scholarly journals Genome-centric metagenomics revealed the spatial distribution and the diverse metabolic functions of lignocellulose degrading uncultured bacteria

2018 ◽  
Author(s):  
Panagiotis G. Kougias ◽  
Stefano Campanaro ◽  
Laura Treu ◽  
Panagiotis Tsapekos ◽  
Andrea Armani ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Plant Biomass ◽  
Cellulose Degradation ◽  
Lignocellulose Degradation ◽  
Pig Manure ◽  
Carbohydrate Binding ◽  
Lignocellulosic Substrate ◽  
Polysaccharide Degradation ◽  
Carbohydrate Binding Modules ◽  
Metabolic Strategies

AbstractThe mechanisms by which specific anaerobic microorganisms remain firmly attached to lignocellulosic material allowing them to efficiently decompose the organic matter are far to be elucidated. To circumvent this issue, the microbiomes collected from anaerobic digesters treating pig manure and meadow grass were fractionated to separate the planktonic microbes from those adhered to lignocellulosic substrate. Assembly of shotgun reads followed by binning process recovered 151 population genomes, 80 out of which were completely new and were not previously deposited in any database. Genome coverage allowed the identification of microbial spatial distribution into the engineered ecosystem. Moreover, a composite bioinformatic analysis using multiple databases for functional annotation revealed that uncultured members of Bacteroidetes and Firmicutes follow diverse metabolic strategies for polysaccharide degradation. The structure of cellulosome in Firmicutes can vary depending on the number and functional roles of carbohydrate-binding modules. On contrary, members of Bacteroidetes are able to adhere and degrade lignocellulose due to the presence of multiple carbohydrate-binding family 6 modules in beta-xylosidase and endoglucanase proteins or S-layer homology modules in unknown proteins. This study combines the concept of variability in spatial distribution with genome-centric metagenomics allowing a functional and taxonomical exploration of the biogas microbiome.ImportanceThis work contributes new knowledge about lignocellulose degradation in engineered ecosystems. Specifically, the combination of the spatial distribution of uncultured microbes with genome-centric metagenomics provides novel insights into the metabolic properties of planktonic and firmly attached to plant biomass bacteria. Moreover, the knowledge obtained in this study enabled us to understand the diverse metabolic strategies for polysaccharide degradation in different species of Bacteroidetes and Clostridiales. Even though structural elements of cellulosome were restricted to Clostridiales, our study identified in Bacteroidetes a putative mechanism for biomass decomposition based on a gene cluster responsible for cellulose degradation, disaccharide cleavage to glucose and transport to cytoplasm.

scholarly journals Spatial Distribution and Diverse Metabolic Functions of Lignocellulose-Degrading Uncultured Bacteria as Revealed by Genome-Centric Metagenomics

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Panagiotis G. Kougias ◽  
Stefano Campanaro ◽  
Laura Treu ◽  
Panagiotis Tsapekos ◽  
Andrea Armani ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Plant Biomass ◽  
Cellulose Degradation ◽  
Lignocellulose Degradation ◽  
Pig Manure ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Polysaccharide Degradation ◽  
Carbohydrate Binding Modules ◽  
Metabolic Strategies

