scholarly journals Quantifying oxidation of cellulose-associated glucuronoxylan by two lytic polysaccharide monooxygenases from Neurospora crassa

2021 ◽  
Author(s):  
Olav A. Hegnar ◽  
Heidi Østby ◽  
Dejan M. Petrović ◽  
Lisbeth Olsson ◽  
Anikó Várnai ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Hydrolytic Enzymes ◽  
Structural Features ◽  
Cell Wall Polysaccharides ◽  
Functional Variation ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Oxidized Products

Family AA9 lytic polysaccharide monooxygenases (LPMOs) are abundant in fungi where they catalyze oxidative depolymerization of recalcitrant plant biomass. These AA9 LPMOs cleave cellulose, and some also act on hemicelluloses, primarily other (substituted) β-(1→4)-glucans. Oxidative cleavage of xylan has been shown for only a handful AA9 LPMOs, and it remains unclear whether this activity is a minor side reaction or primary function. Here, we show that Nc LPMO9F and the phylogenetically related, hitherto uncharacterized Nc LPMO9L from Neurospora crassa are active on both cellulose and cellulose-associated glucuronoxylan, but not on glucuronoxylan alone. A newly developed method for simultaneous quantification of xylan-derived and cellulose-derived oxidized products showed that Nc LPMO9F preferentially cleaves xylan when acting on a cellulose–beechwood glucuronoxylan mixture, yielding about three times more xylan-derived than cellulose-derived oxidized products. Interestingly, under similar conditions, Nc LPMO9L and previously characterized Mc LPMO9H from Malbranchea cinnamomea showed different xylan-to-cellulose preferences, giving oxidized product ratios of about 0.5:1 and 1:1, respectively, indicative of functional variation among xylan-active LPMOs. Phylogenetic and structural analysis of xylan-active AA9 LPMOs led to the identification of characteristic structural features, including unique features that do not occur in phylogenetically remote AA9 LPMOs, such as four AA9 LPMOs whose lack of activity towards glucuronoxylan was demonstrated in the present study. Taken together, the results provide a path towards discovery of additional xylan-active LPMOs and show that the huge family of AA9 LPMOs has members that preferentially act on xylan. These findings shed new light on the biological role and industrial potential of these fascinating enzymes. Importance Plant cell wall polysaccharides are highly resilient to depolymerization by hydrolytic enzymes, partly due to cellulose chains being tightly packed in microfibrils that are covered by hemicelluloses. Lytic polysaccharide monooxygenases (LPMOs) seem well suited to attack these resilient co-polymeric structures, but the occurrence and importance of hemicellulolytic activity among LPMOs remains unclear. Here we show that certain AA9 LPMOs preferentially cleave xylan when acting on a cellulose–glucuronoxylan mixture, and that this ability is the result of protein evolution that has resulted in a clade of AA9 LPMOs with specific structural features. Our findings strengthen the notion that the vast arsenal of AA9 LPMOs in certain fungal species provides functional versatility, and that AA9 LPMOs may have evolved to promote oxidative depolymerization of a wide variety of recalcitrant, co-polymeric plant polysaccharide structures. These findings have implications for understanding the biological roles and industrial potential of LPMOs.

scholarly journals Deciphering the Regulatory Network between the SREBP Pathway and Protein Secretion in Neurospora crassa

