Trichoderma Secretome

C H A P T E R

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Trichoderma Secretome: An Overview Sunil S. Adav*, Siu Kwan Sze* School of Biological Sciences, Nanyang Technological University, Singapore *Corresponding authors email: [email protected], [email protected]

O U T L I N E Introduction103

New Candidates in Cellulose Degradation

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Proteomic Analysis of Secretory Proteins

Hemicellulose Hydrolyzing Enzymes

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Lignin Degradation by T. reesei

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Extraction of Extracellular Proteins for Proteomic Analysis106 107

Industrial Applications of T. reesei Cellulolytic Enzymes111

Polysaccharide Degradation Machinery of T. reesei 108

Conclusion112

Extracellular Protein Secretion by T. reesei

INTRODUCTION Lignocellulose, a fundamental constituent of plant biomass produced through photosynthesis is the most abundant, renewable, and sustainable bioresource. The biomasses from agricultural crop residues, grasses, wood, forest waste and municipal solid waste are sustainable, cost-effective, abundant renewable resources for foreseeable lignocellulosic biorefinery industry. The replacement of existing fossil fuel with lignocellulosic biofuel could substantially reduce greenhouse gases emission in the atmosphere and mitigate global warming (Farrell et al., 2006). Another advantage of lignocellulosic energy includes its CO2 neutral nature, zero carbon emission, environmentally friendly, does not affect food chain, brings agricultural diversification and many more. The bottleneck of the lignocellulosic bioenergy is centered on the cellulose conversion and sugar extraction technology that could sustain cost-effective and efficient biorefinery process. To convert biomass to sugar, and then to high-value products, different pretreatments including acid hydrolysis, alkali hydrolysis, ammonia freeze explosion, steam or acid/alkali-steam pretreatment, etc., have been tested. However, utilization of various chemicals in biomass pretreatment severely affects further hydrolysis and fermentation process due to the

Biotechnology and Biology of Trichoderma http://dx.doi.org/10.1016/B978-0-444-59576-8.00008-4

generation of process inhibitory compounds. Moreover, selective removal of inhibitory compounds from the hydrolysate considerably increases process cost. On the contrary, enzymatic pretreatments of biomass are efficient and environmentally friendly but the high cost of enzyme production hindered the industrial application; therefore, there is an ever increasing demand for more stable, highly active, specific enzymes. Several studies have been undertaken to isolate novel microbial strains, to investigate novel lignocellulolytic enzymes and to understand underlying lignocellulose degradation mechanism. Trichoderma spp. are filamentous fungi widely distributed in the soil and wood decomposing places. Trichoderma spp. grows rapidly, mostly as bright green conidia (Fig. 8.1) and also characterized as a repetitively branched conidiophore structure. Since they are rich in diverse habitat including tropical rain forest, termite guts (SreeramaandVeerabhadrappa, 1993), marine mussels and shellfish (Sallenave et al., 1999); they grow well on very wide variety of substrates and quickly adjust to existing environmental conditions with regulated growth, conidiation, enzyme production, and hence could be exploited for the benefits of mankind. Recently, the genomes of three Trichoderma species, namely, Trichoderma reesei, Trichoderma virens, and Trichoderma

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Copyright © 2014 Elsevier B.V. All rights reserved.

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FIGURE 8.1  Characteristic features of Trichoderma QM6a grown on plates. Fungal strain was grown on potato-dextrose medium at 30 °C. (For color version of this figure, the reader is referred to the online version of this book.)

atroviride have been sequenced by Department of Energy, Joint Genome Institute (http://www.jgi.doe.gov/). The comparisons and properties of in silico predicted secretome and the unique features of carbohydrate active enzymes of these Trichoderma strains have been reviewed (Druzhinina et al., 2012). Presently, the Index Fungorum database (http://www. indexfungorum.org/Names/ Names.asp) lists 504 different names for Hypocrea species and 196 records for Trichoderma. Several of these species names have been introduced long before molecular methods for species identification were available and some of them are likely to be outdated. International Subcommission on Trichoderma/Hypocrea lists 104 species (http://www.isth. info/biodiversity/index.php), which have been characterized at the molecular level. The qualities and versatility of Trichoderma spp., their defense mechanisms, regulatory mechanisms triggering the defense, and other characteristics have been reviewed (SchmollandSchuster, 2010). The strains like Trichoderma lignorum (also called T. atroviride), Trichoderma harzianum, T. virens, and Trichoderma asperellum act as a parasite on other fungi and hence are developed as biological control agents (­Geremia et al., 1993; Harman et al., 2004; SchmollandSchuster, 2010). Biocontrol mechanisms of Trichoderma strains have been reviewed by Benitez et al. (Benítez et al., 2004) and not in the scope of this chapter. The discovery of wild strain T. reesei QM6a in the Solomon Islands during World War II by the U.S. Army