ABSTRACTThe mechanisms by which specific anaerobic microorganisms remain firmly attached to lignocellulosic material, allowing them to efficiently decompose organic matter, have yet to be elucidated. To circumvent this issue, microbiomes collected from anaerobic digesters treating pig manure and meadow grass were fractionated to separate the planktonic microbes from those adhered to lignocellulosic substrate. Assembly of shotgun reads, followed by a binning process, recovered 151 population genomes, 80 out of which were completely new and were not previously deposited in any database. Genome coverage allowed the identification of microbial spatial distribution in the engineered ecosystem. Moreover, a composite bioinformatic analysis using multiple databases for functional annotation revealed that uncultured members of theBacteroidetesandFirmicutesfollow diverse metabolic strategies for polysaccharide degradation. The structure of cellulosome inFirmicutesspecies can differ depending on the number and functional roles of carbohydrate-binding modules. In contrast, members of theBacteroidetesare able to adhere to and degrade lignocellulose due to the presence of multiple carbohydrate-binding family 6 modules in beta-xylosidase and endoglucanase proteins or S-layer homology modules in unknown proteins. This study combines the concept of variability in spatial distribution with genome-centric metagenomics, allowing a functional and taxonomical exploration of the biogas microbiome.IMPORTANCEThis work contributes new knowledge about lignocellulose degradation in engineered ecosystems. Specifically, the combination of the spatial distribution of uncultured microbes with genome-centric metagenomics provides novel insights into the metabolic properties of planktonic and firmly attached to plant biomass bacteria. Moreover, the knowledge obtained in this study enabled us to understand the diverse metabolic strategies for polysaccharide degradation in different species ofBacteroidetesandClostridiales. Even though structural elements of cellulosome were restricted toClostridialesspecies, our study identified a putative mechanism inBacteroidetesspecies for biomass decomposition, which is based on a gene cluster responsible for cellulose degradation, disaccharide cleavage to glucose, and transport to cytoplasm.

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

2014 ◽  
Vol 70 (9) ◽  
pp. 1232-1235 ◽  
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.

scholarly journals Deletion of a Peptidylprolyl Isomerase Gene Results in the Inability of Caldicellulosiruptor bescii To Grow on Crystalline Cellulose without Affecting Protein Glycosylation or Growth on Soluble Substrates

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Jordan F. Russell ◽  
Matthew L. Russo ◽  
Xuewen Wang ◽  
Neal Hengge ◽  
Daehwan Chung ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Plant Biomass ◽  
Protein Glycosylation ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Unique Gene ◽  
Glycosyl Transferase ◽  
Caldicellulosiruptor Bescii ◽  
Carbohydrate Binding Modules

ABSTRACT Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant enzymes in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted in loss of detectable protein glycosylation in C. bescii, and its activity has been shown to be responsible for the glycosylation of the proline-threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. Here, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild-type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases. IMPORTANCE Caldicellulosiruptor has the ability to digest complex plant biomass without pretreatment and have been engineered to convert biomass, a sustainable, carbon neutral substrate, to fuels. Their strategy for deconstructing plant cell walls relies on an interesting class of cellulases consisting of multiple catalytic modules connected by linker regions and carbohydrate binding modules. The best studied of these enzymes, CelA, has a unique deconstruction mechanism. CelA is located in a cluster of genes that likely allows for optimal expression, secretion, and activity. One of the genes in this cluster is a putative isomerase that modifies the CelA protein. In higher eukaryotes, these isomerases are essential for the proper folding of glycoproteins in the endoplasmic reticulum, but little is known about the role of isomerization in cellulase activity. We show that the stability and activity of CelA is dependent on the activity of this isomerase.

scholarly journals Cell Surface Enzyme Attachment Is Mediated by Family 37 Carbohydrate-Binding Modules, Unique to Ruminococcus albus

2008 ◽  
Vol 190 (24) ◽  
pp. 8220-8222 ◽  
Author(s):  
Anat Ezer ◽  
Erez Matalon ◽  
Sadanari Jindou ◽  
Ilya Borovok ◽  
Nof Atamna ◽  
...  
Keyword(s):  
Cell Surface ◽  
Bacterial Cell ◽  
Cellulose Degradation ◽  
Glycoside Hydrolases ◽  
Rumen Bacterium ◽  
Carbohydrate Binding ◽  
Ruminococcus Albus ◽  
System A ◽  
C Terminus ◽  
Carbohydrate Binding Modules

ABSTRACT The rumen bacterium Ruminococcus albus binds to and degrades crystalline cellulosic substrates via a unique cellulose degradation system. A unique family of carbohydrate-binding modules (CBM37), located at the C terminus of different glycoside hydrolases, appears to be responsible both for anchoring these enzymes to the bacterial cell surface and for substrate binding.

scholarly journals Synergism of Glycoside Hydrolase Secretomes from Two Thermophilic Bacteria Cocultivated on Lignocellulose

