Synergism of Recombinant Podospora anserina PaAA9B with Cellulases Containing AA9s Can Boost the Enzymatic Hydrolysis of Cellulosic Substrates

To achieve a fast and efficient cellulose hydrolysis with low enzyme loading remains a challenge for an economically feasible biomass biorefinery process. The synergism between cellulase and lytic polysaccharide monooxygenases (LPMOs) has demonstrated great promise, but it appears that such synergistic effects are highly substrate-dependent. In addition, the need for an efficient method of LPMO production has also limited its application. In this study, the production of Podospora anserina AA9B (PaAA9B), one of the most active LPMOs, was optimized by using double-plasmid coexpression in Pichia pastoris, and its hydrolysis-boosting effects on cellulases were assessed on model cellulosic materials and on our in-house-optimized atmospheric glycerol organosolv (AGO)-pretreated lignocellulosic substrates. The results showed that the double-plasmid coexpression technique successfully improved the PaAA9B production, where up to three times more PaAA9B (3.34 g L-1) was expressed in the 5 L bioreactor after 4 days of induction. The addition of recombinant PaAA9B to Cellic CTec2 (CTec2) significantly boosted the cellulose hydrolysis of a filter paper and Avicel by 2.3- and 1.4-folds, respectively, while no effects were observed on carboxymethyl cellulose (CMC). When lignocellulosic substrates were assessed, the PaAA9B also successfully enhanced the cellulose hydrolysis of both add-catalyzed (ac) and alkali-catalyzed (al) AGO-pretreated sugarcane bagasses by 30 and 20%, respectively, at a 5% solid loading (w/v). At industrially relevant high-solid loading (20% w/v) hydrolysis of an al-AGO-pretreated substrate, the combination of 1.0 mg of PaAA9B and 3 FPU of cellulase per gram of substrate achieved 83% cellulose hydrolysis with 105 g L-1 of the corresponding glucose concentration after 72 h. These results indicated that the double-plasmid coexpression strategy is viable for the high-yield PaAA9B production. The mixture of PaAA9B and cellulase enzymes containing other AA9s exhibited a strong cosynergistic interaction and further boosted the enzymatic hydrolysis of both model cellulosic substrates and our optimized AGO-pretreated lignocellulosic biomass with industrially relevant enzyme loading. This study sheds light on the industrially relevant PaAA9B utilization in the enzymatic hydrolysis of lignocellulosic substrates.