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Specific Xylan Activity Revealed for AA9 Lytic Polysaccharide Monooxygenases of the Thermophilic Fungus Malbranchea cinnamomea by Functional Characterization.

ABSTRACT: 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.

SUBMITTER: Huttner S

PROVIDER: S-EPMC6856335 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:The thermophilic fungus Malbranchea cinnamomea belongs to the order of Onygenales and is a promising source of thermostable, industrially relevant biocatalysts, as it can grow at temperatures of more than 50°C and is able to utilise many different types of plant biomass. Enzymes from M. cinnamomea that have been characterised so far include an α-amylase, an α-glucosidase, xylanases and an alkaline β-1,3-1,4-glucanase (lichenase), all of which have been reported to have temperature optima between 50°C and 80°C. With this study, we complement the knowledge of the enzymatic repertoire of M. cinnamomea with a transcriptomic analysis of strain FCH 10.5 to provide a more comprehensive view of its lignocellulolytic enzyme system. Genes differentially expressed during growth on two different polymeric substrates, beechwood xylan and wheat bran, point to differences in the fungal response to the deconstruction of a hardwood hemicellulose (beechwood xylan) and a cereal hemicellulose (wheat bran). The data presented here will form the basis for a systematic exploration of the full potential of this fungus as a source of thermostable enzymes. We sequenced the genome of M. cinnamomea FCH 10.5, which was isolated from the compost of a waste treatment plant in Hanoi, Vietnam (PMC5604768, https://www.ncbi.nlm.nih.gov/nuccore/FQSS02000000). For RNAseq, the fungus was grown on three different carbon sources (glucose, wheat bran, beechwood xylan) at 50°C. Mycelium was harvested after 4h and 48h and RNA was extracted. For RNAseq analysis, the RNA of 4h and 48h samples was mixed 1:1, to get information about both early- and late-response genes during growth on the different carbon sources. Two independent duplicate experiments were done for each substrate. Total RNA was extracted using TRIzol (Invitrogen) and chloroform, and further purified with the RNeasy Plant RNA Kit with on-column DNAse digestion (QIAGEN). The quality of the purified RNA was verified by agarose gel electrophoresis, Nanodrop (Thermo Scientific) and Qubit (Life Technologies). The NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) was used to process the samples according to the manufacturer’s instructions. Briefly, mRNA was isolated from total RNA using oligo-dT magnetic beads and used to synthesise cDNA. The cDNA was ligated with sequencing adapters and PCR amplified. The quality and yield after sample preparation were determined with the Fragment Analyzer (Advanced Analytical). The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp). Standard Illumina primers for Illumina cBot and HiSeq 2500, and the HiSeq control software HCS v2.2.58 were used according to manufacturer’s protocols for clustering and DNA sequencing with a concentration of 16.0 pM. The Illumina data analysis pipelines RTA v1.18.64 and Bcl2fastq v2.17 were used for image analysis, base calling, and quality check. Sequencing was performed on an Illumina HiSeq 2500 sequencer. The assembled genome from the DNA sequencing was used as a reference to map the reads using the packages Tophat (v2.0.14. Linux_x86_64) and Bowtie (v2-2.1.0) with a default mismatch rate of 2%. The frequency with which a read was mapped on a transcript was determined based on the mapped locations from the alignment. To normalise for transcript length, fpkm (fragments per kilobase of transcript per million mapped reads) were calculated. For differential expression analysis, the read counts were loaded into the DESeq package v 1.10.1. Genes were considered differentially expressed if they showed a log2 fold change ≥ 1 and the adjusted p-value was < 0.05.

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.

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Specific Xylan Activity Revealed for AA9 Lytic Polysaccharide Monooxygenases of the Thermophilic Fungus Malbranchea cinnamomea by Functional Characterization.

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