2014 ◽  
Vol 80 (8) ◽  
pp. 2592-2601 ◽  
Author(s):  
Kundi Zhang ◽  
Xiaohua Chen ◽  
Wolfgang H. Schwarz ◽  
Fuli Li
Keyword(s):  
Glycoside Hydrolase ◽  
Thermophilic Bacteria ◽  
Xylanase Activity ◽  
Mass Spectrometry Analysis ◽  
Lignocellulose Degradation ◽  
Rrna Gene ◽  
Carbohydrate Binding ◽  
Bacterial Strains ◽  
Content Type ◽  
Carbohydrate Binding Modules

ABSTRACTTwo cellulolytic thermophilic bacterial strains, CS-3-2 and CS-4-4, were isolated from decayed cornstalk by the addition of growth-supporting factors to the medium. According to 16S rRNA gene-sequencing results, these strains belonged to the genusClostridiumand showed 98.87% and 98.86% identity withClostridiumstercorariumsubsp.leptospartumATCC 35414TandClostridiumcellulosiAS 1.1777T, respectively. The endoglucanase and exoglucanase activities of strain CS-4-4 were approximately 3 to 5 times those of strain CS-3-2, whereas the β-glucosidase activity of strain CS-3-2 was 18 times higher than that of strain CS-4-4. The xylanase activity of strain CS-3-2 was 9 times that of strain CS-4-4, whereas the β-xylosidase activity of strain CS-4-4 was 27 times that of strain CS-3-2. The enzyme activities in spent cultures following cocultivation of the two strains with cornstalk as the substrate were much greater than those in pure cultures or an artificial mixture of samples, indicating synergism of glycoside hydrolase secretomes between the two strains. Quantitative measurement of the two strains in the cocultivation system indicated that strain CS-3-2 grew robustly during the initial stages, whereas strain CS-4-4 dominated the system in the late-exponential phase. Liquid chromatography-tandem mass spectrometry analysis of protein bands appearing in the native zymograms showed that ORF3880 and ORF3883 from strain CS-4-4 played key roles in the lignocellulose degradation process. Both these open reading frames (ORFs) exhibited endoglucanase and xylanase activities, but ORF3880 showed tighter adhesion to insoluble substrates at 4, 25, and 60°C owing to its five carbohydrate-binding modules (CBMs).

scholarly journals Novel Family of Carbohydrate-Binding Modules Revealed by the Genome Sequence of Spirochaeta thermophila DSM 6192

2011 ◽  
Vol 77 (15) ◽  
pp. 5483-5489 ◽  
Author(s):  
Angel Angelov ◽  
Christoph Loderer ◽  
Susanne Pompei ◽  
Wolfgang Liebl
Keyword(s):  
Genome Sequence ◽  
Sequence Data ◽  
Cellulose Degradation ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Carbohydrate Binding ◽  
Functional Screening ◽  
Content Type ◽  
C Terminus ◽  
Carbohydrate Binding Modules

ABSTRACTSpirochaeta thermophilais a thermophilic, free-living, and cellulolytic anaerobe. The genome sequence data for this organism have revealed a high density of genes encoding enzymes from more than 30 glycoside hydrolase (GH) families and a noncellulosomal enzyme system for (hemi)cellulose degradation. Functional screening of a fosmid library whose inserts were mapped on theS. thermophilagenome sequence allowed the functional annotation of numerous GH open reading frames (ORFs). Seven different GH ORFs from theS. thermophilaDSM 6192 genome, all putative β-glycanase ORFs according to sequence similarity analysis, contained a highly conserved novel GH-associated module of unknown function at their C terminus. Four of these GH enzymes were experimentally verified as xylanase, β-glucanase, β-glucanase/carboxymethylcellulase (CMCase), and CMCase. Binding experiments performed with the recombinantly expressed and purified GH-associated module showed that it represents a new carbohydrate-binding module (CBM) that binds to microcrystalline cellulose and is highly specific for this substrate. In the course of this work, the new CBM type was only detected inSpirochaeta, but recently we found sequences with detectable similarity to the module in the draft genomes ofCytophaga fermentansandMahella australiensis, both of which are phylogenetically very distant fromS. thermophilaand noncellulolytic, yet inhabit similar environments. This suggests a possibly broad distribution of the module in nature.

