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

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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Genomic Insights into the Fungal Lignocellulolytic Machinery of Flammulina rossica

Microorganisms ◽

10.3390/microorganisms7100421 ◽

2019 ◽

Vol 7 (10) ◽

pp. 421

Author(s):

Young-Jin Park ◽

Chang-Soo Lee ◽

Won-Sik Kong

Keyword(s):

De Novo ◽

Gene Prediction ◽

Gc Content ◽

Industrial Applications ◽

Fungal Species ◽

Glycoside Hydrolases ◽

Carbohydrate Binding ◽

De Novo Genome Assembly ◽

Carbohydrate Binding Modules ◽

Species Specific

Next-generation sequencing (NGS) of the Flammulina rossica (wood-rotting basidiomycete) genome was performed to identify its carbohydrate-active enzymes (CAZymes). De novo genome assembly (31 kmer) revealed a total length of 35,646,506 bp (49.79% GC content). In total, 12,588 gene models of F. rossica were predicted using an ab initio gene prediction tool (AUGUSTUS). Orthologous analysis with other fungal species revealed that 7433 groups contained at least one F. rossica gene. Additionally, 12,033 (95.6%) of 12,588 genes for F. rossica proteins had orthologs among the Dikarya, and F. rossica contained 12 species-specific genes. CAZyme annotation in the F. rossica genome revealed 511 genes predicted to encode CAZymes including 102 auxiliary activities, 236 glycoside hydrolases, 94 glycosyltransferases, 19 polysaccharide lyases, 56 carbohydrate esterases, and 21 carbohydrate binding-modules. Among the 511 genes, several genes were predicted to simultaneously encode two different CAZymes such as glycoside hydrolases (GH) as well as carbohydrate-binding module (CBM). The genome information of F. rossica offers opportunities to understand the wood-degrading machinery of this fungus and will be useful for biotechnological and industrial applications.

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Comparative Analysis of Carbohydrate Active Enzymes in the Flammulina velutipes var. lupinicola Genome

Microorganisms ◽

10.3390/microorganisms9010020 ◽

2020 ◽

Vol 9 (1) ◽

pp. 20

Author(s):

Hye-Won Yu ◽

Ji-Hoon Im ◽

Won-Sik Kong ◽

Young-Jin Park

Keyword(s):

De Novo ◽

Animal Feed ◽

Gc Content ◽

Industrial Applications ◽

Glycoside Hydrolases ◽

Flammulina Velutipes ◽

Carbohydrate Binding ◽

Carbohydrate Active Enzymes ◽

Carbohydrate Binding Modules ◽

Genes Encoding

The purpose of this study was to determine the genome sequence of Flammulina velutipes var. lupinicola based on next-generation sequencing (NGS) and to identify the genes encoding carbohydrate-active enzymes (CAZymes) in the genome. The optimal assembly (71 kmer) based on ABySS de novo assembly revealed a total length of 33,223,357 bp (49.53% GC content). A total of 15,337 gene structures were identified in the F.velutipes var. lupinicola genome using ab initio gene prediction method with Funannotate pipeline. Analysis of the orthologs revealed that 11,966 (96.6%) out of the 15,337 predicted genes belonged to the orthogroups and 170 genes were specific for F. velutipes var. lupinicola. CAZymes are divided into six classes: auxiliary activities (AAs), glycosyltransferases (GTs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycoside hydrolases (GHs), and carbohydrate-binding modules (CBMs). A total of 551 genes encoding CAZymes were identified in the F. velutipes var. lupinicola genome by analyzing the dbCAN meta server database (HMMER, Hotpep, and DIAMOND searches), which consisted of 54–95 AAs, 145–188 GHs, 55–73 GTs, 6–19 PLs, 13–59 CEs, and 7–67 CBMs. CAZymes can be widely used to produce bio-based products (food, paper, textiles, animal feed, and biofuels). Therefore, information about the CAZyme repertoire of the F. velutipes var. lupinicola genome will help in understanding the lignocellulosic machinery and in-depth studies will provide opportunities for using this fungus for biotechnological and industrial applications.

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Comparative Genomic Analysis of Bifidobacterium Catenulatum Reveal the Genetic Divergence of its Two Subspecies from Infant and Adult Gut

10.21203/rs.3.rs-957925/v1 ◽

2021 ◽

Author(s):

Jiaqi Liu ◽

Weicheng Li ◽

Caiqing Yao ◽

Jie Yu ◽

Heping Zhang

Keyword(s):

