Podospora anserina Hemicellulases Potentiate the Trichoderma reesei Secretome for Saccharification of Lignocellulosic Biomass

Abstract

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...

GH62 arabinofuranosidases: Structure, function and applications

“…While the catalytic efficiency of SthAbf62A-m2,3 and AnAbf62A-m2,3 was 75 and 520 fold lower, respectively, on L-arabinan than WAXs (Tsujibo et al, 2002;Wang et al, 2014;Wilkens et al, 2016), both PaAbf62A and UmAbf62A had higher kcat on sugar beet Larabinan than on WAX (Table 1) (Siguier et al, 2014). Debranched arabinan was reported hydrolyzed with low activity by three enzymes, SlAbf62A, SthAbf62A-m2,3 and PaAbf62A (Couturier et al, 2011;Siguier et al, 2014;Tsujibo et al, 2002;Wang et al, 2014).…”

“…Nine of the 11 GH62 ABFs tested on both L-arabinan and AX showed from 9 (Couturier et al, 2011;Siguier et al, 2014) to 10 4 fold (McCleary et al 2015) lower specific activity on L-arabinan than AXs (Table 1). While the catalytic efficiency of SthAbf62A-m2,3 and AnAbf62A-m2,3 was 75 and 520 fold lower, respectively, on L-arabinan than WAXs (Tsujibo et al, 2002;Wang et al, 2014;Wilkens et al, 2016), both PaAbf62A and UmAbf62A had higher kcat on sugar beet Larabinan than on WAX (Table 1) (Siguier et al, 2014).…”

First Structural Insights into α-l-Arabinofuranosidases from the Two GH62 Glycoside Hydrolase Subfamilies

“…PaAbf62A was mined from the genome of the ascomycete P. anserina, a fungus that displays an impressive set of hemicellulose-acting enzymes (17,18). UmAbf62A was identified in the secretome of the phytopathogen U. maydis, a secretome that can beneficially complete that of Trichoderma reesei CL847 for the saccharification of wheat straw (7).…”

Analysis of Wheat Straw Biodiversity for Use as a Feedstock for Biofuel Production

“…The WSF was analyzed by high-performance anion-exchange chromatography [18] coupled to pulsed amperometric detection (HPAEC-PAD) using an Dionex ICS3000 system equipped with a CarboPac PA1 column coupled to a CarboPac PA1 guard column (both from Dionex). The amperometric detector was equipped with a disposable gold electrode (Dionex), and the standard carbohydrate waveform recommended by the manufacturer was applied.…”

“…Eluent A was DDW, eluent B was 0.1 M NaOH. Gradient elution method was adopted from Couturier et al [18]. Gradient program started from isocratic elution at 20 % B for 20 min; then, a linear gradient to 100 % B was applied for 0.2 min, isocratic elution at 100 % B for 14.8 min to flush the column, linear gradient to 20 % B for 0.2 min and reconditioning of the column at 20 % B for 14.8 min (total run time was 50 min).…”