Identification of copper-containing oxidoreductases in the secretomes of three Colletotrichum species with a focus on copper radical oxidases for the biocatalytic production of fatty aldehydes

2021 ◽  
Author(s):  
David Ribeaucourt ◽  
Safwan Saker ◽  
David Navarro ◽  
Bastien Bissaro ◽  
Elodie Drula ◽  
...  
Keyword(s):  
Time Course ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Recombinant Expression ◽  
Primary Objective ◽  
Primary Alcohols ◽  
Fungal Strains ◽  
Fatty Aldehydes ◽  
Initial Objective ◽  
Biocatalytic Production

Copper Radical Alcohol Oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens are attractive for the production of fragrant fatty aldehydes. With the initial objective to investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time-course analyses of the secretomes of three Colletotrichum species ( C. graminicola , C. tabacum and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture in absence of any plant-biomass mimicking compounds to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29-52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11-AA13, AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CRO-AlcOx were thus expressed in Pichia pastoris and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C6 and C8 alcohols into their corresponding fragrant aldehydes. IMPORTANCE In the context of the industrial shift toward greener processes, the biocatalytic production of aldehydes is of utmost interest owing to their importance for their use as flavors and fragrances ingredients. CRO-AlcOx have the potential to become platform enzymes for the oxidation of alcohols to aldehydes. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored, while the use of crude fungal secretomes is an appealing approach for industrial application to alleviate various costs pertaining to biocatalysts production. While investigating this primary objective, the secretomics studies revealed unexpected results showing that under the oxidative-stressful conditions we probed, Colletotrichum species can secrete a broad diversity of copper-containing enzymes (laccases, sugar oxidoreductases, LPMOs) usually assigned to “plant-cell wall degradation”, despite the absence of any plant-biomass mimicking compound, and only little amount of CRO-AlcOx were secreted, pointing out at recombinant expression as the most promising path for their biocatalytic application.

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 Time-resolved secretome analysis of three Colletotrichum species identifies copper radical alcohol oxidases for the production of fatty aldehydes

2021 ◽  
Author(s):  
David Ribeaucourt ◽  
Safwan Saker ◽  
David Navarro ◽  
Bastien Bissaro ◽  
Elodie Drula ◽  
...  
Keyword(s):  
Time Course ◽  
Primary Alcohols ◽  
Carbohydrate Active Enzymes ◽  
Time Resolved ◽  
Ethanol Mixture ◽  
Copper Manganese ◽  
Fungal Phytopathogens ◽  
Fatty Aldehydes ◽  
Glyoxal Oxidase ◽  
Full Conversion

Copper Radical Alcohol Oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens are attractive for the production of fragrant fatty aldehydes. To investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time-course analyses of the secretomes of three Colletotrichum species (C. graminicola, C. tabacum and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29-52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11-AA13, AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CRO-AlcOx were expressed in Pichia pastoris and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C6 and C8 alcohols into their corresponding fragrant aldehydes.

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.

scholarly journals Cork Taint of Wines: Role of the Filamentous Fungi Isolated from Cork in the Formation of 2,4,6-Trichloroanisole by O Methylation of 2,4,6-Trichlorophenol

2002 ◽  
Vol 68 (12) ◽  
pp. 5860-5869 ◽  
Author(s):  
María Luisa Álvarez-Rodríguez ◽  
Laura López-Ocaña ◽  
José Miguel López-Coronado ◽  
Enrique Rodríguez ◽  
María Jesús Martínez ◽  
...  
Keyword(s):  
Time Course ◽  
Wine Industry ◽  
Ammonium Salts ◽  
Chlorinated Phenols ◽  
High Concentration ◽  
Fungal Strains ◽  
Great Economic Importance ◽  
Expression Studies ◽  
Cork Taint

ABSTRACT Cork taint is a musty or moldy off-odor in wine mainly caused by 2,4,6-trichloroanisole (2,4,6-TCA). We examined the role of 14 fungal strains isolated from cork samples in the production of 2,4,6-TCA by O methylation of 2,4,6-trichlorophenol (2,4,6-TCP). The fungal strains isolated belong to the genera Penicillium (four isolates); Trichoderma (two isolates); and Acremonium, Chrysonilia, Cladosporium, Fusarium, Mortierella, Mucor, Paecilomyces, and Verticillium (one isolate each). Eleven of these strains could produce 2,4,6-TCA when they were grown directly on cork in the presence of 2,4,6-TCP. The highest levels of bioconversion were carried out by the Trichoderma and Fusarium strains. One strain of Trichoderma longibrachiatum could also efficiently produce 2,4,6-TCA in liquid medium. However, no detectable levels of 2,4,6-TCA production by this strain could be detected on cork when putative precursors other than 2,4,6-TCP, including several anisoles, dichlorophenols, trichlorophenols, or other highly chlorinated compounds, were tested. Time course expression studies with liquid cultures showed that the formation of 2,4,6-TCA was not affected by a high concentration of glucose (2% or 111 mM) or by ammonium salts at concentrations up to 60 mM. In T. longibrachiatum the O methylation of 2,4,6-TCP was catalyzed by a mycelium-associated S-adenosyl-l-methionine (SAM)-dependent methyltransferase that was strongly induced by 2,4,6-TCP. The reaction was inhibited by S-adenosyl-l-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings increase our understanding of the mechanism underlying the origin of 2,4,6-TCA on cork, which is poorly understood despite its great economic importance for the wine industry, and they could also help us improve our knowledge about the biodegradation and detoxification processes associated with chlorinated phenols.

