Genome and Secretome Analysis of Staphylotrichum longicolleum DSM105789 Cultured on Agro-Residual and Chitinous Biomass

Staphylotrichum longicolleum FW57 (DSM105789) is a prolific chitinolytic fungus isolated from wood, with a chitinase activity of 0.11 ± 0.01 U/mg. We selected this strain for genome sequencing and annotation, and compiled its growth characteristics on four different chitinous substrates as well as two agro-industrial waste products. We found that the enzymatic mixture secreted by FW57 was not only able to digest pre-treated sugarcane bagasse, but also untreated sugarcane bagasse and maize leaves. The efficiency was comparable to a commercial enzymatic cocktail, highlighting the potential of the S. longicolleum enzyme mixture as an alternative pretreatment method. To further characterize the enzymes, which efficiently digested polymers such as cellulose, hemicellulose, pectin, starch, and lignin, we performed in-depth mass spectrometry-based secretome analysis using tryptic peptides from in-gel and in-solution digestions. Depending on the growth conditions, we were able to detect from 442 to 1092 proteins, which were annotated to identify from 134 to 224 putative carbohydrate-active enzymes (CAZymes) in five different families: glycoside hydrolases, auxiliary activities, carbohydrate esterases, polysaccharide lyases, glycosyl transferases, and proteins containing a carbohydrate-binding module, as well as combinations thereof. The FW57 enzyme mixture could be used to replace commercial enzyme cocktails for the digestion of agro-residual substrates.

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High Potential Source for Biomass Degradation Enzyme Discovery and Environmental Aspects Revealed through Metagenomics of Indian Buffalo Rumen

BioMed Research International ◽

10.1155/2014/267189 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 18

Author(s):

K. M. Singh ◽

Bhaskar Reddy ◽

Dishita Patel ◽

A. K. Patel ◽

Nidhi Parmar ◽

...

Keyword(s):

Plant Cell Wall ◽

Glycoside Hydrolases ◽

Carbohydrate Binding ◽

Metagenomic Sequencing ◽

Environmental Aspects ◽

Rumen Microbiome ◽

Polysaccharide Lyases ◽

Effective System ◽

Plant Cell Wall Degradation ◽

Buffalo Rumen

The complex microbiomes of the rumen functions as an effective system for plant cell wall degradation, and biomass utilization provide genetic resource for degrading microbial enzymes that could be used in the production of biofuel. Therefore the buffalo rumen microbiota was surveyed using shot gun sequencing. This metagenomic sequencing generated 3.9 GB of sequences and data were assembled into 137270 contiguous sequences (contigs). We identified potential 2614 contigs encoding biomass degrading enzymes including glycoside hydrolases (GH: 1943 contigs), carbohydrate binding module (CBM: 23 contigs), glycosyl transferase (GT: 373 contigs), carbohydrate esterases (CE: 259 contigs), and polysaccharide lyases (PE: 16 contigs). The hierarchical clustering of buffalo metagenomes demonstrated the similarities and dissimilarity in microbial community structures and functional capacity. This demonstrates that buffalo rumen microbiome was considerably enriched in functional genes involved in polysaccharide degradation with great prospects to obtain new molecules that may be applied in the biofuel industry.

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A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material

Journal of Fungi ◽

10.3390/jof7060426 ◽

2021 ◽

Vol 7 (6) ◽

pp. 426

Author(s):

Ander Peña ◽

Rashid Babiker ◽

Delphine Chaduli ◽

Anna Lipzen ◽

Mei Wang ◽

...

Keyword(s):

Extracellular Enzymes ◽

Fungal Growth ◽

2D Nmr ◽

Pleurotus Eryngii ◽

Glycoside Hydrolases ◽

Oxidative Enzymes ◽

Initial Growth ◽

Lignocellulosic Material ◽

Polysaccharide Lyases ◽

Polysaccharide Monooxygenases

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.

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A unique combination of glycoside hydrolases in Streptococcus suis specifically and sequentially acts on host-derived αGal-epitope glycans

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011977 ◽

2020 ◽

Vol 295 (31) ◽

pp. 10638-10652

Author(s):

Ping Chen ◽

Ran Liu ◽

Mengmeng Huang ◽

Jinlu Zhu ◽

Dong Wei ◽

...

Keyword(s):

Wide Spectrum ◽

Streptococcus Suis ◽

Glycoside Hydrolases ◽

Carbohydrate Binding ◽

Unique Combination ◽

Enzymatic Assays ◽

Tracheal Epithelial Cells ◽

Carbohydrate Binding Modules ◽

Host Signaling ◽

Important Host

Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(β1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(β1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-β-N-acetylhexosaminidase activities, including exo-(β1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mm toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling in S. suis. Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.

