scholarly journals Two New Xylanases with Different Substrate Specificities from the Human Gut Bacterium Bacteroides intestinalis DSM 17393

2014 ◽  
Vol 80 (7) ◽  
pp. 2084-2093 ◽  
Author(s):  
Pei-Ying Hong ◽  
Michael Iakiviak ◽  
Dylan Dodd ◽  
Meiling Zhang ◽  
Roderick I. Mackie ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Cell Wall Polysaccharide ◽  
Active Site Residues ◽  
Human Gastrointestinal Tract ◽  
Genome Sequences ◽  
Human Gut ◽  
Content Type ◽  
Substrate Specificities ◽  
Dominant Component ◽  
Putative Active Site

ABSTRACTXylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacteriumBacteroides intestinalisDSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. Thexyn8Agene encodes an endoxylanase (Xyn8A), andrex8Aencodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X5) and xylohexaose (X6) to a mixture of xylobiose (X2) and xylotriose (X3), while Rex8A hydrolyzed X3through X6to a mixture of xylose (X1) and X2. Moreover,rex8Ais located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit β-xylosidase activity and would be able to further hydrolyze X2to X1. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among theBacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.

scholarly journals Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

2017 ◽  
Vol 83 (18) ◽  
Author(s):  
Daniel Wefers ◽  
Jia Dong ◽  
Ahmed M. Abdel-Hamid ◽  
Hans Müller Paul ◽  
Gabriel V. Pereira ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Recombinant Proteins ◽  
Plant Cell Wall ◽  
Elevated Temperatures ◽  
Thermophilic Bacterium ◽  
Glycoside Hydrolases ◽  
Cell Wall Polysaccharide ◽  
Binding Studies ◽  
Content Type ◽  
Substrate Specificities

ABSTRACT The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-l-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 β-l-arabinopyranosidase (CpAbp27A), and two GH127 β-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para-nitrophenyl (pNP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α-l-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticus. IMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus. The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as β-l-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.

scholarly journals The Homogalacturonan Deconstruction System of Paenibacillus amylolyticus 27C64 Requires No Extracellular Pectin Methylesterase and Has Significant Industrial Potential

2020 ◽  
Vol 86 (12) ◽  
Author(s):  
Christian Keggi ◽  
Joy Doran-Peterson
Keyword(s):  
Plant Cell ◽  
Plant Cell Wall ◽  
Pectate Lyase ◽  
Pectin Methylesterase ◽  
Pectin Lyase ◽  
Cell Wall Polysaccharide ◽  
Positive Bacterium ◽  
High Turnover ◽  
Gram Positive ◽  
Content Type

ABSTRACT Paenibacillus amylolyticus 27C64, a Gram-positive bacterium with diverse plant cell wall polysaccharide deconstruction capabilities, was isolated previously from an insect hindgut. Previous work suggested that this organism’s pectin deconstruction system differs from known systems in that its sole pectin methylesterase is cytoplasmic, not extracellular. In this work, we have characterized the specific roles of key extracellular pectinases involved in homogalacturonan deconstruction, including four pectate lyases and one pectin lyase. We show that one newly characterized pectate lyase, PelC, has a novel substrate specificity, with a lower Km for highly methylated pectins than for polygalacturonic acid. PelC works synergistically with PelB, a high-turnover exo-pectate lyase that releases Δ4,5-unsaturated trigalacturonate as its major product. It is likely that PelC frees internal stretches of demethylated homogalacturonan which PelB can degrade. We also show that the sole pectin lyase has a high kcat value and rapidly depolymerizes methylated substrates. Three cytoplasmic GH105 hydrolases were screened for the ability to remove terminal unsaturated galacturonic acid residues from oligogalacturonide products produced by the action of extracellular lyases, and we found that two are active on demethylated oligogalacturonides. This work confirms that efficient homogalacturonan deconstruction in P. amylolyticus 27C65 does not require extracellular pectin methylesterase activity. Three of the extracellular lyases studied in this work are also thermostable, function well over a broad pH range, and have significant industrial potential. IMPORTANCE Pectin is an important structural polysaccharide found in most plant cell walls. In the environment, pectin degradation is part of the decomposition process that turns over dead plant material and is important to organisms that feed on plants. Industrially, pectinases are used to improve the quality of fruit juices and can also be used to process coffee cherries or tea leaves. These enzymes may also prove useful in reducing the environmental impact of paper and cotton manufacturing. This work is significant because it focuses on a Gram-positive bacterium that is evolutionarily distinct from other well-studied pectin-degrading organisms and differs from known systems in key ways. Most importantly, a simplified extracellular deconstruction process in this organism is able to break down pectins without first removing the methyl groups that inhibit other systems. Moreover, some of the enzymes described here have the potential to improve industrial processes that rely on pectin deconstruction.

