scholarly journals Novel fungal metal-dependent GH54 α-L-arabinofuranosidase: expanded substrate specificity and potential use for plant biomass degradation

2020 ◽  
Author(s):  
Maria Lorenza Leal Motta ◽  
Jaire Alves Ferreira Filho ◽  
Ricardo Rodrigues de Melo ◽  
Leticia Maria Zanphorlin Murakami ◽  
Clelton Aparecido dos Santos ◽  
...  
Keyword(s):  
Functional Diversity ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Biomass Conversion ◽  
Biomass Degradation ◽  
Lignocellulosic Substrate ◽  
Plant Biomass Degradation ◽  
Substrate Conversion ◽  
Wall Decoration

AbstractTrichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme’s biochemical and biophysical properties were assessed. The GH54 members show wide functional diversity and specifically remove plant cell decorations including arabinose and galactose, in the presence of a metallic cofactor. Plant cell wall decoration have a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity within the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.

scholarly journals A novel fungal metal-dependent α-l-arabinofuranosidase of family 54 glycoside hydrolase shows expanded substrate specificity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria Lorenza Leal Motta ◽  
Jaire Alves Ferreira Filho ◽  
Ricardo Rodrigues de Melo ◽  
Leticia Maria Zanphorlin ◽  
Clelton Aparecido dos Santos ◽  
...  
Keyword(s):  
Functional Diversity ◽  
Plant Cell ◽  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Biomass Conversion ◽  
Carbohydrate Active Enzymes ◽  
Lignocellulosic Substrate ◽  
Free Sugars ◽  
Substrate Conversion

AbstractTrichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme’s biochemical and biophysical properties were assessed. GH54 members show wide functional diversity and specifically remove plant cell substitutions including arabinose and galactose in the presence of a metallic cofactor. Plant cell wall substitution has a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity of the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.

scholarly journals Thermophilic Degradation of Hemicellulose, a Critical Feedstock in the Production of Bioenergy and Other Value-Added Products

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Isaac Cann ◽  
Gabriel V. Pereira ◽  
Ahmed M. Abdel-Hamid ◽  
Heejin Kim ◽  
Daniel Wefers ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Animal Feed ◽  
Value Added ◽  
Building Blocks ◽  
Economic Feasibility ◽  
Renewable Fuels ◽  
Microbial Enzymes

ABSTRACT Renewable fuels have gained importance as the world moves toward diversifying its energy portfolio. A critical step in the biomass-to-bioenergy initiative is deconstruction of plant cell wall polysaccharides to their unit sugars for subsequent fermentation to fuels. To acquire carbon and energy for their metabolic processes, diverse microorganisms have evolved genes encoding enzymes that depolymerize polysaccharides to their carbon/energy-rich building blocks. The microbial enzymes mostly target the energy present in cellulose, hemicellulose, and pectin, three major forms of energy storage in plants. In the effort to develop bioenergy as an alternative to fossil fuel, a common strategy is to harness microbial enzymes to hydrolyze cellulose to glucose for fermentation to fuels. However, the conversion of plant biomass to renewable fuels will require both cellulose and hemicellulose, the two largest components of the plant cell wall, as feedstock to improve economic feasibility. Here, we explore the enzymes and strategies evolved by two well-studied bacteria to depolymerize the hemicelluloses xylan/arabinoxylan and mannan. The sets of enzymes, in addition to their applications in biofuels and value-added chemical production, have utility in animal feed enzymes, a rapidly developing industry with potential to minimize adverse impacts of animal agriculture on the environment.

scholarly journals The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus

2020 ◽  
Vol 117 (11) ◽  
pp. 6003-6013 ◽  
Author(s):  
Vincent W. Wu ◽  
Nils Thieme ◽  
Lori B. Huberman ◽  
Axel Dietschmann ◽  
David J. Kowbel ◽  
...  
Keyword(s):  
Cell Wall ◽  
Transcription Factors ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Carbon Sources ◽  
Cell Wall Degrading Enzymes ◽  
Degrading Enzymes ◽  
Genes Encoding

Filamentous fungi, such asNeurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling ofN. crassaon 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors inN. crassaand characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.

scholarly journals A Novel Cys2His2 Zinc Finger Homolog of AZF1 Modulates Holocellulase Expression in Trichoderma reesei

