Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina

Abstract

Background: The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood.

Results: In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).

Conclusions: This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.

Keywords: AA9; Biomass; Biorefinery; Cellobiose dehydrogenase; Cellulose; Hemicellulose; LPMO; Lignocellulose; Oxidative cleavage; Oxidized cello-oligosaccharides.

Fig. 1

Oxidative cleavage of cellulose by P. anserina AA9 LPMOs. a Schematic representation of the modularity of P. anserina AA9 LPMOs based on CAZy annotation. b Quantification by HPAEC analysis of the soluble sugars released by 4.4 μM PaLPMO and 1.2 U.ml⁻¹ of PaCDHB, at 50 °C for 16 h. The concentration of each sugar was determined by integration of the peak area and comparison with a standard curve. Values are the mean of three biological replicates (n = 3). Error bars correspond to one cumulated SD (error bar = ±SDtot; with SDtot = √(SD₁ ² + SD₂ ² + …). c HPAEC chromatograms showing products generated from cellulose with PaLPMO9A, PaLPMO9H, and PaLPMO9E. The peak annotations are based on comparison with oligosaccharides standards oxidized at the C1 position (DP2ox-DP5 ox)

Fig. 2

Analysis of degradation products generated by PaLPMO9A and PaLPMO9H. The HPAEC chromatograms of the oligosaccharides released upon degradation of 0.1 % PASC with 4.4 μM PaLPMO in the presence of 1 mM ascorbate, at 50 °C for 16 h (in black) followed by the incubation with 1.2 U.ml⁻¹ of PaCDHB at 50 °C for 8 h (in red). The peak annotations are based on comparison with oligosaccharides standards oxidized at the C1 position (DP2ox-DP5ox). Coelution of DP1ox with DP3 and DP2ox with DP6 was observed. Peaks eluting at 27, 38, and 41 min are annotated with dotted lines

Fig. 3

Mass spectrometry analysis of degradation products generated by PaLPMO9H. a Analysis was performed after 16 h of cellulose degradation. The main panel shows the full spectrum of sample with peaks corresponding to native and oxidized cello-oligosaccharides. Fragmented peaks are indicated by arrows. The panels below b, c, and d show the DP4 peaks with m/z value of 687.21, 705.22, and 721.21, respectively, that were fragmented using ESI MS. The oxidized oligosaccharides product species are represented in panels b, c, and d based on the fragmentation patterns. In panel d, the different product species corresponding to the fragmentation pattern are indicated by blue and red dotted bonds

Fig. 4

Time-course analysis of the products released from cellulose by PaLPMO9H. a The HPAEC chromatograms show products generated from cellulose with 4.4 μM PaLPMO9H and 1.2 U.ml⁻¹ of PaCDHB, at 50 °C for 1, 2, 3, 5, 7, 9, 24, 30, and 48 h of incubation. b A quantification by HPAEC analysis of the soluble sugars (aldonic acid and non-oxidized oligosaccharides) released by PaLPMO9H over time has been conducted. The concentration of each sugar was determined by integration of the peak area and comparison with a standard curve. Values are the mean of three biological replicates (n = 3). Error bars correspond to one cumulated SD (error bar = ±SDtot; with SDtot = √(SD₁ ² + SD₂ ² + …)

Fig. 5

PaLPMO9H activity on oligosaccharide substrates. a Generation of H₂O₂ by PaLPMO9H in the presence and/or absence of various oligosaccharides substrates. Glc4, cellotetraose; Glc5, cellopentaose; Glc6, cellohexaose; Lam6, laminarinhexaose; Man6, mannohexaose; β(1,3;1,4)Glc4, β(1,3;1,4)-glucotetraose (G4G3G4G); XXXG, xyloglucan-derived heptasaccharide. b HPAEC chromatogram of products released from cellohexaose by action of PaLPMO9H in the presence of ascorbic acid (in black) or PaCDHB (in red) with the same labeling of peaks as Fig. 2

Fig. 6

PaLPMO9H activity on polysaccharide substrates. a Relative production of H₂O₂ by PaLPMO9H in the presence and/or absence of various polysaccharides substrates. b HPAEC chromatogram of products released from xyloglucan (XG) by action of PaLPMO9H in the presence of ascorbic acid. The C1-oxidized xyloglucan-derived heptasaccharide standard (XXXGox in blue) was prepared from XXXG (based on the nomenclature defined by [49]) using the PaCDHB as described in Material and Methods