Dataset Information

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 ?-(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.

SUBMITTER: Bennati-Granier C

PROVIDER: S-EPMC4487207 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Bennati-Granier Chloé C Garajova Sona S Champion Charlotte C Grisel Sacha S Haon Mireille M Zhou Simeng S Fanuel Mathieu M Ropartz David D Rogniaux Hélène H Gimbert Isabelle I Record Eric E Berrin Jean-Guy JG

Biotechnology for biofuels 20150620

<h4>Background</h4>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.<h4>Results</h4>In this study, we investigated a s ...[more]

PMID: 26136828

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Project description:Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes found in viruses, archaea, and bacteria as well as eukaryotes, such as fungi, algae and insects, actively contributing to the degradation of different polysaccharides. In Aspergillus nidulans, LPMOs from family AA9 (AnLPMO9s), along with an AA3 cellobiose dehydrogenase (AnCDH1), are cosecreted upon growth on crystalline cellulose and lignocellulosic substrates, indicating their role in the degradation of plant cell wall components. Functional analysis revealed that three target LPMO9s (AnLPMO9C, AnLPMO9F and AnLPMO9G) correspond to cellulose-active enzymes with distinct regioselectivity and activity on cellulose with different proportions of crystalline and amorphous regions. AnLPMO9s deletion and overexpression studies corroborate functional data. The abundantly secreted AnLPMO9F is a major component of the extracellular cellulolytic system, while AnLPMO9G was less abundant and constantly secreted, and acts preferentially on crystalline regions of cellulose, uniquely displaying activity on highly crystalline algae cellulose. Single or double deletion of AnLPMO9s resulted in about 25% reduction in fungal growth on sugarcane straw but not on Avicel, demonstrating the contribution of LPMO9s for the saprophytic fungal lifestyle relies on the degradation of complex lignocellulosic substrates. Although the deletion of AnCDH1 slightly reduced the cellulolytic activity, it did not affect fungal growth indicating the existence of alternative electron donors to LPMOs. Additionally, double or triple knockouts of these enzymes had no accumulative deleterious effect on the cellulolytic activity nor on fungal growth, regardless of the deleted gene. Overexpression of AnLPMO9s in a cellulose-induced secretome background confirmed the importance and applicability of AnLPMO9G to improve lignocellulose saccharification. IMPORTANCE Fungal lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that boost plant biomass degradation in combination with glycoside hydrolases. Secretion of LPMO9s arsenal by Aspergillus nidulans is influenced by the substrate and time of induction. These findings along with the biochemical characterization of novel fungal LPMO9s have implications on our understanding of their concerted action, allowing rational engineering of fungal strains for biotechnological applications such as plant biomass degradation. Additionally, the role of oxidative players in fungal growth on plant biomass was evaluated by deletion and overexpression experiments using a model fungal system.

Project description:BackgroundUnderstanding the diversity of lignocellulose-degrading enzymes in nature will provide insights for the improvement of cellulolytic enzyme cocktails used in the biofuels industry. Two families of enzymes, fungal AA9 and bacterial AA10, have recently been characterized as crystalline cellulose or chitin-cleaving lytic polysaccharide monooxygenases (LPMOs). Here we analyze the sequences, structures, and evolution of LPMOs to understand the factors that may influence substrate specificity both within and between these enzyme families.ResultsComparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families demonstrated that, although these two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues. Phylogenetic analysis of the AA10 family identified clades with putative chitinolytic and cellulolytic activities. Estimation of the rate of synonymous versus non-synonymous substitutions (dN/dS) within two major AA10 subclades showed distinct selective pressures between putative cellulolytic genes (subclade A) and CBP21-like chitinolytic genes (subclade D). Estimation of site-specific selection demonstrated that changes in the active sites were strongly negatively selected in all subclades. Furthermore, all codons in the subclade D had dN/dS values of less than 0.7, whereas codons in the cellulolytic subclade had dN/dS values of greater than 1.5. Positively selected codons were enriched at sites localized on the surface of the protein adjacent to the active site.ConclusionsThe structural similarity but absence of significant sequence similarity between AA9 and AA10 families suggests that these enzyme families share an ancient ancestral protein. Combined analysis of amino acid sites under Darwinian selection and structural homology modeling identified a subclade of AA10 with diversifying selection at different surfaces, potentially used for cellulose-binding and protein-protein interactions. Together, these data indicate that AA10 LPMOs are under selection to change their function, which may optimize cellulolytic activity. This work provides a phylogenetic basis for identifying and classifying additional cellulolytic or chitinolytic LPMOs.

Dataset Information

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

Publications

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

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