Fungal enzyme sets for plant polysaccharide degradation

Joost van den Brink Ronald P. de Vries 0 ) CBS-KNAW Fungal Biodiversity Centre , Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands Enzymatic degradation of plant polysaccharides has many industrial applications, such as within the paper, food, and feed industry and for sustainable production of fuels and chemicals. Cellulose, hemicelluloses, and pectins are the main components of plant cell wall polysaccharides. These polysaccharides are often tightly packed, contain many different sugar residues, and are branched with a diversity of structures. To enable efficient degradation of these polysaccharides, fungi produce an extensive set of carbohydrate-active enzymes. The variety of the enzyme set differs between fungi and often corresponds to the requirements of its habitat. Carbohydrate-active enzymes can be organized in different families based on the amino acid sequence of the structurally related catalytic modules. Fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase families, three carbohydrate esterase families and six polysaccharide lyase families. This mini-review will discuss the enzymes needed for complete degradation of plant polysaccharides and will give an overview of the latest developments concerning fungal carbohydrate-active enzymes and their corresponding families. - Plant polysaccharides have applications in many industrial sectors, such as biofuel, pulp and paper, and food and feed. Cellulose, hemicelluloses, and pectin are the main components of plant cell walls representing up to 70% of the biomass (Jorgensen et al. 2007). Of the three, cellulose is the least complex polymer with a linear structure of -1,4linked D-glucose residues. The long glucose chains are tightly bundled together in microfibrils and are noncovalently linked together by hemicelluloses (Kolpak and Blackwell 1976; Carpita and Gibeaut 1993). Hemicelluloses are classified according to the main sugar in the backbone of the polymer, i.e., xylan (-1,4linked D-xylose), mannan (-1,4-linked D-mannose), or xyloglucan (-1,4-linked D-glucose). The backbone of hemicelluloses has many branches composed of monomers such as D-galactose, D-xylose, L-arabinose, and D-glucuronic acid. The precise composition of hemicellulose is strongly dependent on the plant species and tissue (Scheller and Ulvskov 2010). For instance, hard wood xylans often have D-glucuronic acid attached to their backbone, whereas Larabinose is the most common branching residue in cereal xylans (de Vries and Visser 2001). Moreover, hemicelluloses are often acetylated and to a lesser extent ester-linked with feruloyl or p-coumaroyl residues (Ebringerova et al. 1990; Xu et al. 2010). Pectin is less prominently present in most plant biomass compared to cellulose and hemicellulose. However, some plant biomass types (e.g., citrus peels) are very rich in pectin (Angel Siles Lopez et al. 2010; Ridley et al. 2001; Grohmann and Bothast 1994). The backbone of pectin consists mainly of alpha-1,4-linked D-galacturonic acid residues that can be methyl-esterified or substituted with acetyl groups. Pectins are classified in three general groups, homogalacturonan (linear polymer), xylogalacturonan (branched by -1,3-linked D-xylose), and rhamnogalacturonan (Ridley et al. 2001; Wong 2008; Caffall and Mohnen 2009). The latter polysaccharide is the most complex pectin structure. Its backbone consists of alternating L-rhamnose and D-galacturonic acid residues, while branches with 1,4-linked D-galactose and different -linked L-arabinose residues are connected to the L-rhamnose residues (Ridley et al. 2001; Wong 2008). In nature, fungi play a central role in the degradation of plant biomass. Plant-biomass-degrading fungi produce an extensive set of carbohydrate-active enzymes specifically dedicated to degrade plant polysaccharides. However, these sets differ between fungal species. For instance, Trichoderma reesei has a highly efficient set of enzymes involved in cellulose degradation (Martinez et al. 2008; Kubicek et al. 2011), while Aspergillus species produce many enzymes to degrade pectin (Martens-Uzunova and Schaap 2009). The industrial importance of polysaccharide-degrading enzymes and the availability of many fungal genomes have strongly deepened our understanding of fungal biodiversity with respect to plant cell wall degradation. This minireview will give an overview of the latest developments and insights into fungal enzymes involved in plant polysaccharide degradation. Dedicated fungal toolboxes for the degradation of specific plant polysaccharides Carbohydrate-active enzymes can be organized in different families based on amino acid sequence of the structurally related catalytic modules (www.cazy.org) (Cantarel et al. 2009; Henrissat 1991). Fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase (GH) families, three carbohydrate esterase (CE) families, and six polysaccharide lyase (PL) families (Battaglia et al. 2011; Coutinho et al. 2009). Even though enzymes within the same family share sequence similarity, some families can contain multiple activities. For example, GH5 contains many catalytic activities, including exoglucanases, endoglucanases, and endomannanases (Dias et al. 2004). In addition, a specific enzyme activity can be present in several CAZy families. This is important for efficient degradation of plant polysaccharides as enzymes of each family have often complementary substrate specificity. For instance, endoxylanases in GH10 have lower substrate specificity and can degrade xylan backbones with many substitutions, while GH11 endoxylanases have higher substrate specificity with preference for unsubstituted xylan chains (Pollet et al. 2010; Biely et al. 1997). Annotation of carbohydrate-active enzymes has been done for many fungal genomes (Pel et al. 2007; Espagne et al. 2008; Battaglia et al. 2011; Ohm et al. 2010; Martinez et al. 2004; Martinez et al. 2008). As an illustration, Table 1 shows a comparison of carbohydrate-active enzymes involved in plant polysaccharide degradation of 13 fungal genomes, including industrial fungi such as Aspergillus oryzae, Aspergillus niger, Penicillium chrysogenum, T. reesei, and Saccharomyces cerevisiae. Most apparent from this table is the correlation between habitat and the amount of carbohydrate-active enzymes. For example, the Saccharomycete S. cerevisiae does not require extracellular enzymes for polysaccharide degradation to survive in its natural niches like surfaces of rotting fruits (Liti et al. 2009; Cherry et al. 1997). This fungus has therefore hardly any carbohydrate-active enzymes. Another Saccharomycete Pichia stipitis can be found, among other places, in the guts of termites that inhabit and degrade white-rotted hardwood (Jeffries et al. 2007). The genome of this fungus contains only a few -glucosidases and -mannosidases to degrade glucan and mannan oligosaccharid (...truncated)