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Enzymatic Adaptation of Bifidobacterium bifidum to Host Glycans, Viewed from Glycoside Hydrolyases and Carbohydrate-Binding Modules

Microorganisms ◽

10.3390/microorganisms8040481 ◽

2020 ◽

Vol 8 (4) ◽

pp. 481 ◽

Cited By ~ 3

Author(s):

Toshihiko Katoh ◽

Miriam N. Ojima ◽

Mikiyasu Sakanaka ◽

Hisashi Ashida ◽

Aina Gotoh ◽

...

Keyword(s):

Carbon Sources ◽

Bifidobacterium Bifidum ◽

Glycoside Hydrolases ◽

Altruistic Behavior ◽

Carbohydrate Binding ◽

Human Gut ◽

Genetic Characteristics ◽

Beneficial Effects ◽

Carbohydrate Binding Modules ◽

Enzymatic Machinery

Certain species of the genus Bifidobacterium represent human symbionts. Many studies have shown that the establishment of symbiosis with such bifidobacterial species confers various beneficial effects on human health. Among the more than ten (sub)species of human gut-associated Bifidobacterium that have significantly varied genetic characteristics at the species level, Bifidobacterium bifidum is unique in that it is found in the intestines of a wide age group, ranging from infants to adults. This species is likely to have adapted to efficiently degrade host-derived carbohydrate chains, such as human milk oligosaccharides (HMOs) and mucin O-glycans, which enabled the longitudinal colonization of intestines. The ability of this species to assimilate various host glycans can be attributed to the possession of an adequate set of extracellular glycoside hydrolases (GHs). Importantly, the polypeptides of those glycosidases frequently contain carbohydrate-binding modules (CBMs) with deduced affinities to the target glycans, which is also a distinct characteristic of this species among members of human gut-associated bifidobacteria. This review firstly describes the prevalence and distribution of B. bifidum in the human gut and then explains the enzymatic machinery that B. bifidum has developed for host glycan degradation by referring to the functions of GHs and CBMs. Finally, we show the data of co-culture experiments using host-derived glycans as carbon sources, which underpin the interesting altruistic behavior of this species as a cross-feeder.

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Multimodular fused acetyl–feruloyl esterases from soil and gut Bacteroidetes improve xylanase depolymerization of recalcitrant biomass

Biotechnology for Biofuels ◽

10.1186/s13068-020-01698-9 ◽

2020 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Cathleen Kmezik ◽

Cyrielle Bonzom ◽

Lisbeth Olsson ◽

Scott Mazurkewich ◽

Johan Larsbrink

Keyword(s):

Esterase Activity ◽

Glycoside Hydrolase ◽

Full Length ◽

Glycoside Hydrolases ◽

Corn Cob ◽

Biomass Degradation ◽

Carbohydrate Esterase ◽

Catalytic Domains ◽

Cob Biomass ◽

Carbohydrate Esterases

Abstract Background Plant biomass is an abundant and renewable carbon source that is recalcitrant towards both chemical and biochemical degradation. Xylan is the second most abundant polysaccharide in biomass after cellulose, and it possesses a variety of carbohydrate substitutions and non-carbohydrate decorations which can impede enzymatic degradation by glycoside hydrolases. Carbohydrate esterases are able to cleave the ester-linked decorations and thereby improve the accessibility of the xylan backbone to glycoside hydrolases, thus improving the degradation process. Enzymes comprising multiple catalytic glycoside hydrolase domains on the same polypeptide have previously been shown to exhibit intramolecular synergism during degradation of biomass. Similarly, natively fused carbohydrate esterase domains are encoded by certain bacteria, but whether these enzymes can result in similar synergistic boosts in biomass degradation has not previously been evaluated. Results Two carbohydrate esterases with similar architectures, each comprising two distinct physically linked catalytic domains from families 1 (CE1) and 6 (CE6), were selected from xylan-targeting polysaccharide utilization loci (PULs) encoded by the Bacteroidetes species Bacteroides ovatus and Flavobacterium johnsoniae. The full-length enzymes as well as the individual catalytic domains showed activity on a range of synthetic model substrates, corn cob biomass, and Japanese beechwood biomass, with predominant acetyl esterase activity for the N-terminal CE6 domains and feruloyl esterase activity for the C-terminal CE1 domains. Moreover, several of the enzyme constructs were able to substantially boost the performance of a commercially available xylanase on corn cob biomass (close to twofold) and Japanese beechwood biomass (up to 20-fold). Interestingly, a significant improvement in xylanase biomass degradation was observed following addition of the full-length multidomain enzyme from B. ovatus versus the addition of its two separated single domains, indicating an intramolecular synergy between the esterase domains. Despite high sequence similarities between the esterase domains from B. ovatus and F. johnsoniae, their addition to the xylanolytic reaction led to different degradation patterns. Conclusion We demonstrated that multidomain carbohydrate esterases, targeting the non-carbohydrate decorations on different xylan polysaccharides, can considerably facilitate glycoside hydrolase-mediated hydrolysis of xylan and xylan-rich biomass. Moreover, we demonstrated for the first time a synergistic effect between the two fused catalytic domains of a multidomain carbohydrate esterase.