scholarly journals Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module from family 64 (StX)

2015 ◽  
Vol 71 (3) ◽  
pp. 311-314 ◽  
Author(s):  
Bruna Medeia Campos ◽  
Marcelo Vizona Liberato ◽  
Igor Polikarpov ◽  
Ana Carolina de Mattos Zeri ◽  
Fabio Marcio Squina
Keyword(s):  
Plant Biomass ◽  
Functional Characterization ◽  
Diffraction Method ◽  
Glycoside Hydrolases ◽  
Diffusion Method ◽  
Carbohydrate Binding ◽  
Thermophilic Microorganisms ◽  
X Ray ◽  
Cell Parameters ◽  
Carbohydrate Binding Modules

In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost-effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM-encoding module named StX was expressed, purified and crystallized, and X-ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Å resolution, respectively. The crystals, which were obtained by the hanging-drop vapour-diffusion method, belonged to space groupP3121, with unit-cell parametersa=b= 43.42,c= 100.96 Å for the native form. The phases were found using the single-wavelength anomalous diffraction method.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of a trimodular endo-β-1,4-glucanase (Cel5B) fromBacillus halodurans

2014 ◽  
Vol 70 (12) ◽  
pp. 1628-1630 ◽  
Author(s):  
Immacolata Venditto ◽  
Helena Santos ◽  
James Sandy ◽  
Juan Sanchez-Weatherby ◽  
Luis M. A. Ferreira ◽  
...  
Keyword(s):  
Diffraction Analysis ◽  
Plant Biomass ◽  
Organic Polymer ◽  
Major Constituent ◽  
Bacillus Halodurans ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
X Ray Diffraction ◽  
X Ray ◽  
Carbohydrate Binding Modules

Cellulases catalyze the hydrolysis of cellulose, the major constituent of plant biomass and the most abundant organic polymer on earth. Cellulases are modular enzymes containing catalytic domains connected,vialinker sequences, to noncatalytic carbohydrate-binding modules (CBMs). A putative modular endo-β-1,4-glucanase (BhCel5B) is encoded at locus BH0603 in the genome ofBacillus halodurans. It is composed of an N-terminal glycoside hydrolase family 5 catalytic module (GH5) followed by an immunoglobulin-like module and a C-terminal family 46 CBM (BhCBM46). Here, the crystallization and preliminary X-ray diffraction analysis of the trimodularBhCel5B are reported. The crystals ofBhCel5B belonged to the orthorhombic space groupP21212 and data were processed to a resolution of 1.64 Å. A molecular-replacement solution has been found.

scholarly journals CalkGH9T: A Glycoside Hydrolase Family 9 Enzyme from Clostridium alkalicellulosi

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1011
Author(s):  
Paripok Phitsuwan ◽  
Sengthong Lee ◽  
Techly San ◽  
Khanok Ratanakhanokchai
Keyword(s):  
Glycoside Hydrolase ◽  
Cellulose Degradation ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Type I ◽  
Amorphous Cellulose ◽  
Carbohydrate Binding Modules ◽  
Glycoside Hydrolase Family 9 ◽  
Hydrolysis Of ◽  
Hydrolase Family

Glycoside hydrolase family 9 (GH9) endoglucanases are important enzymes for cellulose degradation. However, their activity on cellulose is diverse. Here, we cloned and expressed one GH9 enzyme (CalkGH9T) from Clostridium alkalicellulosi in Escherichia coli. CalkGH9T has a modular structure, containing one GH9 catalytic module, two family 3 carbohydrate binding modules, and one type I dockerin domain. CalkGH9T exhibited maximal activity at pH 7.0–8.0 and 55 °C and was resistant to urea and NaCl. It efficiently hydrolyzed carboxymethyl cellulose (CMC) but poorly degraded regenerated amorphous cellulose (RAC). Despite strongly binding to Avicel, CalkGH9T lacked the ability to hydrolyze this substrate. The hydrolysis of CMC by CalkGH9T produced a series of cello-oligomers, with cellotetraose being preferentially released. Similar proportions of soluble and insoluble reducing ends generated by hydrolysis of RAC indicated non-processive activity. Our study extends our knowledge of the molecular mechanism of cellulose hydrolysis by GH9 family endoglucanases with industrial relevance.