laboratories, Natick (Reese, 1976) and its outstanding efficiency of cellulases led to extensive research toward industrial applications of these enzymes. These enzymes not only have wide applications in food, animal feed, textile, pulp and paper, grain alcohol fermentation, starch processing, pharmaceutical, malting and brewing industries but also are most vital for the saccharification of cellulosic plant material to simple sugars for biofuel production (RyuandMandels, 1980; Mandels, 1985). Due to the cellulolytic enzyme production potential of T. reesei QM6a, this strain has been extensively subjected to random mutagenesis to generate hypercellulolytic mutant strains (EveleighandMontenecourt, 1979; Durand et al., 1988). The high cellulolytic mutant QM9123 was isolated by irradiating conidia of QM6a in a linear particle accelerator and it was further transformed to QM 9414 by high-voltage electrons. Again, different series such as L, VTTD, MG and MHG was prepared from QM9414. While, mutants developed at Rutgers University, USA, form a separate series of high cellulose-producing mutants. Rut C30, the best-characterized and one of the most widely used T. reesei strain, is a member of this series. As shown in Fig. 8.2, T. reesei Rut C30 was generated by three mutagenesis steps with major objective to isolate catabolite derepressed mutants. The detailed results from biochemical-, microscopic-, genomic-, transcriptomic-, glycomic- and proteomic-based research on the Rut C30 strain have been reviewed by Peterson and Nevalainen (PetersonandNevalainen, 2012). Moreover, necrophytic nature of this

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Proteomic Analysis of Secretory Proteins

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FIGURE 8.3  SDS-PAGE of secretome by T. reesei on different sub-

FIGURE 8.2  Trichoderma reesei mutants development by different mutagens. (For color version of this figure, the reader is referred to the online version of this book.)

fungus demonstrated its safe use for industrial enzyme production and as an important model system for studying lignocellulose degradation (Nevalainen, 1994). Plenty of literature is documented on cellulolytic activities of numerous microbial strains using traditional colorimetric methods. However, colorimetric techniques are incapable in precisely differentiating individual enzymes in complex secretome secreted by microbial strains during soluble or complex cellulosic substrate utilization. The other limitations of these techniques are lower limit of detection, low limit of quantitation, inability of detecting various isoforms, reagent cross reactivity, etc. On the contrary, proteomics technology is well advanced, sensitive, free of colorimetric reagents, cross reactivity, have ability to detects low abundant proteins, detect various isoforms. Yet again, proteomic and genomic technologies are advancing and getting better day by day. Therefore, this chapter focuses on proteomic profiling of secretory lignocellulolytic enzymes by Trichoderma with major emphasis on wild T.reesei and its mutants using label-free and isobaric tags for relative and absolute quantification (iTRAQ) quantitative proteomic approach by liquid chromatography tandem mass spectrometry (LC–MS/MS). This chapter attempts to shed light on the fundamental understanding in lignocellulolytic enzyme secretion and their quantitative expressions during lignocellulosic biomass hydrolysis.

PROTEOMIC ANALYSIS OF SECRETORY PROTEINS Proteomic technologies are powerful tools for investigating alterations in protein profiles with time and with environmental factors such as in response to different carbon sources. The techniques such as 1D and 2D gel electrophoresis (2DGE) and MS have been recently

strates (M: protein marker, 1: T. reesei Rut C30 with carboxymethyl cellulose, 2: T. reesei Rut C30 with fibrous insoluble cellulose, 3: T. reesei Rut C30 with corn stover, 4: T. reesei Rut C30 with saw dust, 5. T. reesei QM6a with fibrous insoluble cellulose, 6: T. reesei QM6a with corn stover, 7: T. reesei QM6a with saw dust). (For color version of this figure, the reader is referred to the online version of this book.)

applied to investigate the secreted and/or intracellular proteins produced by filamentous fungi (CarberryandDoyle, 2007; Kim et al., 2007; González-Fernández et al., 2010; Gonzalez-FernandezandJorrin-Novo, 2012). Extracellular proteins produced by T. reesei under different culture conditions can be separated on 1D gel electrophoresis (Fig. 8.3). This 1D technique is classical protein analysis methods for separating, identifying and visualizing individual proteins in complex samples or to examine multiple proteins within a single sample based on their mass or charge. Again, 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) is often utilized as the last purification step for proteins of interest. Proteomics technology also adopt advanced separation technique such as 2DGE, highly sensitive identification tool like mass spectrometry and bioinformatics to understand protein expression profile in different physiological states of microbes. Protein profile by 2DGE for both qualitative and quantitative analysis of T. reesei has been well established (Herpoël-Gimbert et al., 2008). However, 2DGE have several limitations such as its costly, fairly insensitive to low abundant proteins, low recovery of 2DGE spots, nonsuitability for the entire proteome. Again, for comparison of targeted proteins at different conditions requires multiple 2DGE gels, expensive software, and expertise. Both gel-based and gel-free methods demonstrate advantages and disadvantages and choice of method depends on the ultimate goal of investigation. With sequencing of genomes of more than 150 microbes, both microbiology and proteomics has got revolutionized, and hence instead of studying single protein at a given time, researchers started focusing whole proteome or secretome under different physiological conditions. The advances in genomics and also to fill the gaps left by 2DGE, several gel-free proteomic techniques have been adopted and proteome coverage has been improved. Further, to reduce sample complexity,

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several high performance liquid chromatography fractionation techniques have been introduced prior to mass spectrometric analysis of proteome samples.