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Amanda Cristina Campos Antonieto ◽  
Karoline Maria Vieira Nogueira ◽  
Renato Graciano de Paula ◽  
Luísa Czamanski Nora ◽  
Murilo Henrique Anzolini Cassiano ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Filamentous Fungi ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Regulatory Elements ◽  
Biomass Degradation ◽  
Control Of Gene Expression ◽  
Content Type ◽  
Encoding Genes

ABSTRACT Filamentous fungi are remarkable producers of enzymes dedicated to the degradation of sugar polymers found in the plant cell wall. Here, we integrated transcriptomic data to identify novel transcription factors (TFs) related to the control of gene expression of lignocellulosic hydrolases in Trichoderma reesei and Aspergillus nidulans. Using various sets of differentially expressed genes, we identified some putative cis-regulatory elements that were related to known binding sites for Saccharomyces cerevisiae TFs. Comparative genomics allowed the identification of six transcriptional factors in filamentous fungi that have corresponding S. cerevisiae homologs. Additionally, a knockout strain of T. reesei lacking one of these TFs (S. cerevisiae AZF1 homolog) displayed strong reductions in the levels of expression of several cellulase-encoding genes in response to both Avicel and sugarcane bagasse, revealing a new player in the complex regulatory network operating in filamentous fungi during plant biomass degradation. Finally, RNA sequencing (RNA-seq) analysis showed the scope of the AZF1 homologue in regulating a number of processes in T. reesei, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) provided evidence for the direct interaction of this TF in the promoter regions of cel7a, cel45a, and swo. Therefore, we identified here a novel TF which plays a positive effect in the expression of cellulase-encoding genes in T. reesei. IMPORTANCE In this work, we used a systems biology approach to map new regulatory interactions in Trichoderma reesei controlling the expression of genes encoding cellulase and hemicellulase. By integrating transcriptomics related to complex biomass degradation, we were able to identify a novel transcriptional regulator which is able to activate the expression of these genes in response to two different cellulose sources. In vivo experimental validation confirmed the role of this new regulator in several other processes related to carbon source utilization and nutrient transport. Therefore, this work revealed novel forms of regulatory interaction in this model system for plant biomass deconstruction and also represented a new approach that could be easy applied to other organisms.

Analysis of the Structural and Functional Diversity of Plant Cell Wall Specific Family 6 Carbohydrate Binding Modules

Biochemistry ◽  
2009 ◽  
Vol 48 (43) ◽  
pp. 10395-10404 ◽  
Author(s):  
D. Wade Abbott ◽  
Elizabeth Ficko-Blean ◽  
Alicia Lammerts van Bueren ◽  
Artur Rogowski ◽  
Alan Cartmell ◽  
...  
Keyword(s):  
Cell Wall ◽  
Functional Diversity ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules

scholarly journals Directed plant cell-wall accumulation of iron: embedding co-catalyst for efficient biomass conversion

2016 ◽  
Vol 9 (1) ◽  
Author(s):  
Chien-Yuan Lin ◽  
Joseph E. Jakes ◽  
Bryon S. Donohoe ◽  
Peter N. Ciesielski ◽  
Haibing Yang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Biomass Conversion ◽  
Co Catalyst

scholarly journals Proteomics reveals synergy between biomass degrading enzymes and inorganic Fenton chemistry in leaf-cutting ant colonies

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Morten Schiøtt ◽  
Jacobus J Boomsma
Keyword(s):  
Hydrogen Peroxide ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Biomass Degradation ◽  
Ant Colonies ◽  
Fenton Chemistry ◽  
Degrading Enzymes ◽  
Biochemical Assays ◽  
Oxidized Iron ◽  
Leaf Cutting

The symbiotic partnership between leaf-cutting ants and fungal cultivars processes plant biomass via ant fecal fluid mixed with chewed plant substrate before fungal degradation. Here we present a full proteome of the fecal fluid of Acromyrmex leaf-cutting ants, showing that most proteins function as biomass degrading enzymes and that ca. 85% are produced by the fungus and ingested, but not digested, by the ants. Hydrogen peroxide producing oxidoreductases were remarkably common in the proteome, inspiring us to test a scenario in which hydrogen peroxide reacts with iron to form reactive oxygen radicals after which oxidized iron is reduced by other fecal-fluid enzymes. Our biochemical assays confirmed that these so-called Fenton reactions do indeed take place in special substrate pellets, presumably to degrade plant cell wall polymers. This implies that the symbiotic partnership manages a combination of oxidative and enzymatic biomass degradation, an achievement that surpasses current human bioconversion technology.

