Comparison of three seemingly similar lytic polysaccharide monooxygenases from Neurospora crassa suggests different roles in plant biomass degradation

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01652-21 ◽

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.

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Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay

Biotechnology for Biofuels ◽

10.1186/1754-6834-5-79 ◽

2012 ◽

Vol 5 (1) ◽

pp. 79 ◽

Cited By ~ 166

Author(s):

Roman Kittl ◽

Daniel Kracher ◽

Daniel Burgstaller ◽

Dietmar Haltrich ◽

Roland Ludwig

Keyword(s):

Pichia Pastoris ◽

Neurospora Crassa ◽

Fluorimetric Assay ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

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Genomic and transcriptomic analysis of the thermophilic lignocellulose-degrading fungus Thielavia terrestris LPH172

10.21203/rs.3.rs-96298/v1 ◽

2020 ◽

Author(s):

Monika Tõlgo ◽

Silvia Hüttner ◽

Nguyen Than Thuy ◽

Vu Nguyen Than ◽

Johan Larsbrink ◽

...

Keyword(s):

Gc Content ◽

Low Ph ◽

Biomass Degradation ◽

Carbohydrate Active Enzymes ◽

Thielavia Terrestris ◽

Biomass Saccharification ◽

Lignocellulosic Substrates ◽

Lytic Polysaccharide Monooxygenases ◽

Beechwood Xylan ◽

Polysaccharide Monooxygenases

Abstract Background: Biomass-degrading enzymes with improved activity and stability can ameliorate substrate saccharification and make biorefineries economically feasible. Filamentous fungi are a rich source of carbohydrate-active enzymes (CAZymes) for biomass degradation. The newly isolated LPH172 strain of the thermophilic Ascomycete Thielavia terrestris has been shown to possess high xylanase and cellulase activities and tolerate well low pH and high temperatures. Here, we aimed to illuminate the lignocellulose degrading machinery and novel carbohydrate-active enzymes in LPH172 in detail.Results: We sequenced and analysed the 36.6-Mb genome and transcriptome of LPH172 during growth on glucose, cellulose, rice straw, and beechwood xylan. In total, 411 CAZy domains were found among 10,128 predicted genes. Compared to other fungi, auxiliary activity (AA) enzymes were particularly enriched. GC content was higher in coding sequences than in the overall genome. A high GC3 content was hypothesised to contribute to thermophilicity. T. terrestris employed mainly lytic polysaccharide monooxygenases (LPMOs) and glycoside hydrolase (GH) family 7 glucanases to attack cellulosic substrates, and conventional hemicellulases (GH10 and GH11) to degrade xylan. The observed co-expression and co-upregulation of AA9 LPMOs, other AA CAZymes, and (hemi)cellulases points to a complex and nuanced degradation strategy. Growth on more complex and heterogeneous substrates resulted in a more varied but generally lower gene expression. Conclusions: Our analysis of the genome and transcriptome of T. terrestris LPH172 elucidates the enzyme arsenal the fungus uses to degrade lignocellulosic substrates. The study provides the basis for future characterisation of potential new enzymes for industrial biomass saccharification.

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Insight into the Lytic Polysaccharide Monooxygenases for Plant Biomass Valorization

Authorea ◽

10.22541/au.158379523.32546673 ◽

2020 ◽

Author(s):

Shweta Srivastava ◽

Nishant Dafale ◽

Hemant Purohit

Keyword(s):

Plant Biomass ◽

Biomass Valorization ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases ◽

Insight Into

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Inter- and intra-domain functional redundancy in the rumen microbiome during plant biomass degradation

10.1101/254672 ◽

2018 ◽

Author(s):

Andrea Söllinger ◽

Alexander Tøsdal Tveit ◽

Morten Poulsen ◽

Samantha Joan Noel ◽

Mia Bengtsson ◽

...

Keyword(s):

Temporal Dynamics ◽

Plant Biomass ◽

Expression Profiles ◽

Functional Redundancy ◽

Mitigation Strategies ◽

Biomass Degradation ◽

Strong Response ◽

Plant Biomass Degradation ◽

Rumen Microbiome ◽

Domain Functional

AbstractBackgroundRuminant livestock is a major source of the potent greenhouse gas methane (CH4), produced by the complex rumen microbiome. Using an integrated approach, combining quantitative metatranscriptomics with gas- and volatile fatty acid (VFA) profiling, we gained fundamental insights into temporal dynamics of the cow rumen microbiome during feed degradation.ResultsThe microbiome composition was highly individual and remarkably stable within each cow, despite similar gas emission and VFA profiles between cows. Gene expression profiles revealed a fast microbial growth response to feeding, reflected by drastic increases in microbial biomass, CH4emissions and VFA concentrations. Microbiome individuality was accompanied by high inter- and intra-domain functional redundancy among pro- and eukaryotic microbiome members in the key steps of anaerobic feed degradation. Methyl-reducing but not CO2-reducing methanogens were correlated with increased CH4emissions during plant biomass degradation.ConclusionsThe major response of the rumen microbiome to feed intake was a general growth of the whole community. The high functional redundancy of the cow-individual microbiomes was possibly linked to the robust performance of the anaerobic degradation process. Furthermore, the strong response of methylotrophic methanogens is suggesting that they might play a more important role in ruminant CH4emissions than previously assumed, making them potential targets for CH4mitigation strategies.

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“Integrative Genomic Analysis for the Bioprospection of Regulators and Accessory Enzymes Associated with Cellulose Degradation in a Filamentous Fungus (Trichoderma harzianum)”

10.1101/731323 ◽

2019 ◽

Author(s):

Jaire A. Ferreira Filho ◽

Maria Augusta C. Horta ◽

Clelton A. dos Santos ◽

Deborah A. Almeida ◽

Natália F. Murad ◽

...

Keyword(s):

Trichoderma Harzianum ◽

Plant Biomass ◽

Cellulose Degradation ◽

Genome Structure ◽

Hydrolytic Enzyme ◽

Differentially Expressed ◽

Fungal Genome ◽

Biomass Degradation ◽

Plant Biomass Degradation ◽

Genomic Regions

AbstractBackgroundUnveiling fungal genome structure and function reveals the potential biotechnological use of fungi. Trichoderma harzianum is a powerful CAZyme-producing fungus. We studied the genomic regions in T. harzianum IOC3844 containing CAZyme genes, transcription factors and transporters.ResultsWe used bioinformatics tools to mine the T. harzianum genome for potential genomics, transcriptomics, and exoproteomics data and coexpression networks. The DNA was sequenced by PacBio SMRT technology for multi-omics data analysis and integration. In total, 1676 genes were annotated in the genomic regions analyzed; 222 were identified as CAZymes in T. harzianum IOC3844. When comparing transcriptome data under cellulose or glucose conditions, 114 genes were differentially expressed in cellulose, with 51 CAZymes. CLR2, a transcription factor physically and phylogenetically conserved in T. harzianum spp., was differentially expressed under cellulose conditions. The genes induced/repressed under cellulose conditions included those important for plant biomass degradation, including CIP2 of the CE15 family and a copper-dependent LPMO of the AA9 family.ConclusionsOur results provide new insights into the relationship between genomic organization and hydrolytic enzyme expression and regulation in T. harzianum IOC3844. Our results can improve plant biomass degradation, which is fundamental for developing more efficient strains and/or enzymatic cocktails for the production of hydrolytic enzymes.

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