EXTRACTION OF EXTRACELLULAR PROTEINS FOR PROTEOMIC ANALYSIS Recent literature has perceived a significant increase in the number of reports that attempts the determination of expressed protein abundances during different substrate utilization by microbes. These reports provide an important data for the emerging system biology, modeling or designing enzyme cocktail for efficient biomass hydrolysis. Therefore, it's important to extract extracellular proteins with suitable techniques keeping in mind the major objectives like a quantitative extraction and solubilization of all secretory proteins, and a minimum manual intervention in order to make the procedure easily applicable, repeatable, reproducible and amenable to high-throughput experimental approaches. The best

approach is to harvest the supernatant at mid-exponential phase from a liquid culture by centrifugation and subject it for further filtration through 0.2 μm filters. Similarly, secretome from solid state fermentation can be extracted using suitable buffers. The culture filtrate contains some metabolites and if the lignocellulosic biomass is used as a substrate then it may contain brown colored extractives-probably lignin derived. The attempts to identify proteins from brown-extractive samples were unsuccessful (Abbas et al., 2005). Washing lignocellulosic substrate prior to use for fungal cultivation improves the protein quality and also minimizes brown coloration. The concentration of extracellular proteins (supernatant) by freeze drying technique enhances total protein coverage and also increases possibility of identification or quantification of low abundant proteins. Further, extraction of proteins from lyophilized sample with 10% trichloroacetic acid or acetone decreases the brown coloration and improves protein coverage in proteomic analysis. To remove low molecular weight metabolites, SDS-PAGE gel electrophoresis could be adopted. Further,

FIGURE 8.4  Peptide elution profile with high performance liquid chromatography gradient used for ERLIC fractionation. (For color version of this figure, the reader is referred to the online version of this book.)

B. SECRETION AND PROTEIN PRODUCTION

EXTRACELLULAR PROTEIN SECRETION BY T. REESEI

subjecting this gel to “in gel-digestion” using trypsin helps to improve LC–MS/MS analysis (Ravindran et al., 2012). For quantitative protein profiling, tryptic peptides have been labeled with iTRAQ reagents which are available in 4-plex or 8-plex kits (Adav et al., 2011a, 2012a). To improve protein identification, quantification and total coverage of proteome/secretome, an electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) technique has been developed and adopted for iTRAQ labeled peptides (Adav et al., 2010b Hao et al., 2010). The peptide elution profile using ERLIC technique together with high-performance liquid chromatography gradient is shown in Fig. 8.4. Absolute quantification and absolute stable isotope labeling with amino acids (SILAC) have also been successfully applied for fungal secretome analysis (Austin et al., 2011; Phillips et al., 2011).

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FIGURE 8.5  Molecular weight and pI values of secretory protein by T. reesei when lignocellulosic biomasses were used as a major carbon source.

EXTRACELLULAR PROTEIN SECRETION BY T. REESEI Trichoderma reesei is an efficient cell factory for protein production and also acts as a host for homologous and heterologous protein production, and hence is exploited by enzyme industry. As reviewed by Saloheimo and Pakula (Saloheimo and Pakula, 2012), protein production yield by this strain during industrial fermentation ranges around 100 g secreted proteins per litter. To study its enzyme system, complete genome sequencing was undertaken and released to the public early in 2005 (http://gsphere.lanl .gov/trire1/trire1.home.html). Being a potent cellulose degrader, it was expected that T. reesei would encode large number of glycoside hydrolases (GHs) but comparatively it encodes lesser GH genes than Aspergillus nidulans (strain FGSC A4), Aspergillus fumigatus (clinical isolate Af293), Aspergillus oryzae (strain RIB40), Magnaporhe grisea (strain 70-15), Fusarium graminearum (strain PH-1) (Martinez et al., 2008). Although it encodes 200 GH genes but their expression depends on the carbon source, pH, temperature and other environmental factors. When T. reesei was cultivated with cellulosic substrates and lignocellulosic biomasses as a carbon sources, enzymes like cellulases, amylases, hemicellulases, lignin degrading enzymes, peptidases and proteinases, transport proteins, hypothetical proteins and proteins involved in cell morphogenesis were identified and quantified using iTRAQ technique (Adav et al., 2012a). These enzymes secreted in response to lignocellulosic biomasses revealed molecular weight in the range 20–160 kDa and pI between 3.8 and 7.8 (Fig. 8.5). However, protein like polyphenoloxidase, manganese superoxide dismutase, reduced form of Nicotinamide Adenine Dinucleotide-ubiquinone oxidoreductase etc. showed pI values more than 8.0. Further, pH dependent expression of lignocellulolytic enzymes of T. reesei QM6a, T. reesei QM9414, T. reesei Rut C30, and

