To improve the enzymatic hydrolysis (saccharification) of lignocellulosic biomass by Trichoderma reesei, a set of genes encoding putative polysaccharide-degrading enzymes were selected from the coprophilic fungus Podospora anserina using comparative genomics. Five hemicellulase-encoding genes were successfully cloned and expressed as secreted functional proteins in the yeast Pichia pastoris. These novel fungal CAZymes belonging to different glycoside hydrolase families (PaMan5A and PaMan26A mannanases, PaXyn11A xylanase, and PaAbf51A and PaAbf62A arabinofuranosidases) were able to break down their predicted cognate substrates. Although PaMan5A and PaMan26A displayed similar specificities toward a range of mannan substrates, they differed in their end products, suggesting differences in substrate binding. The N-terminal CBM35 module of PaMan26A displayed dual binding specificity toward xylan and mannan. PaXyn11A harboring a C-terminal CBM1 module efficiently degraded wheat arabinoxylan, releasing mainly xylobiose as end product. PaAbf51A and PaAbf62A arabinose-debranching enzymes exhibited differences in activity toward arabinose-containing substrates. Further investigation of the contribution made by each P. anserina auxiliary enzyme to the saccharification of wheat straw and spruce demonstrated that the endo-acting hemicellulases (PaXyn11A, PaMan5A, and PaMan26A) individually supplemented the secretome of the industrial T. reesei CL847 strain. The most striking effect was obtained with PaMan5A that improved the release of total sugars by 28% and of glucose by 18%, using spruce as lignocellulosic substrate.Lignocellulosic biomass is a desirable feedstock for the supply of second-generation bioethanol, being the largest renewable source of carbohydrates, but the recalcitrance of raw materials makes biotechnological conversion complex and costly (34).The main components of plant cell walls are cellulose, lignin, and hemicellulose, which form a tight complex with varying proportions between plants (for a review, see reference 45). Cellulose microfibrils are formed by crystalline-like combination of linear and highly ordered polymer chains of -1,4-linked D-glucose residues, and lignin consists of an amorphous aromatic heteropolymer with hydrophobic properties. Hemicellulose is a combined designation of a diverse set of noncrystalline carbohydrate polymers from plant tissues that form closely associated networks with cellulose microfibrils and lignin (6, 24). Typically, the most abundant component of hemicellulose in the cell walls of monocots (e.g., cereals) is -1,4-xylan, which consists of -1,4-linked D-xylose residues substituted with L-arabinosyl, 4-O-methyl-glucuronosyl and acetyl side chains (46,53). Arabinofuranosyl residues of arabinoxylan may also be esterified with hydroxycinnamic acid residues, e.g., ferulic and p-coumaric acids (53). In softwoods (gymnosperms), the main hemicellulose component is -1,4-mannan, which consists of a backbone formed by -1,4-linked D-mannose and D-glucose residues substitu...