scholarly journals Specific Xylan Activity Revealed for AA9 Lytic Polysaccharide Monooxygenases of the Thermophilic Fungus Malbranchea cinnamomea by Functional Characterization

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Silvia Hüttner ◽  
Anikó Várnai ◽  
Dejan M. Petrović ◽  
Cao Xuan Bach ◽  
Dang Thi Kim Anh ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Functional Characterization ◽  
Cellulose Microfibrils ◽  
Cell Wall Polysaccharide ◽  
Poor Growth ◽  
Content Type ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

ABSTRACT The thermophilic biomass-degrader Malbranchea cinnamomea exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight (M. cinnamomea AA9s) McAA9s, namely, McAA9A, McAA9B, McAA9F, and McAA9H, to gain a deeper understanding about their roles in the fungus. The characterized McAA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the McAA9s, McAA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends, McAA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO. McAA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers’ physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall. IMPORTANCE The Malbranchea cinnamomea LPMOs (McAA9s) showed activity on a broad range of soluble and insoluble substrates, suggesting their involvement in various steps of biomass degradation besides cellulose decomposition. Our results indicate that the fungal AA9 family is more diverse than originally thought and able to degrade almost any kind of plant cell wall polysaccharide. The discovery of an AA9 that preferentially cleaves xylan enhances our understanding of the physiological roles of LPMOs and enables the use of xylan-specific LPMOs in future applications.

scholarly journals Genomewide and Enzymatic Analysis Reveals Efficient D-Galacturonic Acid Metabolism in the Basidiomycete Yeast Rhodosporidium toruloides

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Ryan J. Protzko ◽  
Christina A. Hach ◽  
Samuel T. Coradetti ◽  
Magdalena A. Hackhofer ◽  
Sonja Magosch ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Galacturonic Acid ◽  
Plant Cell Wall ◽  
Agricultural Waste ◽  
Rhodosporidium Toruloides ◽  
Cell Wall Polysaccharide ◽  
Content Type ◽  
Aromatic Molecules ◽  
Renewable Feedstocks

ABSTRACT Biorefining of renewable feedstocks is one of the most promising routes to replace fossil-based products. Since many common fermentation hosts, such as Saccharomyces cerevisiae, are naturally unable to convert many component plant cell wall polysaccharides, the identification of organisms with broad catabolism capabilities represents an opportunity to expand the range of substrates used in fermentation biorefinery approaches. The red basidiomycete yeast Rhodosporidium toruloides is a promising and robust host for lipid- and terpene-derived chemicals. Previous studies demonstrated assimilation of a range of substrates, from C5/C6 sugars to aromatic molecules similar to lignin monomers. In the current study, we analyzed the potential of R. toruloides to assimilate d-galacturonic acid, a major sugar in many pectin-rich agricultural waste streams, including sugar beet pulp and citrus peels. d-Galacturonic acid is not a preferred substrate for many fungi, but its metabolism was found to be on par with those of d-glucose and d-xylose in R. toruloides. A genomewide analysis by combined transcriptome sequencing (RNA-seq) and RB-TDNA-seq revealed those genes with high relevance for fitness on d-galacturonic acid. While R. toruloides was found to utilize the nonphosphorylative catabolic pathway known from ascomycetes, the maximal velocities of several enzymes exceeded those previously reported. In addition, an efficient downstream glycerol catabolism and a novel transcription factor were found to be important for d-galacturonic acid utilization. These results set the basis for use of R. toruloides as a potential host for pectin-rich waste conversions and demonstrate its suitability as a model for metabolic studies with basidiomycetes. IMPORTANCE The switch from the traditional fossil-based industry to a green and sustainable bioeconomy demands the complete utilization of renewable feedstocks. Many currently used bioconversion hosts are unable to utilize major components of plant biomass, warranting the identification of microorganisms with broader catabolic capacity and characterization of their unique biochemical pathways. d-Galacturonic acid is a plant component of bioconversion interest and is the major backbone sugar of pectin, a plant cell wall polysaccharide abundant in soft and young plant tissues. The red basidiomycete and oleaginous yeast Rhodosporidium toruloides has been previously shown to utilize a range of sugars and aromatic molecules. Using state-of-the-art functional genomic methods and physiological and biochemical assays, we elucidated the molecular basis underlying the efficient metabolism of d-galacturonic acid. This study identified an efficient pathway for uronic acid conversion to guide future engineering efforts and represents the first detailed metabolic analysis of pectin metabolism in a basidiomycete fungus.