T. reesei QM9414MG5 profiling by LC–MS/MS provided pH sensitive and resistance enzyme targets for industrial lignocellulose hydrolysis(Adav et al., 2011b). Due to the intricate regulation of cellulolytic enzyme systems, and the complex nature of lignocellulosic materials, the physiological responses of fungi, in terms of type of secreted enzyme, their abundances, enzyme titers, and so on, are quite variable. It has also been confirmed that the production of cellulases by Trichoderma is transcriptionally regulated and carbon source dependent (Stricker et al., 2008). The influence of medium components including delignified steam-exploded sugarcane bagasse, sucrose, and soybean flour on the production of cellulolytic enzymes by T. harzianum P49P11 strain has been reported (da Silva Delabona et al., 2013). Comparative secretome analyses of two T. reesei RUT C30 and CL847 by 2D and Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) or LC–MS/MS using lactose as a carbon source revealed significant differences in terms of both spot numbers and protein composition (Herpoël-Gimbert et al., 2008). Trichoderma reesei grown in a medium with cellulose or complex lignocellulosic biomass as a substrate secretes complex enzyme mixture composed mostly of cellulases and hemicellulases (Saloheimo and Pakula, 2012). This secretome contains numerous endoglucanases which cleaves internal β-1,4-glucosidic bonds and exoglucanases that cut the dissaccharide cellobiose from the nonreducing end of the cellulose polymer chain and their abundance corresponds to approximately 80% of the total secreted protein; however, its β-glucosidase, which hydrolyzes the cellobiose to glucose remains very low (Saloheimo and Pakula, 2012). Hence, using mutagenesis, catabolite-derepressed Rut C30 has been developed in which two major genetic changes have been described: (1) truncation in the cre1 gene encoding CRE1, the carbon catabolite repressor protein; (2) a frameshift mutation in

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the glycoprotein processing β-glucosidase II encoding gene (Ilmén et al., 1996; Geysens et al., 2005; Seidl et al., 2008). Thus, as confirmed by Seidl et al. (Seidl et al., 2008), Rut C30 lacks 85 kb genomic fragment, and consequently misses additional 29 genes comprising transcription factors, enzymes of the primary metabolism and transport proteins. According to Gallo et al. (Gallo et al., 1979), Rut C30 outperformed a high cellulase producing mutant MCG77 and its parent strain QM9414 and hence this mutant has been used in developing processes for the production of cellulolytic enzymes (Olsson et al., 2003).

POLYSACCHARIDE DEGRADATION MACHINERY OF T. REESEI Lignocellulose is composed of cellulose, hemicellulose, pectin, lignin and other substances in minor quantities. Cellulose, composed of β-1,4-linked units of the glucose; and hemicellulose, a widely distributed heteropolysaccharides such as xylan and mannan, are the most abundant polymer in biosphere and the major structural components of plant biomass. Hence, plant biomass represents the key natural raw material for many current biotechnological processes and a sustainable source of future fuels, chemicals and materials. Using solar energy, terrestrial plants produce yearly 1.3 × 1010 metric tons biomass (on dry weight basis) by photosynthetic fixation of carbon dioxide (Kumar et al., 2008). This biomass has the energetic equivalent of 7 × 109 metric tons of coal or about two-thirds of the world's energy requirement (Kumar et al., 2008). Again, cellulosic feedstock from agriculture and other sources represent about 180 million tons per year (Kumar et al., 2008). The cellulose microfibrils are embedded in a matrix of hemicellulose and lignin to form a strong, yet flexible biocomposite (Jeffries, 1996). In nature, many different fungi and bacteria contribute to the degradation of these natural polymers and play major role in recycling carbon. Cellulose and hemicellulose are hydrolyzed by cellulases and hemicellulases, whereas lignin is oxidized by various oxidases and peroxidases. Hydrolysis of biomass by chemical, enzymatic pretreatment or combinations of both methods are under consideration for extraction of energy from biomass. Although chemical methods such as sulfuric acid pretreatment solubilizes hemicellulose content of biomass and thereby disrupt the lignocellulosic composite material but it results in the formation of process inhibitory products that often inhibit further hydrolysis and also downstream fermentation lowering the overall process yield. On the contrary, enzymatic pretreatment processes are highly efficient and environmentally friendly. Lignocellulose degradation by fungi requires the secretion of proteins involved in depolymerization of

cell wall constituents. According to exo–endo model, the main categories of cellulose degrading enzymes are:   

1. e ndoglucanases that catalyzes random cleavage of internal bonds of the cellulose chain, 2. exoglucanases that attack the chain ends of cellulose, releasing cellobiose and 3. β-glucosidases converts cellobiose into glucose.   