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Lina Qin ◽  
Vincent W. Wu ◽  
N. Louise Glass
Keyword(s):  
Neurospora Crassa ◽  
Unfolded Protein Response ◽  
Molecular Oxygen ◽  
Protein Secretion ◽  
Plant Biomass ◽  
Unfolded Protein ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACT Sterol regulatory element binding proteins (SREBPs) are conserved from yeast to mammalian cells and function in the regulation of sterol homeostasis. In fungi, the SREBP pathway has been implicated in the adaptation to hypoxia and in virulence. In Neurospora crassa and Trichoderma reesei, the SREBP pathway also negatively regulates protein secretion under lignocellulolytic conditions. Here we utilized global transcriptional profiling combined with genetic and physiological analyses to address the regulatory link between the SREBP pathway and protein secretion in N. crassa. Our results demonstrated that the function of the SREBP pathway in ergosterol biosynthesis and adaptation to hypoxia was conserved in N. crassa. Under lignocellulolytic conditions, the SREBP pathway was highly activated, resulting in the reduced expression of lytic polysaccharide monooxygenases, which require molecular oxygen for catalytic activity. Additionally, activation of the SREBP pathway under lignocellulolytic conditions repressed a set of genes predicted to be involved in the endoplasmic reticulum stress response. Here we show that the inability of a hac-1 mutant, which bears a deletion of the major regulator of the unfolded protein response (UPR), to efficiently produce cellulases and utilize cellulose was suppressed by mutations in the SREBP pathway. The analyses presented here demonstrated new SREBP pathway functions, including linkages to the UPR, and provide new clues for genetic engineering of filamentous fungi to improve their production of extracellular proteins. IMPORTANCE The role of SREBP transcription factors in the regulation of sterol biosynthesis is conserved from humans to yeast. In filamentous fungi, this pathway regulates the secretion of lignocellulolytic enzymes during plant biomass deconstruction. Here we show that the SREBP pathway in Neurospora crassa regulates the production of specific cellulases, lytic polysaccharide monooxygenases that utilize molecular oxygen. Via global transcriptional profile and genetic analyses, a relationship between the SREBP pathway and the unfolded protein response (UPR) pathway was revealed, suggesting a regulatory interplay of these two pathways in the trafficking of plant biomass-degrading enzymes. These findings have implications for our understanding of the cross talk of the SREBP and UPR pathways in other organisms and will guide the rational engineering of fungal strains to improve cellulolytic enzyme production. IMPORTANCE The role of SREBP transcription factors in the regulation of sterol biosynthesis is conserved from humans to yeast. In filamentous fungi, this pathway regulates the secretion of lignocellulolytic enzymes during plant biomass deconstruction. Here we show that the SREBP pathway in Neurospora crassa regulates the production of specific cellulases, lytic polysaccharide monooxygenases that utilize molecular oxygen. Via global transcriptional profile and genetic analyses, a relationship between the SREBP pathway and the unfolded protein response (UPR) pathway was revealed, suggesting a regulatory interplay of these two pathways in the trafficking of plant biomass-degrading enzymes. These findings have implications for our understanding of the cross talk of the SREBP and UPR pathways in other organisms and will guide the rational engineering of fungal strains to improve cellulolytic enzyme production.

scholarly journals Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs)

2020 ◽  
pp. jbc.RA120.015545
Author(s):  
Kristian E. H. Frandsen ◽  
Mireille Haon ◽  
Sacha Grisel ◽  
Bernard Henrissat ◽  
Leila Lo Leggio ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Time Course ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Substrate Binding ◽  
Glycoside Hydrolases ◽  
Sequence Alignments ◽  
Lytic Polysaccharide Monooxygenases ◽  
Molecular Determinants ◽  
Polysaccharide Monooxygenases

Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall-derived hemicellulosic polysaccharides and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharides degradation suggests that the subtle differences in amino acids sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO sub-clusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)-linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries.

scholarly journals Network reconstruction and systems analysis of plant cell wall deconstruction byneurospora crassa

2017 ◽  
Author(s):  
Areejit Samal ◽  
James P. Craig ◽  
Samuel T. Coradetti ◽  
J. Philipp Benz ◽  
James A. Eddy ◽  
...  
Keyword(s):  
Cell Wall ◽  
Neurospora Crassa ◽  
Filamentous Fungi ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Degradation Pathway ◽  
Heterogeneous Data ◽  
Data Types ◽  
Cell Wall Polysaccharides

AbstractPlant biomass degradation by fungal derived enzymes is rapidly expanding in economic importance as a clean and efficient source for biofuels. The ability to rationally engineer filamentous fungi would facilitate biotechnological applications for degradation of plant cell wall polysaccharides. However, incomplete knowledge of biomolecular networks responsible for plant cell wall deconstruction impedes experimental efforts in this direction. To expand this knowledge base, a detailed network of reactions important for deconstruction of plant cell wall polysaccharides into simple sugars was constructed for the filamentous fungusNeurospora crassa. To reconstruct this network, information was integrated from five heterogeneous data types: functional genomics, transcriptomics, proteomics, genetics, and biochemical characterizations. The combined information was encapsulated into a feature matrix and the evidence weighed to assign annotation confidence scores for each gene within the network. Comparative analyses of RNA-seq and ChIP-seq data shed light on the regulation of the plant cell wall degradation network (PCWDN), leading to a novel hypothesis for degradation of the hemicellulose mannan. The transcription factor CLR-2 was subsequently experimentally shown to play a key role in the mannan degradation pathway ofNeurospora crassa. Our network serves as a scaffold for integration of diverse experimental data, leading to elucidation of regulatory design principles for plant cell wall deconstruction by filamentous fungi, and guiding efforts to rationally engineer industrially relevant hyper-production strains.