scholarly journals Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen

2014 ◽  
Vol 81 (4) ◽  
pp. 1375-1386 ◽  
Author(s):  
Xin Dai ◽  
Yan Tian ◽  
Jinting Li ◽  
Xiaoyun Su ◽  
Xuewei Wang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Average Length ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Cell Wall Polysaccharide ◽  
Cell Wall Polysaccharides ◽  
Content Type ◽  
Carbohydrate Binding Modules

ABSTRACTThe bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for ∼1% and ∼0.1% of the total non-rRNAs, respectively. The majority (∼98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized byRuminococcusandFibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the generaRuminococcus,Prevotella, andFibrobacter. Most (∼82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the generaRuminococcus,Fibrobacter, andPrevotellaare predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen.

Substrate use prioritization by a coculture of five species of gut bacteria fed mixtures of arabinoxylan, xyloglucan, β-glucan, and pectin

2020 ◽  
Author(s):  
Y Liu ◽  
AL Heath ◽  
B Galland ◽  
N Rehrer ◽  
L Drummond ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Dietary Fiber ◽  
Plant Cell Wall ◽  
Bacterial Species ◽  
Short Chain ◽  
Emergent Properties ◽  
Human Gut ◽  
Fermentation Products ◽  
Plant Polysaccharides ◽  
Human Gut Microbiota

© 2020 American Society for Microbiology. Dietary fiber provides growth substrates for bacterial species that belong to the colonic microbiota of humans. The microbiota degrades and ferments substrates, producing characteristic short-chain fatty acid profiles. Dietary fiber contains plant cell wall-associated polysaccharides (hemicelluloses and pectins) that are chemically diverse in composition and structure. Thus, depending on plant sources, dietary fiber daily presents the microbiota with mixtures of plant polysaccharides of various types and complexity. We studied the extent and preferential order in which mixtures of plant polysaccharides (arabinoxylan, xyloglucan, β-glucan, and pectin) were utilized by a coculture of five bacterial species (Bacteroides ovatus, Bifidobacterium longum subspecies longum, Megasphaera elsdenii, Ruminococcus gnavus, and Veillonella parvula). These species are members of the human gut microbiota and have the biochemical capacity, collectively, to degrade and ferment the polysaccharides and produce short-chain fatty acids (SCFAs). B. ovatus utilized glycans in the order β-glucan, pectin, xyloglucan, and arabinoxylan, whereas B. longum subsp. longum utilization was in the order arabinoxylan, arabinan, pectin, and β-glucan. Propionate, as a proportion of total SCFAs, was augmented when polysaccharide mixtures contained galactan, resulting in greater succinate production by B. ovatus and conversion of succinate to propionate by V. parvula. Overall, we derived a synthetic ecological community that carries out SCFA production by the common pathways used by bacterial species for this purpose. Systems like this might be used to predict changes to the emergent properties of the gut ecosystem when diet is altered, with the aim of beneficially affecting human physiology. This study addresses the question as to how bacterial species, characteristic of the human gut microbiota, collectively utilize mixtures of plant polysaccharides such as are found in dietary fiber. Five bacterial species with the capacity to degrade polymers and/or produce acidic fermentation products detectable in human feces were used in the experiments. The bacteria showed preferential use of certain polysaccharides over others for growth, and this influenced their fermentation output qualitatively. These kinds of studies are essential in developing concepts of how the gut microbial community shares habitat resources, directly and indirectly, when presented with mixtures of polysaccharides that are found in human diets. The concepts are required in planning dietary interventions that might correct imbalances in the functioning of the human microbiota so as to support measures to reduce metabolic conditions such as obesity.