The cellulolytic machinery of T. reesei constituting CBHs, different EGs, and β-glycosidases act synergistically on substrates by multiple cooperation, including exo/endo synergism and exo/exo synergism. Although, 10 cellulases (one CBH1, CBH2, EG1, EG3, and EG5; two EG2s; and three EG4s) belonging to different GH families have been encoded in T. reesei genome (­Martinez et al., 2008), but only four major cellulases [CBHI (Cel7A), CBH II (Cel6A), EG I (Cel7B) and EG II (Cel5A)] have usually been secreted in notable quantities by this fungus (Foreman et al., 2003; Markov et al., 2005). When soluble substrates like crystalline/fibrous cellulose, complex lignocellulosic biomasses such as corn stover and saw dust were used for cultivation of T. reesei QM6a and Rut C30; one each CBHI, CBHII, EG1, EG3, and EG5; two EG2s; and two EG4s were identified and quantified in the secretome (Adav et al., 2012a). Further, these authors found upregulation of these enzymes in lignocellulosic culture condition. Similarly, culturing of Phanerochaete chrysosporium with cellulosic substrate exhibited expression of genes encoding Cel7B, Cel7C, Cel7D, Cel7F/G, and Cel6A (Suzuki et al., 2010). Vanden Wymelenberg et al. (Wymelenberg et al., 2009) also reported differential regulation of carbohydrate active enzymes by P. chrysosporium in response to different carbon sources. The genome of T. reesei encodes eight endoglucases (one Cel7B, Cel12 and Cel45, two Cel5, and three Cel61) that have endo-1,4-β-D-glucanase activities and these all enzymes except Cel61 were secreted when T. reesei QM6a and Rut C30 were cultivated using lignocellulosic biomass (Adav et al., 2012a). Cel61 of T. reesei is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. Cel61B lacks a carbohydrate-binding module and is a single domain protein that folds into a twisted β-sandwich. In addition to enzymes with endo-1,4-β-d-glucanase activity, endo-1,6-β-d-glucanase was also detected and quantified in the secretome of these fungal strains. Again, 1,6-β-d-glucanase has been shown to lyse yeast and fungal cell walls. Two extracellular β-1,6-glucanases from T. harzianum, when it was grown on chitin as the sole carbon source, emphasized its role as biological control agent against several fungal plant pathogens (De la Cruz et al., 1995; De La C ­ ruzandLlobell, 1999). Further, culturing of T. harzianum on cellulose medium, and analyzing it's secretome by a combination of 2DGE and MALDI–MS or MS/MS, and LC-MS/MS revealed

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New Candidates in Cellulose Degradation

major hydrolytic enzymes as chitinases and endochitinases, which may reflect the biocontrol feature of T. ­harzianum (Do Vale et al., 2012). Their study identified chitinases, endo-N-acetylglucosaminidases, hexosaminidases, galactosidases, xylanases, exo-1,3-glucanases, endoglucanases, xylosidases, α-l-arabinofuranosidase, N-acetylhexosaminidases, and other enzymes that represented 51.36% of the total secretome; while other proteins were proteases, hypothetical and intracellular proteins. In addition to the biocontrol competence, T. harzianum also secrete other glycosyl hydrolases such as cellulases, xylanases, and mannanases, under appropriate culture conditions supporting its role in biomass recycling (Deschamps et al., 1985; De Castro et al., 2010a; De Castro et al., 2010b). Trichoderma harzianum, cultivated in medium containing sugarcane bagasse secreted multienzymatic complexes bearing cellulolytic and xylanolytic activities (Silva et al., 2012). Trichoderma reesei genome data revealed only one GH7 cellobiohydrolase, whereas A. nidulans, A. fumigatus, A. oryzae, and Neurospora crassa encodes two GH7 cellobiohydrolase (Martinez et al., 2008). Although, one GH7 cellobiohydrolase has been encoded in T. reesei, but yet this fungus is potent cellulolytic fungus possibly due to unique nature of GH7 and GH6 cellobiohydrolases that hydrolyzes crystalline cellulose in the absence of endoglucanases. Cellobiohydrolase remain abundant (more than 75%) in the secretome with 50–60% CBHI and about 20% CBHII (Margeot et al., 2009; Gusakov, 2011). CBHI of T. reesei is known to bear a C-terminal cellulose-­ binding module (CBM) attached to its CD of GH7, and CBHII belonging to GH6 contains an N-terminal CBM (­Ouyang et al., 2006). Majority of cellulases' structure (except EGIII) consists of catalytic and CBM linked with a flexible peptide linker. Enzymes with CBM posses higher activity on crystalline cellulose than those lacking this module (Lynd et al., 2002) since CBM helps enzyme to bind with insoluble cellulose surface. The role of each CBHI module has been investigated by using a single molecule approach that combines optical total internal reflection fluorescence microscopy and nonoptical atomic force microscopy (Liu et al., 2010). Analysis of secretome of T. reesei Rut C30 with alkaline- or acid-treated rice straw as a carbon source by 2DGE revealed dominancy of cellobiohydrolases (Sun et al., 2008). Similarly, the extracellular cellulolytic system of T. reesei in response to 1 mM sophorose was dominated by cellobiohydrolases, and was quite different from the enzyme mixtures produced in lactose-based media (Juhász et al., 2005; Jun et al., 2011). Based on the iTRAQ ratios of GH7 cellobiohydrolase in the secretome of T. reesei QM6a and Rut C30, Adav et al. (Adav et al., 2012a) concluded the strain-dependent expression of this protein. 2DGE analysis of T. reesei Rut C30 secretome grown either on a spent hydrolysate or on a