scholarly journals Structural studies of biomass degrading enzyme systems

2014 ◽  
Vol 70 (a1) ◽  
pp. C1812-C1812
Author(s):  
Vladimir Lunin ◽  
Markus Alahuhta ◽  
Gregg Beckham ◽  
Roman Brunecky ◽  
Larry Taylor ◽  
...  
Keyword(s):  
Plant Cell ◽  
Fossil Fuels ◽  
Plant Cell Wall ◽  
Biomass Conversion ◽  
Hydrolytic Enzymes ◽  
Transportation Fuels ◽  
Self Assembling ◽  
Enzyme Systems ◽  
Molecular Machinery ◽  
The Cost

Renewable energy today comprises wind, photovoltaics, geothermal, and biofuels. Biomass is the leading source of renewable, sustainable energy used for the production of liquid transportation fuels. While the focus is shifting today from the ethanol towards next generation or advanced biofuels the real challenge however remains the same: reducing the recalcitrance of biomass to deconstruction, which yields the sugars needed for further processing. NREL's Biosciences Center conducts studies of the fundamental nature of the plant cell wall; as well as those enzyme systems utilized in Nature to deconstruct it. These systems could be classified in two ways: the "free enzymes" and the "cellulosomes." Cellulosomes are self-assembling, multi-enzyme machinery that can include dozens and hundreds of catalytic domains and cellulose binding modules interconnected by linker peptides. We will present a structural overview of the biomass degrading enzymes from fungi using Trichoderma reesei and Penicillum funiculosum as examples. The bacterial cellulosome system discussed will be from a thermophile Clostridium thermocellum and bacterial free enzyme example will be the hyperthermophile, Caldicellulosiruptor bescii. To study these systems, we combined classical biochemistry and molecular biology, mass spectrometry, electron microscopy, high throughput robotics, macromolecular crystallography, and molecular dynamics. We seek to understand the properties and structure of biomass and plant cell walls, the structure-function relationships of the relevant hydrolytic enzymes, and the ways these enzymes interact with and alter the biomass during the degradation. Thorough understanding of the details of the molecular machinery at work has led to the development of improved enzyme cocktails that have reduced the cost of biomass conversion to renewable fuels so that today, this technology is becoming competitive with traditional fossil fuels.

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

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.

scholarly journals Comparative Analysis of Herbaceous and Woody Cell Wall Digestibility by Pathogenic Fungi

Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7220
Author(s):  
Yanhua Dou ◽  
Yan Yang ◽  
Nitesh Kumar Mund ◽  
Yanping Wei ◽  
Yisong Liu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Fungal Pathogens ◽  
Plant Cell Wall ◽  
Apple Tree ◽  
Plant Biomass ◽  
Genomic Analysis ◽  
Pathogenic Fungi ◽  
Cell Wall Degrading Enzymes ◽  
Enzyme Activity Assay

Fungal pathogens have evolved combinations of plant cell-wall-degrading enzymes (PCWDEs) to deconstruct host plant cell walls (PCWs). An understanding of this process is hoped to create a basis for improving plant biomass conversion efficiency into sustainable biofuels and bioproducts. Here, an approach integrating enzyme activity assay, biomass pretreatment, field emission scanning electron microscopy (FESEM), and genomic analysis of PCWDEs were applied to examine digestibility or degradability of selected woody and herbaceous biomass by pathogenic fungi. Preferred hydrolysis of apple tree branch, rapeseed straw, or wheat straw were observed by the apple-tree-specific pathogen Valsa mali, the rapeseed pathogen Sclerotinia sclerotiorum, and the wheat pathogen Rhizoctonia cerealis, respectively. Delignification by peracetic acid (PAA) pretreatment increased PCW digestibility, and the increase was generally more profound with non-host than host PCW substrates. Hemicellulase pretreatment slightly reduced or had no effect on hemicellulose content in the PCW substrates tested; however, the pretreatment significantly changed hydrolytic preferences of the selected pathogens, indicating a role of hemicellulose branching in PCW digestibility. Cellulose organization appears to also impact digestibility of host PCWs, as reflected by differences in cellulose microfibril organization in woody and herbaceous PCWs and variation in cellulose-binding domain organization in cellulases of pathogenic fungi, which is known to influence enzyme access to cellulose. Taken together, this study highlighted the importance of chemical structure of both hemicelluloses and cellulose in host PCW digestibility by fungal pathogens.

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