12 cellulases have been isolated and characterized (Table 3). The group of Bukhtojarov et al. (2004) investigated the properties of individual cellulases from the multienzyme complex produced by a mutant strain of M. thermophila C1 (Visser et al., 2011). Among EGs, the highest saccharification activity was displayed by EG60 and EG51, representing enzymes of 60 and 51 kDa, respectively, which exhibited pI values of 3.6 and 5.0, respectively. It has been shown later that the EG51 and EG60 represent the GH5 and GH7 EGs from M. thermophila, respectively, (Gusakov et al., 2011). A different EG (StCel5A) displays a typical GH5 domain, exhibiting optimal activity at pH 6.0 and 70°C and retained greater than 50% of its activity following 2 h of incubation at 55°C, diluted in 10 mM citrate buffer pH 4.5 (Tambor et al., 2012). A GH7 EG gene was functionally expressed in methylotrophic yeast P. pastoris and subsequently characterized (Karnaouri et al., 2014). Substrate specificity analysis revealed that the enzyme is one of the most thermostable fungal enzymes reported up to now and exhibits high activity on substrates containing β-1,4-glycosidic bonds as well as activity on xylan-containing substrates. Moreover, MtEG7a was proved to liquefy rapidly and efficiently pretreated wheat straw, indicating EGs' key role to the initial step of hydrolysis of high-solids lignocellulose substrates (Karnaouri et al., 2014). This change in viscosity of these substrates is probably due to the gradual reduction of the average chain length of cellulose polysaccharides by endo-acting enzymes, such as endoglucanases. Totally, four CBHs and two BGs have been isolated from M. thermophila crude supernatant and studied. CBH IIb is the product of MYCTH_66729 gene that represents an enzyme of GH6 family, which is attached to polysaccharide substrate through a CBM and exhibits high levels of activity in comparison to other CBHs (Gusakov et al., 2007). In the same study, the isolation of CBH Ib, a GH7 family enzyme (MYCTH_2140736) is reported, which acts mainly against microcrystalline cellulose and CMC. Bukhtojarov et al. (2004) studied the properties of CBH Ia and CBH IIa, which are classified to GH7 and GH6 family, respectively. CBH Ia is the product of MYCTH_109566 gene, and seems to be expressed in two isoforms with distinct molecular weights, one exhibiting the catalytic domain owing a CMB and the other only the catalytic domain and part of the linker, after proteolysis. This enzyme is produced as a major protein of fungi's secretome (20–25% of the total extracellular protein) and adsorbed strongly on microcrystalline cellulose. It has been shown that there is a significant synergism between CBH IIb and CBH Ia enzymes during substrate hydrolysis (Gusakov et al., 2007).

Recently, another type of specific activity was revealed. Xyloglucan specific exo-β-1,4-glucanase (Xgl74A; EC 3.2.1.155) is classified to GH74 family and catalyzes the hydrolysis of (1-4)-D-glucosidic linkages in xyloglucans aiming in the successful removal of oligosaccharides from the chain end (Grishutin et al., 2004). Xyloglucan is a major structural polysaccharide found in the primary cell walls of higher plants that interact with cellulose microfibrils via hydrogen bonds to form a structural network that is assumed to play a key role in cell wall integrity. It consists of a cellulose-like backbone of β-1,4-linked D-glucopyranose (D-Glcp) residues, which most of them are substituted at C-6 with α-d-Xylp-(1→6) residues, to which other saccharides may be attached (most frequently, d-Galp and l-Fucp). M. thermophila was found to produce an exo-β-1,4-glucanase (Xgl74A) (Grishutin et al., 2004) with high specific activity toward tamarind xyloglucan, and very low or absent activity against carboxymethylcellulose (CMC) and barley β-glucan. Due to its unique substrate specificity the enzyme was given a new number in the Enzyme Nomenclature (EC 3.2.1.155). Apart from Xgl74A, two out of the seven cellulases reported from M. thermophila (Cel12A and Cel45A) possess a notable activity against xyloglucan, together with their major activities toward CMC and barley β-glucan (Bukhtojarov et al., 2004).