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lactose-based media also showed higher abundance of cellobiohydrolases (Juhász et al., 2005). The cultivation of T. reesei QM9414, T. reesei Rut C30, T. reesei QM9414MG5 and T. reesei QM6a in cellulosic medium having pH within 3.0–9.0 demonstrated expression of GH7 cellobiohydrolase but emPAI values suggested its pH and strain-dependent abundances (Adav et al., 2011b). The secretion of at least two β-glucosidases by T. reesei facilitates the hydrolysis of cellobiose as well as oligosaccharides to glucose. However, cellobiose is a stronger inhibitor for cellulases, and its potency of inhibition is greater than that of glucose (LeeandFan, 1982, 1983). To overcome this inhibition, external supplement of β-glucosidases brought commercial cellulase preparations from companies like Novozymes, Genencor International Inc. into the market. On other side, to conquer this inhibitory effect by cellobiose, catabolite-depressed T. reesei Rut C30 was generated. About 12 β-glucosidases have been identified in the T. reesei genome but some of them are intracellular in nature (Foreman et al., 2003; Ouyang et al., 2006). When Trichoderma citrinoviride was grown on delignified Lantana camara produced a β-glucosidase and secreted it out in the medium. This extracellular secreted enzyme was 90 kDa, monomeric, optimally active at pH 5.5 and insensitive to inhibition by glucose (up to 5 mM) (Chandra et al., 2012). Of several iTRAQ quantified glucosidases in the secretome of T. reesei QM6a and Rut C30 during lignocellulose ­utilization, proteins like GH3 β-glucosidase Cel3b, GH17 glucan1,3-β-glucosidase and GH71 glucan endo-1,3-β-glucosidase were upregulated. To improve biomass hydrolysis, considerable improvements have been made in T. reesei cellulases, their thermostability, pH, etc., by direct protein engineering (Wang et al., 2005; Nakazawa et al., 2009).

NEW CANDIDATES IN CELLULOSE DEGRADATION Plant cell wall proteins named “expansins” disrupt hydrogen bonding between cell wall polysaccharides without hydrolyzing them (Cosgrove, 2000). Similarly, in T. reesei secretome, a protein with endoglucanase activity having sequence similarity to expansin called “swollenin” has been iTRAQ quantified. This protein has N-terminal fungal type cellulose-binding domain connected by a linker region to the expansin-like domain. It has been well documented that the regulation of swollenin gene resembles with regulation of T. reesei cellulase genes (Saloheimo et al., 2002). The biological role of swollenin has been studied by disrupting the swo1 gene from T. reesei (Saloheimo et al., 2002). Further, the swollenin gene was expressed in yeast and Aspergillus niger var. awamori and also showed that the activities of SWOI-containing yeast supernatant disrupts the

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structure of the cotton fibers without detectable formation of reducing sugars (Saloheimo et al., 2002). It has been presumed that these fungal strains encode three to four swollenin-like proteins that vary in their mode of action, but contribute in polysaccharide hydrolysis. On the contrary, plant parasitic roundworm, Globodera rostochiensis, can produce a functional expansin (Gr-EXPB1) used to loosen cell walls when invading its host plant (Qin et al., 2004). The upregulation of swollenin in the secretome of T. reesei QM6a and Rut C30 during lignocellulose degradation indicated its possible role in deconstructing lignocellulose structure (Adav et al., 2012a). It has also been speculated that cell wall disruption by Trichoderma are more efficient due to swollenin, which could facilitate the access to other cellulolytic enzymes at less accessible areas of the substrate. It is quite fascinating that swollenin homologs are found only in Trichoderma species, A. fumigatus, or its close relative Nassarius fischeri, and not in other fungal phytopathogens. Trichoderma reesei also secrete hydrophobins that are surface active proteins and perform a wide variety of functions. Hydrophobins self-assembles in rodlet-like structures on the outer surfaces of fungal cell walls, and mediate interactions between the fungi and their environment, enabling aerial growth and conidiation, recognition of the host surface, and also in symbiosis. Hydrophobin 1 and hydrophobin 2 were upregulated when T. reesei QM6a and Rut C30 cultured with biomass. According to Kubicek et al. (Kubicek et al., 2008), T. virens and T. atroviride had a much higher number of class II hydrophobin genes compared to other ascomycetes. While, a novel set of hydrophobins from Trichoderma spp. that differs in cysteine spacing and protein surface pattern from earlier reported protein have also been reported (Seidl-Seiboth et al., 2011). Based on their characteristic surface activity and capability to form amphiphilic protein films these proteins are also treated as microbial surfactants. Due to high surface activity, hydrophobins reduces the surface tension of the medium or the substratum in/on which fungi grow. This further allows fungi to breach the air– water interface or preventing water logging while maintaining permeability to gaseous exchange. Hydrophobins also play a major role in masking the immunogenicity of airborne fungal spores (Bayry et al., 2012). By covering the spore surface, hydrophobins impart immunological inertness to the spores and prevent activation of host immune system (Aimanianda et al., 2009).