scholarly journals Genus-Wide Assessment of Lignocellulose Utilization in the Extremely Thermophilic GenusCaldicellulosiruptorby Genomic, Pangenomic, and Metagenomic Analyses

2018 ◽  
Vol 84 (9) ◽  
Author(s):  
Laura L. Lee ◽  
Sara E. Blumer-Schuette ◽  
Javier A. Izquierdo ◽  
Jeffrey V. Zurawski ◽  
Andrew J. Loder ◽  
...  
Keyword(s):  
Microcrystalline Cellulose ◽  
Plant Biomass ◽  
Thermophilic Bacteria ◽  
Cellulose Degradation ◽  
Glycoside Hydrolases ◽  
Metagenomic Data ◽  
Lignocellulose Degradation ◽  
Sequence Information ◽  
Metagenomic Sequence ◽  
Content Type

ABSTRACTMetagenomic data from Obsidian Pool (Yellowstone National Park, USA) and 13 genome sequences were used to reassess genus-wide biodiversity for the extremely thermophilicCaldicellulosiruptor. The updated core genome contains 1,401 ortholog groups (average genome size for 13 species = 2,516 genes). The pangenome, which remains open with a revised total of 3,493 ortholog groups, encodes a variety of multidomain glycoside hydrolases (GHs). These include three cellulases with GH48 domains that are colocated in the glucan degradation locus (GDL) and are specific determinants for microcrystalline cellulose utilization. Three recently sequenced species,Caldicellulosiruptorsp. strain Rt8.B8 (renamed hereCaldicellulosiruptor morganii),Thermoanaerobacter cellulolyticusstrain NA10 (renamed hereCaldicellulosiruptor naganoensis), andCaldicellulosiruptorsp. strain Wai35.B1 (renamed hereCaldicellulosiruptor danielii), degraded Avicel and lignocellulose (switchgrass).C. morganiiwas more efficient thanCaldicellulosiruptor besciiin this regard and differed from the other 12 species examined, both based on genome content and organization and in the specific domain features of conserved GHs. Metagenomic analysis of lignocellulose-enriched samples from Obsidian Pool revealed limited new information on genus biodiversity. Enrichments yielded genomic signatures closely related to that ofCaldicellulosiruptor obsidiansis, but there was also evidence for other thermophilic fermentative anaerobes (Caldanaerobacter,Fervidobacterium,Caloramator, andClostridium). One enrichment, containing 89.8%Caldicellulosiruptorand 9.7%Caloramator, had a capacity for switchgrass solubilization comparable to that ofC. bescii. These results refine the known biodiversity ofCaldicellulosiruptorand indicate that microcrystalline cellulose degradation at temperatures above 70°C, based on current information, is limited to certain members of this genus that produce GH48 domain-containing enzymes.IMPORTANCEThe genusCaldicellulosiruptorcontains the most thermophilic bacteria capable of lignocellulose deconstruction, which are promising candidates for consolidated bioprocessing for the production of biofuels and bio-based chemicals. The focus here is on the extant capability of this genus for plant biomass degradation and the extent to which this can be inferred from the core and pangenomes, based on analysis of 13 species and metagenomic sequence information from environmental samples. Key to microcrystalline hydrolysis is the content of the glucan degradation locus (GDL), a set of genes encoding glycoside hydrolases (GHs), several of which have GH48 and family 3 carbohydrate binding module domains, that function as primary cellulases. Resolving the relationship between the GDL and lignocellulose degradation will inform efforts to identify more prolific members of the genus and to develop metabolic engineering strategies to improve this characteristic.

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