Hemicellulose polymers have a much more diverse structure than cellulose and consequently several enzymes are needed to completely degrade the polysaccharide into monosaccharides. Xylan that is the major component of hemicellulose in the plant cell wall, is consisted of a β-D-(1,4)-linked xylopyranosyl backbone, which, depending on the origin, can be substituted with arabinofuranosyl, 4-0-methylglucopyranosyl, feruloyl and acetyl groups (Shibuya and Iwasaki, 1985). Feruloyl groups can form strong networks through peroxidase-catalyzed oxidative coupling forming diferuloyl bridges (Topakas et al., 2007). The main enzymes needed for depolymerization are xylanases, assisted by accessory enzymes such as β-xylosidases and different arabinofuranosidases making the xylan backbone more accessible (Sørgensen et al., 2007). Other accessible enzymes that enhance xylan degradation are acetyl-xylanesterases (Poutanen et al., 1990), ferulic acid esterases (Topakas et al., 2007), and α-glucuronidases (De Vries et al., 1998). M. thermophila's hemicellulase genes are organized in 10 GH families (3, 10, 11, 30, 43, 51, 62, and 67) (Figure 2) and nine carbohydrate esterase (CE) families (1, 3, 4, 5, 8, 9, 12, 15, and 16) (Figure 3). Many of the encoding proteins have been isolated from the WT culture supernatant or expressed in heterologous hosts and finally characterized in terms of specific activity and physicochemical properties. The majority of them are predicted to follow the secretion pathway, while modified with N- and/or O- glucans, comprising a total amount of 66 enzymes that act synergistically for the degradation of hemicellulose.

In spite of the cooperative activity exhibited by the cellulolytic and hemicellulolytic enzymes, the impressive hydrolytic ability of various microorganisms in nature cannot be attributed only to this endo–exo mechanism. Apart from the hydrolytic system responsible for carbohydrate degradation, it seems that an oxidative system catalyze lignin depolymerization and oxidation of plant cell wall components, yielding reactive molecules (e.g., H₂O₂). Recent evidence highlights the critical role of alternative enzymatic partners involved in the oxidation of cell wall components. Among these enzymes, outstanding role during hydrolysis exhibit the originally described as cellulases LPMO enzymes, CDH and multicopper enzymes such as laccases. The genome of M. thermophila possesses more than 30 genes that encode proteins with such auxiliary activities (Figure 4). Members of the LPMO family AA9, have been shown to be copper-dependent monooxygenases that enhance cellulose degradation in concert with classical cellulases, as aforementioned before and reviewed by Dimarogona et al. (2013). These enzymes catalyze the cleavage of cellulose by an oxidative mechanism provided that reduction equivalents are available. These equivalents either involve low molecular weight reducing agents (e.g., ascorbate) or are produced by CDH activity (Langston et al., 2011). CDHs are extracellular enzymes produced by various wood-degrading fungi that oxidize soluble cellodextrins, mannodextrins and lactose efficiently to their corresponding lactones by a ping-pong mechanism using a wide spectrum of electron acceptors (Henriksson et al., 2000). Throughout the genome of M. thermophila, two genes encoding proteins classified to AA3 and 8 families have been identified (Figure 4). Both of them are predicted to be secreted in the culture supernatant and have potential glycosylation sites. The translated CDH MYCTH_111388 exhibits a C-terminal CBM and a cDNA clone of this sequence has been isolated and biochemically characterized by screening an expression library of M. thermophila (Subramaniam et al., 1999). Canevascini et al. (1991) purified a monomeric (91 kDa) and a dimeric (192 kDa) form of CDH that differed not only in molecular weight, but amino acid composition and carbohydrate content. Both forms oxidized cellobiose in the presence of cytochrome c or dichlorophenol–indophenol.