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Secretome analysis of Ganoderma lucidum cultivated in sugarcane bagasse

Journal of Proteomics ◽

10.1016/j.jprot.2012.09.004 ◽

2012 ◽

Vol 77 ◽

pp. 298-309 ◽

Cited By ~ 46

Author(s):

Tamilvendan Manavalan ◽

Arulmani Manavalan ◽

Kalaichelvan P. Thangavelu ◽

Klaus Heese

Keyword(s):

Sugarcane Bagasse ◽

Ganoderma Lucidum ◽

Secretome Analysis

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Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module (CBM_E1) derived from sugarcane soil metagenome

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14015520 ◽

2014 ◽

Vol 70 (9) ◽

pp. 1232-1235 ◽

Cited By ~ 2

Author(s):

Bruna Medeia Campos ◽

Thabata Maria Alvarez ◽

Marcelo Vizona Liberato ◽

Igor Polikarpov ◽

Harry J. Gilbert ◽

...

Keyword(s):

Fossil Fuels ◽

Plant Biomass ◽

Metagenomic Library ◽

Glycoside Hydrolases ◽

Diffusion Method ◽

Carbohydrate Binding ◽

X Ray Diffraction ◽

X Ray ◽

Cell Parameters ◽

Carbohydrate Binding Modules

In recent years, owing to the growing global demand for energy, dependence on fossil fuels, limited natural resources and environmental pollution, biofuels have attracted great interest as a source of renewable energy. However, the production of biofuels from plant biomass is still considered to be an expensive technology. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases for polysaccharide degradation, is attracting growing attention. Aiming at the identification of new CBMs, a sugarcane soil metagenomic library was analyzed and an uncharacterized CBM (CBM_E1) was identified. In this study, CBM_E1 was expressed, purified and crystallized. X-ray diffraction data were collected to 1.95 Å resolution. The crystals, which were obtained by the sitting-drop vapour-diffusion method, belonged to space groupI23, with unit-cell parametersa=b=c= 88.07 Å.

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Genome Sequencing and Carbohydrate-Active Enzyme (CAZyme) Repertoire of the White Rot Fungus Flammulina elastica

International Journal of Molecular Sciences ◽

10.3390/ijms19082379 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2379 ◽

Cited By ~ 12

Author(s):

Young-Jin Park ◽

Yong-Un Jeong ◽

Won-Sik Kong

Keyword(s):

Gene Prediction ◽

Gc Content ◽

Industrial Applications ◽

Fungal Species ◽

Amino Acid Sequences ◽

Glycoside Hydrolases ◽

White Rot ◽

White Rot Fungus ◽

Carbohydrate Binding ◽

Carbohydrate Binding Modules

Next-generation sequencing (NGS) of the Flammulina elastica (wood-rotting basidiomycete) genome was performed to identify carbohydrate-active enzymes (CAZymes). The resulting assembly (31 kmer) revealed a total length of 35,045,521 bp (49.7% GC content). Using the AUGUSTUS tool, 12,536 total gene structures were predicted by ab initio gene prediction. An analysis of orthologs revealed that 6806 groups contained at least one F. elastica protein. Among the 12,536 predicted genes, F. elastica contained 24 species-specific genes, of which 17 genes were paralogous. CAZymes are divided into five classes: glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycosyltransferases (GTs), and auxiliary activities (AA). In the present study, annotation of the predicted amino acid sequences from F. elastica genes using the dbCAN CAZyme database revealed 508 CAZymes, including 82 AAs, 218 GHs, 89 GTs, 18 PLs, 59 CEs, and 42 carbohydrate binding modules in the F. elastica genome. Although the CAZyme repertoire of F. elastica was similar to those of other fungal species, the total number of GTs in F. elastica was larger than those of other basidiomycetes. This genome information elucidates newly identified wood-degrading machinery in F. elastica, offers opportunities to better understand this fungus, and presents possibilities for more detailed studies on lignocellulosic biomass degradation that may lead to future biotechnological and industrial applications.

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The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation

Biochemical Journal ◽

10.1042/bj20021860 ◽

2003 ◽

Vol 371 (3) ◽

pp. 1027-1043 ◽

Cited By ~ 82

Author(s):

Deborah HOGG ◽

Gavin PELL ◽

Paul DUPREE ◽

Florence GOUBET ◽

Susana M. MARTÍN-ORÚE ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Plant Cell Wall ◽

Catalytic Domain ◽

Glycoside Hydrolases ◽

Crystalline Cellulose ◽

Carbohydrate Binding ◽

Molecular Architecture ◽

Catalytic Domains ◽

Carbohydrate Binding Modules ◽

Cellvibrio Japonicus

β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

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