Time-course accumulation of flavonoids in hydroponically grown Achillea millefolium L.

2014 ◽  
Vol 94 (2) ◽  
pp. 383-395 ◽  
Author(s):  
Karine Pedneault ◽  
Martine Dorais ◽  
Sébastien Léonhart ◽  
Paul Angers ◽  
André Gosselin
Keyword(s):  
Time Course ◽  
Plant Biomass ◽  
Dry Weight ◽  
Hydroponic Culture ◽  
Weight Basis ◽  
Early Flowering ◽  
Achillea Millefolium ◽  
Phenological Stage ◽  
Stage 4 ◽  
Hydroponically Grown

Pedneault, K., Dorais, M., Léonhart, S., Angers, P. and Gosselin, A. 2014. Time-course accumulation of flavonoids in hydroponically grown Achillea millefolium L. Can. J. Plant. Sci. 94: 383–395. In recent decades, the use of plant-based medicines as health products has increased considerably all over the world. As greenhouse hydroponic culture allows standardized cultural methods to be used, it may be valuable for reducing the risks associated with harvesting medicinal plants from the wild, such as species dissemination, species misidentification, adulteration, and non-hygienic handling, while allowing the production of high yields of clean, standardized biomass year-round. To evaluate the potential of hydroponic culture for medicinal plant production, the present study investigated the accumulation patterns of apigenin, luteolin, apigenin glycosides, and the chlorogenic acid 5-caffeoylquinic acid in the plant organs of A. millefolium at five phenological stages from 35 to 102 d after sowing, and drew a comparison with outdoor-grown plants at 122 d after sowing. The results showed two flavonoid accumulation peaks: one at the early growth stage (35 d after sowing) and one at early flowering (87 d after sowing). At 87 d after sowing, most of the apigenin glycosides were concentrated in the roots (3.80% wt/wt, dry weight basis), whereas free apigenin and luteolin were located mainly in the flower heads (1.25 and 0.86% wt/wt, dry weight basis, respectively). Early flowering was the best harvesting stage for optimal flavonoid production in terms of active compounds per plant and kilograms of plant biomass per cultivated area. At 122 d after sowing (phenological stage 4), the outdoor-grown plants were nine times smaller than the early flowering plants (87 d after sowing) from the hydroponic system and had a root-tissue apigenin glycoside level that was five times lower than that of the hydroponically grown plants. In conclusion, the use of a hydroponic growing system reduced by 29% the time required to reach phenological stage 4, which corresponds to maximum plant bioactive concentration, in comparison with field production. Therefore, hydroponic culture represents an effective alternative to outdoor production and can result in standardized, high-quality medicinal plant biomass with potential flavonoid yields approximating 515 mg per plant.

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 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 Clostridium thermocellum as a Promising Source of Genetic Material for Designer Cellulosomes: An Overview

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 996
Author(s):  
Dung Minh Ha-Tran ◽  
Trinh Thi My Nguyen ◽  
Chieh-Chen Huang
Keyword(s):  
Clostridium Thermocellum ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Consolidated Bioprocessing ◽  
Genetic Material ◽  
Economic Feasibility ◽  
Biofuel Production ◽  
Cellulolytic Bacterium ◽  
Microbial Conversion ◽  
Promising Source

Plant biomass-based biofuels have gradually substituted for conventional energy sources thanks to their obvious advantages, such as renewability, huge quantity, wide availability, economic feasibility, and sustainability. However, to make use of the large amount of carbon sources stored in the plant cell wall, robust cellulolytic microorganisms are highly demanded to efficiently disintegrate the recalcitrant intertwined cellulose fibers to release fermentable sugars for microbial conversion. The Gram-positive, thermophilic, cellulolytic bacterium Clostridium thermocellum possesses a cellulolytic multienzyme complex termed the cellulosome, which has been widely considered to be nature’s finest cellulolytic machinery, fascinating scientists as an auspicious source of saccharolytic enzymes for biomass-based biofuel production. Owing to the supra-modular characteristics of the C. thermocellum cellulosome architecture, the cellulosomal components, including cohesin, dockerin, scaffoldin protein, and the plentiful cellulolytic and hemicellulolytic enzymes have been widely used for constructing artificial cellulosomes for basic studies and industrial applications. In addition, as the well-known microbial workhorses are naïve to biomass deconstruction, several research groups have sought to transform them from non-cellulolytic microbes into consolidated bioprocessing-enabling microbes. This review aims to update and discuss the current progress in these mentioned issues, point out their limitations, and suggest some future directions.

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