HEMICELLULOSE HYDROLYZING ENZYMES Hemicelluloses, the second most common polysaccharides in nature, represent about 20–35% of lignocellulosic biomass. Structurally, hemicelluloses are

heterogeneous polymers of pentoses (xylose, arabinose), hexoses (mannose, glucose, galactose), and sugar acids. Hardwood hemicelluloses contain mostly xylans, whereas softwood hemicelluloses are prosperous with glucomannans. Xylans from different sources, such as grasses, cereals, softwood, and hardwood, differ significantly in composition. For example, birch wood (Roth) xylan contains 89.3% xylose, 1% arabinose, 1.4% glucose, and 8.3% anhydrouronic acid; while rice bran contains 46% xylose, 44.9% arabinose, 6.1% galactose, 1.9% glucose, and 1.1% anhydrouronic acid (ShibuyaandIwasaki, 1985; KormelinkandVoragen, 1993). Corn fiber xylan is one of the complex heteroxylans containing β-1,4-linked xylose residues. It contains 48–54% xylose, 33–35% arabinose, 5–11% galactose, and 3–6% glucuronic acid (DonerandHicks, 1997). Thus, complete degradation of hemicelluloses need endoxylanases and endomannanases that cleave the main chains of xylan and mannan, respectively; and the side chain and substitution cleaving activities including arabinofuranosidases, α-glucuronidases, α-galactosidases, acetyl xylan esterases, ferulic acid esterase, and p-coumaric acid esterase, etc. Thus, α-arabinofuranosidase and α-glucuronidase confiscate the arabinose and methyl glucuronic acid substituent, respectively, from the xylan backbone. While, acetylxylan esterase break the ester linkages between xylose units and acetic acid, and ferulic acid esterase hydrolyzes ester linkage between arabinose side chain residues and ferulic acid. Due to several side chain residues, structurally xylan is more complex than cellulose and need several different enzymes with specific specificities for complete hydrolysis. Several microorganisms such as Thermomonospora fusca, T. reesei or Aspergillus sp., Chrysosporium lucknowense, Penicillium capsulatum, Talaromyces emersonii, etc. possess xylan degrading enzyme systems (Filho et al., 1991; Olsson et al., 2003; Martinez et al., 2008; Adav et al., 2010a, 2012b, 2012c). Synergistic interactions among endoxylanase, β-xylosidase, α-arabinofuranosidase, and acetylxylan esterase produced by thermophilic actinomycete T. fusca have been studied (Bachmann and McCarthy, 1991). The hemicellulases including endo-1,4-β-xylanase, α-L-arabinofuranosidase, acetyl xylan esterase were expressed when T. fusca was cultivated with cellulose as a major carbon source indicating cellulose alone can stimulate the expression of hemicellulolytic enzymes. Similarly, comparison of endoxylanase produced by T. reesei during its cultivations on cellulose, sugar beet pulp and alkaline extracted sugar beet pulp showed the highest total activity on cellulose eliminating necessity of hemicellulose to induce the expression of endoxylanse (Olsson et al., 2003). Although T. reesei has low inventory of hemicellulases (16 hemicellulase genes), variable regulation of enzymes like xylanases (GH11 and GH30), arabinofuranosidases (GH54 and GH62), β-xylosidases

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(GH52, GH43), β-glucuronidase (GH79), acetylxylan esterase, acetyl esterase, etc. were iTRAQ quantified when this fungus was cultivated with cellulose, saw dust, and corn stover. The use of spectral counting methods in the proteomic characterization has been shown to determine relative abundances of different hemicellulases (­Chundawat et al., 2011). The correlation between the secreted amount of xylanase and mannanase by T. reesei Rut C30 and their induced production in presence of xylan has been established (Sipos et al., 2010). Induction of cellulase and hemicellulase secretion has already been examined both on the levels of transcription and secretion (Juhász et al., 2005; Nakazawa et al., 2009). Many microorganisms, such as P. capsulatum and T. emersonii, possess complete xylan degrading enzyme systems (Filho et al., 1991).

LIGNIN DEGRADATION BY T. REESEI Numerous microorganisms are evolved with the capability to degrade and utilize cellulose and hemicellulose as carbon and energy source, however, only limited group of fungi have potential to breakdown recalcitrant lignin. The white rot fungi have evolved with unique enzyme system that can degrade lignin to CO2 to gain access to cellulose and hemicelluloses. In addition to major lignin degrading peroxidases, a group of oxidases and proteins belonging to oxidoreductase family plays major role in generating highly reactive free radicals that undergo complex series of reactions and cleaves several bonds. The culturing T. reesei with lignocellulosic biomasses resulted into expression of proteins belonging to oxidoreductase family, peroxidase/catalase, glyoxal oxidase, glutathione reductase, and glutathione S-­transferase glyoxalase (Adav et al., 2012a, 2012b). Laccase, a multicopper containing enzyme that catalyzes oxidation of phenolic compounds with concomitant reduction of oxygen to water was noted abundantly in the secretome of T. reesei and other Trichoderma strains (Gianfreda et al., 1999; Hölker et al., 2002). In addition to their role in lignin degradation, laccases have wide commercial applications within food industry, pulp and paper industries, textile industry, synthetic chemistry, cosmetics, soil bioremediation and biodegradation of environmental phenolic pollutants (­BhatandBhat, 1997; Kuhad et al., 2011). Fungal strains belonging to various classes such as Ascomycetes, Basidiomycetes and Deuteromycetes, have capability to produce laccase (Gianfreda et al., 1999). Extracellular laccases from different Trichoderma strains including T. atroviride, T. harzianum, Trichoderma longibrachiatum have been isolated and characterized (Hölker et al., 2002; Chakroun et al., 2010). Lignin degradation mechanism is reliant on H2O2 as oxidant in the peroxidative reactions. A number of oxidases

