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Deletion of AA9 Lytic Polysaccharide Monooxygenases Impacts A. nidulans Secretome and Growth on Lignocellulose.

ABSTRACT: 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.

SUBMITTER: Terrasan CRF

PROVIDER: S-EPMC9241910 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes that can enhance polysaccharide depolymerization through an oxidative mechanism and hence boost generation of biofuel from e.g. cellulose. By employing density functional theory in a combination of quantum mechanics and molecular mechanics (QM/MM), we report a complete description of the molecular mechanism of LPMOs. The QM/MM scheme allows us to describe all reaction steps with a detailed protein environment and we show that this is necessary. Several active species capable of abstracting a hydrogen from the substrate have been proposed previously and starting from recent crystallographic work on a substrate-LPMO complex, we investigate previously suggested paths as well as new ones. We describe the generation of the reactive intermediates, the abstraction of a hydrogen atom from the polysaccharide substrate, as well as the final recombination step in which OH is transferred back to the substrate. We show that a superoxo [CuO2]+ complex can be protonated by a nearby histidine residue (suggested by recent mutagenesis studies and crystallographic work) and, provided an electron source is available, leads to formation of an oxyl-complex after cleavage of the O-O bond and dissociation of water. The oxyl complex either reacts with the substrate or is further protonated to a hydroxyl complex. Both the oxyl and hydroxyl complexes are also readily generated from a reaction with H2O2, which was recently suggested to be the true co-substrate, rather than O2. The C-H abstraction by the oxyl and hydroxy complexes is overall favorable with activation barriers of 69 and 94 kJ mol-1, compared to the much higher barrier (156 kJ mol-1) obtained for the copper-superoxo species. We obtain good structural agreement for intermediates for which structural data are available and the estimated reaction energies agree with experimental rate constants. Thus, our suggested mechanism is the most complete to date and concur with available experimental evidence.

Project description:The thermophilic biomass-degrader Malbranchea cinnamomea exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight (M. cinnamomea AA9s) McAA9s, namely, McAA9A, McAA9B, McAA9F, and McAA9H, to gain a deeper understanding about their roles in the fungus. The characterized McAA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the McAA9s, McAA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends, McAA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO. McAA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers' physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall.IMPORTANCE The Malbranchea cinnamomea LPMOs (McAA9s) showed activity on a broad range of soluble and insoluble substrates, suggesting their involvement in various steps of biomass degradation besides cellulose decomposition. Our results indicate that the fungal AA9 family is more diverse than originally thought and able to degrade almost any kind of plant cell wall polysaccharide. The discovery of an AA9 that preferentially cleaves xylan enhances our understanding of the physiological roles of LPMOs and enables the use of xylan-specific LPMOs in future applications.

Project description:BackgroundStarch is the second most abundant plant-derived biomass and a major feedstock in non-food industrial applications and first generation biofuel production. In contrast to lignocellulose, detailed insight into fungal degradation of starch is currently lacking. This study explores the secretomes of Aspergillus nidulans grown on cereal starches from wheat and high-amylose (HA) maize, as well as legume starch from pea for 5 days.ResultsAspergillus nidulans grew efficiently on cereal starches, whereas growth on pea starch was poor. The secretomes at days 3-5 were starch-type dependent as also reflected by amylolytic activity measurements. Nearly half of the 312 proteins in the secretomes were carbohydrate-active enzymes (CAZymes), mostly glycoside hydrolases (GHs) and oxidative auxiliary activities (AAs). The abundance of the GH13 α-amylase (AmyB) decreased with time, as opposed to other starch-degrading enzymes, e.g., the GH13 AmyF, GH15 glucoamylases (GlaA and GlaB), and the GH31 α-glucosidase (AgdE). Two AA13 LPMOs displayed similar secretion patterns as amylolytic hydrolases and were among the most abundant CAZymes. The starch-active AnLPMO13A that possesses a CBM20 carbohydrate-binding module dominated the starch-binding secretome fraction. A striking observation is the co-secretion of several redox-active enzymes with the starch-active AA13 LPMOs and GHs, some at high abundance. Notably nine AA9 LPMOs, six AA3 sub-family 2 (AA_2) oxidoreductases, and ten AA7 glyco-oligosaccharide oxidases were identified in the secretomes in addition to other non-CAZyme oxidoreductases.ConclusionsThe co-secretion and high abundance of AA13 LPMOs are indicative of a key role in starch granule deconstruction. The increase in AA13 LPMO abundance with culture time may reflect accumulation of a more resistant starch fraction towards the later stages of the culture. The identification of AmyR sites upstream AA13 LPMOs unveils co-regulation of LPMOs featuring in starch utilization. Differential deployment of amylolytic hydrolases and LPMOs over time suggests additional regulatory mechanisms. The abundant co-secretion of distinct AA3 and AA7 oxidoreductases merits further studies into their roles and possible interplay with LPMOs and other enzymes in the deconstruction of starchy substrates. The study reports for the first time the biological significance of LPMOs in starch degradation and the temporal interplay between these and amylolytic hydrolases.

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Deletion of AA9 Lytic Polysaccharide Monooxygenases Impacts A. nidulans Secretome and Growth on Lignocellulose.

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