Laccases (EC 1.10.3.1) are multicopper enzymes that catalyze the oxidation of a variety of phenolic compounds, with concomitant reduction of O₂ to H₂O. These polyphenol oxidases are produced by most ligninolytic basidiomycetes (Baldrian, 2006) and can degrade lignin and other recalcitrant compounds in the presence of redox mediators (Ruiz-Dueñas and Martínez, 2009). The genome of the M. thermophila encodes eight putative enzymes with multicopper oxidase activity. Four of them have been annotated and one (MYCTH_51627) matches the lcc1 gene product encoding an extracellular laccase (Berka et al., 1997). Four sequences are predicted to possess a secretion signal, while one appears to remain membrane-bound. Lcc1 gene has been isolated from fungi's genome, heterologously expressed in A. oryzae and the produced 85 kDa enzyme (MtL) was characterized as a thermostable low oxidation potential laccase with high reactivity in aqueous medium at room temperature and neutral pH. MtL was tested for its capacity to catalyze enzymatic oxidation of several phenolic and polyphenolic compounds (ferulic acid, gallic acid, caffeic acid, and catechin) (Mustafa et al., 2005). M. thermophila laccases have been reported to oxidize lignin surface, by increasing the amount of radicals during thermomechanical pulp fiber material bleaching (Grönqvist et al., 2003) and promote oxidative polymerization of Kraft lignin from back liquor, which is the main by-product of pulp and paper industry (Gouveia et al., 2013).

M. thermophila is a powerful lignocellulolytic organism, which secretes a complex system of carbohydrate hydrolases for the breakdown of cellulose and hemicellulose, as well as oxidoreductases embedded in lignin degradation. Genome analysis in this review revealed 30 genes encoding cellulases classified to 10 GH families, 66 genes encoding hemicellulases classified to 10 GHs, 9 CEs and 35 genes encoding auxiliary enzymes. The latter include CDHs (AA3/AA8 family), LPMOs (AA9 family) and multicopper oxidases (laccases or laccase-like enzymes). Out of the total consortium of M. thermophila sequences encoding proteins with putative lignocellulosic activity, 80.2% are predicted to have a secretion signal peptide. Almost 76% of cellulases, hemicellulases and 88% of the accessory redox enzymes are targeted to secretion pathway, while only a very small amount remain inside the cell or represent membrane cell—bound macromolecules. Only 15.8% of the secreted enzymes in this review are predicted to possess a CBM and the majority of them comprise of auxiliary enzyme activities. The theoretical average molecular weight of secreted enzymes is 41.36 ± 15.9, varying between 10 and 97 kDa. The majority of secreted enzymes have molecular weight varying between 20 and 50 kDa, whereas β-xylosidases and β-glycosidases (GH3 family), and arabinofuranosidases (GH43 and GH51) appear to be high molecular weight proteins (Figure 5). The theoretical average isoelectric point of secretory enzymes is calculated 5.27 ± 0.8, at a range 4.34–7.9. In vivo expression and study of these enzymes would give different results, as the proteins are glycosylated, so size and pI value tend to moderate.

Rapid depolymerization of lignocellulosic material is a distinguishing feature of thermophilic fungi, such as M. thermophila, which was isolated from soil and self-heating masses of composted vegetable matter (Domsch et al., 1993). However, the precise biochemical mechanisms and underlying genetics of this procedure are not completely understood. Systematic examination of the M. thermophila genome revealed a unique enzymatic system comprising of an unusual repertoire of auxiliary enzymes, especially those classified to AA9 family, and provided insights into its extraordinary capacity for protein secretion. The current review constitutes, to the best of our knowledge, the first genomic analysis of the lignocellulolytic system of M. thermophila. The genomic data, along with the observed enzymatic activity of several isolated and characterized enzymes suggest that this fungus possesses a complete set of enzymes, including 30 cellulases, 66 hemicellulases, and 35 proteins with auxiliary auxiliary enzymes, covering the most of the recognized CAZy families. From its cellulases to its oxido-reductases and multicopper enzymes, M. thermophila gene complement represents several avenues for further research and its diverse array of enzymatic capabilities will contribute to the study of lignocellulose degradation and the subsequent ethanol biofuel production.