Recent insights into lytic polysaccharide monooxygenases (LPMOs)

2018 ◽  
Vol 46 (6) ◽  
pp. 1431-1447 ◽  
Author(s):  
Tobias Tandrup ◽  
Kristian E. H. Frandsen ◽  
Katja S. Johansen ◽  
Jean-Guy Berrin ◽  
Leila Lo Leggio
Keyword(s):  
Active Site ◽  
Room Temperature ◽  
Experimental Studies ◽  
Binding Mode ◽  
Structural Features ◽  
Oxygen Species ◽  
Animal Kingdom ◽  
X Ray ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

scholarly journals Re-routing of Sugar Catabolism Provides a Better Insight Into Fungal Flexibility in Using Plant Biomass-Derived Monomers as Substrates

2021 ◽  
Vol 9 ◽  
Author(s):  
Tania Chroumpi ◽  
Mao Peng ◽  
Lye Meng Markillie ◽  
Hugh D. Mitchell ◽  
Carrie D. Nicora ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Plant Biomass ◽  
Value Added ◽  
Sugar Metabolism ◽  
Cell Factory ◽  
Cell Wall Polysaccharides ◽  
Filamentous Ascomycete ◽  
Extensive Metabolism ◽  
Beet Pulp ◽  
A Cell

The filamentous ascomycete Aspergillus niger has received increasing interest as a cell factory, being able to efficiently degrade plant cell wall polysaccharides as well as having an extensive metabolism to convert the released monosaccharides into value added compounds. The pentoses D-xylose and L-arabinose are the most abundant monosaccharides in plant biomass after the hexose D-glucose, being major constituents of xylan, pectin and xyloglucan. In this study, the influence of selected pentose catabolic pathway (PCP) deletion strains on growth on plant biomass and re-routing of sugar catabolism was addressed to gain a better understanding of the flexibility of this fungus in using plant biomass-derived monomers. The transcriptome, metabolome and proteome response of three PCP mutant strains, ΔlarAΔxyrAΔxyrB, ΔladAΔxdhAΔsdhA and ΔxkiA, grown on wheat bran (WB) and sugar beet pulp (SBP), was evaluated. Our results showed that despite the absolute impact of these PCP mutations on pure pentose sugars, they are not as critical for growth of A. niger on more complex biomass substrates, such as WB and SBP. However, significant phenotypic variation was observed between the two biomass substrates, but also between the different PCP mutants. This shows that the high sugar heterogeneity of these substrates in combination with the high complexity and adaptability of the fungal sugar metabolism allow for activation of alternative strategies to support growth.

scholarly journals In vitro biosynthesis of poly-β-1,4-glucan derivatives using a pro-miscuous glycosyltransferase

2020 ◽  
Author(s):  
Gregory S. Bulmer ◽  
Ashley P. Mattey ◽  
Fabio Parmeggiani ◽  
Ryan Williams ◽  
Helene Ledru ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Chemical Probes ◽  
Cell Wall Degrading Enzymes ◽  
Lytic Polysaccharide Monooxygenases ◽  
Natural Cellulose ◽  
Polysaccharide Monooxygenases ◽  
In Vitro Biosynthesis ◽  
New Applications ◽  
Biosynthetic Machinery

AbstractThe β-1,4-glucose linkage of cellulose is the most abundant polymeric linkage on earth and as such is of considerable interest in biology and biotechnology. It remains challenging to synthesize this linkage in vitro due to a lack of suitable biocatalysts; the natural cellulose biosynthetic machinery is a membrane-associated complex with processive activity that cannot be easily manipulated to synthesize tailor-made oligosaccharides and their derivatives. Here we identify a promiscuous activity of a soluble recombinant biocatalyst, Neisseria meningitidis glycosyltransferase LgtB, suitable for the polymerization of glucose from UDP-glucose via the generation of β-1,4-glycosidic linkages. We employed LgtB to synthesize natural and derivatized cello-oligosaccharides and we demonstrate how LgtB can be incorporated in biocatalytic cascades and chemo-enzymatic strategies to synthesize cello-oligosaccharides with tailored functionalities. We also show how the resulting glycan structures can be applied as chemical probes to report on activity and selectivity of plant cell wall degrading enzymes, including lytic polysaccharide monooxygenases. We anticipate that this biocatalytic approach to derivatized cello-oligosaccharides via glucose polymerization will open up new applications in biology and nanobiotechnology.

scholarly journals Secreted pectin monooxygenases drive plant infection by pathogenic oomycetes

Science ◽  
2021 ◽  
Vol 373 (6556) ◽  
pp. 774-779
Author(s):  
Federico Sabbadin ◽  
Saioa Urresti ◽  
Bernard Henrissat ◽  
Anna O. Avrova ◽  
Lydia R. J. Welsh ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Crop Protection ◽  
Model Organism ◽  
Plant Infection ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Encoding Gene ◽  
Plant Pathogen Interactions ◽  
Encoding Genes ◽  
Secreted Enzymes

The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans. We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.

Sign in / Sign up

Export Citation Format

Share Document