Genetic Divergence ◽

Gc Content ◽

Genomic Analysis ◽

Glycoside Hydrolases ◽

Comparative Genomic ◽

Carbohydrate Binding ◽

Functional Difference ◽

Carbohydrate Utilization ◽

Carbohydrate Binding Modules ◽

Genomic Studies

Abstract Background: Bifidobacterium catenulatum, which includes two subspecies that B. catenulatum subsp. kashiwanohense and B. catenulatum subsp. catenulatum are usually from infant and adult gut respectively, while the genomic studies of functional difference and genetic divergence in them have been rarely reported. In this study, we analyzed 16 B. catenulatum strains through comparative genomics, including two novel sequenced strains. Results: A phylogenetic tree based on 785 core genes indicated that the two subspecies of B. catenulatum were significantly separated and confirmed their colonizing bias in infants and adults. Comparison of general genomic characteristics revealed that the two subspecies had significantly different genomic sizes but similar GC content. Functional annotations found that they peculiarly differ in utilization of carbohydrates and amino acid. Among them, we found that carbohydrate metabolism seems to play an important role in the divergence because of their carbohydrate-active enzymes (CAZyme) present two different clustering patterns. B. catenulatum subsp. kashiwanohense have functional genes that specifically adapted to the infant gut for glycoside hydrolases 95 (GH95) and carbohydrate-binding modules 51 (CBM51), which specifically participated in the metabolism of Human Milk Oligosaccharides (HMOs), and specific genes fuc that related to HMOs were also detected. While B. catenulatum subsp. catenulatum rich in GH3 and glycosyltransferases 4 (GT4) tended to metabolize plant-derived glycan that may help it metabolize more complex carbohydrates (eg. xylan) in the adult intestine. Conclusions: Our findings revealed genomic evidence of carbohydrate utilization bias which may be a key leading to the genetic divergence of two subspecies of B. catenulatum.

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Introducing a Thermo-Alkali-Stable, Metallic Ion-Tolerant Laccase Purified From White Rot Fungus Trametes hirsuta

Frontiers in Microbiology ◽

10.3389/fmicb.2021.670163 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jing Si ◽

Hongfei Ma ◽

Yongjia Cao ◽

Baokai Cui ◽

Yucheng Dai

Keyword(s):

Specific Activity ◽

Endocrine Disrupting Chemicals ◽

Environmental Pollutants ◽

Fold Increase ◽

Industrial Applications ◽

White Rot ◽

White Rot Fungus ◽

Metallic Ions ◽

Trametes Hirsuta ◽

Ph Range

This study introduces a valuable laccase, designated ThLacc-S, purified from white rot fungus Trametes hirsuta. ThLacc-S is a monomeric protein in nature with a molecular weight of 57.0 kDa and can efficiently metabolize endocrine disrupting chemicals. The enzyme was successfully purified to homogeneity via three consecutive steps consisting of salt precipitation and column chromatography, resulting in a 20.76-fold increase in purity and 46.79% yield, with specific activity of 22.111 U/mg protein. ThLacc-S was deciphered as a novel member of the laccase family and is a rare metalloenzyme that contains cysteine, serine, histidine, and tyrosine residues in its catalytic site, and follows Michaelis-Menten kinetic behavior with a Km and a kcat/Km of 87.466 μM and 1.479 s–1μM–1, respectively. ThLacc-S exerted excellent thermo-alkali stability, since it was markedly active after a 2-h incubation at temperatures ranging from 20 to 70°C and retained more than 50% of its activity after incubation for 72 h in a broad pH range of 5.0–10.0. Enzymatic activities of ThLacc-S were enhanced and preserved when exposed to metallic ions, surfactants, and organic solvents, rendering this novel enzyme of interest as a green catalyst for versatile biotechnological and industrial applications that require these singularities of laccases, particularly biodegradation and bioremediation of environmental pollutants.

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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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Investigating the effects of symbiotic fungi on the flight behaviour ofSirex noctilio(Hymenoptera: Siricidae)

The Canadian Entomologist ◽

10.4039/tce.2016.10 ◽

2016 ◽

Vol 148 (5) ◽

pp. 543-551 ◽

Cited By ~ 5

Author(s):

Mark A. Sarvary ◽

Ann E. Hajek ◽

Katalin Böröczky ◽

Robert A. Raguso ◽

Miriam F. Cooperband

Keyword(s):

Fungal Species ◽

Flight Tunnel ◽

White Rot ◽

White Rot Fungus ◽

Sirex Noctilio ◽

Flight Behaviour ◽

Native North American ◽

Complete Chemical Analysis ◽

Volatile Profiles ◽

Symbiotic Fungi

AbstractThe invasive woodwaspSirex noctilioFabricius (Hymenoptera: Siricidae) is obligately associated with the symbiotic white rot fungusAmylostereum areolatum(Chaillet ex Fries) Boidin (Basidiomycota: Amylosteraceae), and shows positive chemotaxis to volatiles emitted by this symbiont. After introduction to North America,S. noctiliowas collected carrying another fungus speciesAmylostereum chailletii(Persoon) Boidin, used symbiotically by native North AmericanSirexLinnaeus. We conducted flight behaviour studies in a walk-in flight tunnel to evaluate specificity of the attraction of mated and unmatedS. noctilioto its primary symbiont,A. areolatum, versus the alternative symbiont,A. chailletii. Fewer unmated than matedS. noctiliofemales responded to either of the fungi. Unmated females showed no landing preference but matedS. noctiliofemales were attracted toA. areolatumalthough avoidance ofA. chailletiiwas not complete. Chemical analysis demonstrated major differences in the volatile profiles of the two fungal species. Sesquiterpenes dominated theA. areolatumsamples, whereas only two aromatic volatiles were consistently present in the nativeA. chailletii.

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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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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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