scholarly journals Draft Genome Sequences of 12 Mycolicibacterium smegmatis Strains Resistant to Imidazo[1,2-b][1,2,4,5]Tetrazines

2019 ◽  
Vol 8 (16) ◽  
Author(s):  
Aleksey A. Vatlin ◽  
Kirill V. Shur ◽  
Valery N. Danilenko ◽  
Dmitry A. Maslov
Keyword(s):  
Draft Genome ◽  
Transcriptional Repressor ◽  
Antituberculosis Drug ◽  
Genome Sequences ◽  
Content Type ◽  
Drug Candidates

Here, we report 12 draft genome sequences of mutant Mycolicibacterium smegmatis strains resistant to imidazo[1,2-b][1,2,4,5]tetrazines, which are antituberculosis drug candidates. We have identified 7 different mutations in the MSMEG_1380 gene, which encodes the AcrR/TetR_N transcriptional repressor, which may activate efflux-mediated resistance.

scholarly journals Draft Genome Sequences of Three Flavobacterium psychrophilum Strains Isolated from Diseased Ayu (Plecoglossus altivelis altivelis) Caught at Three Sites in the Kagami River in Kochi, Japan

2019 ◽  
Vol 8 (34) ◽  
Author(s):  
Hazuki Yamashita ◽  
Takayuki Wada ◽  
Yusuke Kato ◽  
Takuji Ikeda ◽  
Masayuki Imajoh
Keyword(s):  
Draft Genome ◽  
Psychrophilic Bacterium ◽  
Genome Sequences ◽  
Plecoglossus Altivelis ◽  
Gram Negative ◽  
Content Type ◽  
Flavobacterium Psychrophilum ◽  
Skin Ulcers ◽  
The Family

Flavobacterium psychrophilum is a Gram-negative, psychrophilic bacterium within the family Flavobacteriaceae. Here, we report the draft genome sequences of three F. psychrophilum strains isolated from skin ulcers of diseased ayu caught by tomozuri angling at three sites in the Kagami River in Japan.

scholarly journals PUL-Mediated Plant Cell Wall Polysaccharide Utilization in the Gut Bacteroidetes

2021 ◽  
Vol 22 (6) ◽  
pp. 3077
Author(s):  
Zhenzhen Hao ◽  
Xiaolu Wang ◽  
Haomeng Yang ◽  
Tao Tu ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Microbial Community ◽  
Gut Microbiota ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Cell Wall Polysaccharide ◽  
Prominent Feature ◽  
Cell Wall Polysaccharides ◽  
Gut Microbial Community

Plant cell wall polysaccharides (PCWP) are abundantly present in the food of humans and feed of livestock. Mammalians by themselves cannot degrade PCWP but rather depend on microbes resident in the gut intestine for deconstruction. The dominant Bacteroidetes in the gut microbial community are such bacteria with PCWP-degrading ability. The polysaccharide utilization systems (PUL) responsible for PCWP degradation and utilization are a prominent feature of Bacteroidetes. In recent years, there have been tremendous efforts in elucidating how PULs assist Bacteroidetes to assimilate carbon and acquire energy from PCWP. Here, we will review the PUL-mediated plant cell wall polysaccharides utilization in the gut Bacteroidetes focusing on cellulose, xylan, mannan, and pectin utilization and discuss how the mechanisms can be exploited to modulate the gut microbiota.

scholarly journals Draft Genome Sequences of Escherichia coli Strains FP2 and FP3, Isolated from the Canada Goose (Branta canadensis)

2018 ◽  
Vol 7 (9) ◽  
Author(s):  
Allison L. Denny ◽  
Susan E. Arruda
Keyword(s):  
Escherichia Coli ◽  
Draft Genome ◽  
Branta Canadensis ◽  
Canada Goose ◽  
Genome Sequences ◽  
Content Type

Draft genomes of two strains of Escherichia coli, FP2 and FP3, isolated from the feces of the Canada goose (Branta canadensis), were sequenced. Genome sizes were 5.26 Mb with a predicted G+C content of 50.54% (FP2) and 5.07 Mb with a predicted G+C content of 50.41% (FP3).

Sign in / Sign up

Export Citation Format

Share Document