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have been considered to play major role in H2O2 generation. The culturing of T. reesei QM6a and Rut C30 with lignocellulosic biomasses resulted secretion of glyoxal oxidase that was identified and iTRAQ quantified. In addition to these cellulases, hemicellulases and lignin degrading proteins, peptidases, chitinases, phosphatase, transport proteins, and hypothetical proteins have been reported in the secretome of T. reesei QM6a and its mutant strains like Rut C30, QM9414 and QM9414MG5 (Adav et al., 2011b, 2012a). A limited literature report exists on exact role of peptidases and protease in lignocellulose degradation. The protein contents in the plant cell wall, their composition and role have been documented (Cassab, 1998). According to Cassab (Cassab, 1998), plant proteins are structural components of plant cell wall and play a major role in the formation of β-pleated sheets. Thus, presence of proteins in plant cell wall supports the expression of peptidases and protesases in the secretome of T. reesei during lignocellulose hydrolysis.

INDUSTRIAL APPLICATIONS OF T. REESEI CELLULOLYTIC ENZYMES Microbial cellulases have a wide range of potential applications in biotechnology. In several applications, they are used with supplement of hemicellulases, pectinases, ligninases and associated enzymes. In addition to lignocellulosic bioenergy, some most important applications of cellulases are in food, brewery and wine, animal feed, textile and laundry, pulp and paper industries, as well as in agriculture and many more (RyuandMandels, 1980; Mandels, 1985; BhatandBhat, 1997). Humans lack the ability to digest cellulose fiber hence a digestive enzyme “Digestin” that contains cellulase has been commercialized. Cellulases have been applied successfully in textile wet processing and finishing of cellulose based textile to improve final quality of the products. Microbial cellulases and polysaccharides play important roles in fermentation processes to produce alcoholic beverages including beer and wine. Uses of cellulases improve both quality and yield of the fermented products (­BhatandBhat, 1997; Bamforth, 2009; Kuhad et al., 2011), hence, cellulases are supplemented during mashing or preliminary fermentation to hydrolyze glucan that help to reduce viscosity of wort and improve the filterability. In wine industry, cellulases, hemicellulases and pectinases have been adopted since their use improves color extraction, skin maceration, clarification and filterability and finally the quality of wine. Malting of barley, preparation of grape juice for wine production and several other processes use cellulases derived from T. reesei. Cellulases and hemicellulases are immensely useful in animal feed to improve feed value by pretreatment of agricultural silage and grains by cellulases and hemicellulases

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(Dhiman et al., 2002; Kuhad et al., 2011). Application of these enzymes eliminates antinutritional factors of feed grains, enhance feed quality and also provide supplementary digestive enzymes such as proteases, amylases, glucanases and many more (Dhiman et al., 2002; Kuhad et al., 2011). In agricultural field, to control various crop diseases and pest, to enhance crop growth, mixture of cellulases, hemicellulases and pectinases has been used (BaileyandLumsden, 1998; Kuhad et al., 2011). The cellulolytic fungi T. reesei play a major role in agricultural industry by facilitating enhanced seed germination, plant growth and ultimately enhance crop yield (­BaileyandLumsden, 1998; Kuhad et al., 2011). Trichoderma reesei cellulases are also widely used in detergent industry and waste management.

CONCLUSION Trichoderma sp. can adapt to various environmental condition, carbon and nitrogen sources and also produce wide range of extracellular hydrolytic enzymes. Several fungal strains like T. reesei can serve as cell factory for protein production and also a potential host for homologous and heterologous protein expression hence they are exploited by enzyme industries. Fungal enzymes have wide applications in various industries including pulp and paper, textile, laundry, biofuel production, food and feed industry, brewing, and agriculture. Due to their potential in biomass conversion, the secretome of Trichoderma sp. including their mutant stains have been explored using proteomics. Proteomic technology is powerful, sensitive and well advanced tool to study quantitative expressions of proteins. The techniques like 1D and 2DGE have been successfully established in fungal proteomics. While, recently, highthroughput iTRAQ technique that profile quantitative expression of proteins has been applied to profile relative expression of cellulolytic, hemicellulolytic, ligninolytic and proteolytic enzymes. Further development in absolute quantitation of lignocellulolytic enzymes would shed light on designing of biomass hydrolyzing enzyme cocktail. With the advances in accurate absolute quantitation of microbial lignocellulosic enzymes from potent biomass degrading strain and microbial consortium, it is most likely that the lignocellulosic biorefinery, by producing multiple value-added products and biofuel, may take advantage of the various biomass structural components and maximize importance and economic value of biomass feedstock.

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B. SECRETION AND PROTEIN PRODUCTION