scholarly journals Sift-PULs: A public repository for specific functional polysaccharide utilization loci

2021 ◽  
Author(s):  
Tao Song ◽  
Congchong Wei ◽  
Dezhi Yuan ◽  
Shengwei Xiang ◽  
Lin Liu ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Gene Clusters ◽  
Public Repository ◽  
Function Annotation ◽  
Bacterial Gene ◽  
Bacterial Phyla ◽  
Signature Genes ◽  
Specific Polysaccharide ◽  
Encoding Genes ◽  
Polysaccharide Utilization Loci

Background Polysaccharide utilization loci (PULs) were bacterial gene clusters encoding genes responsible for polysaccharide utilization process. PUL studies are blooming in recent years but the biochemical characterization speed is relative slow. There is a growing demand for PUL database with function annotations. Results Using signature genes corresponding for specific polysaccharide, 10422 PULs specific for 6 polysaccharides (agar, alginate, pectin, carrageenan, chitin and β-manan) from various bacterial phyla were predicted. Then online website of specific functional polysaccharide utilization loci (Sift-PULs) was constructed. Sift-PULs provides a repository where users could browse, search and download interested PULs without registration. Conclusions The key advantage of Sift-PULs is to assign a function annotation of each PUL, which is not available in existing PUL databases. PUL's functional annotation lays a foundation for studying novel enzymes, new pathways, PUL evolution or bioengineering. The website is available on http://sift-puls.org

scholarly journals Polysaccharide utilization loci-driven enzyme discovery reveals BD-FAE: a bifunctional feruloyl and acetyl xylan esterase active on complex natural xylans

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lisanne Hameleers ◽  
Leena Penttinen ◽  
Martina Ikonen ◽  
Léa Jaillot ◽  
Régis Fauré ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Structural Information ◽  
Gene Clusters ◽  
Biochemical Properties ◽  
Corn Fiber ◽  
Feruloyl Esterase ◽  
Acetyl Xylan Esterase ◽  
Acetyl Esterase ◽  
Enzyme Discovery ◽  
Polysaccharide Utilization Loci

Abstract Background Nowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g., saccharification) can offer a sustainable route due to avoidance of harsh chemicals and high temperatures. For novel enzyme discovery, physically linked gene clusters targeting carbohydrate degradation in bacteria, polysaccharide utilization loci (PULs), are recognized ‘treasure troves’ in the era of exponentially growing numbers of sequenced genomes. Results We determined the biochemical properties and structure of a protein of unknown function (PUF) encoded within PULs of metagenomes from beaver droppings and moose rumen enriched on poplar hydrolysate. The corresponding novel bifunctional carbohydrate esterase (CE), now named BD-FAE, displayed feruloyl esterase (FAE) and acetyl esterase activity on simple, synthetic substrates. Whereas acetyl xylan esterase (AcXE) activity was detected on acetylated glucuronoxylan from birchwood, only FAE activity was observed on acetylated and feruloylated xylooligosaccharides from corn fiber. The genomic contexts of 200 homologs of BD-FAE revealed that the 33 closest homologs appear in PULs likely involved in xylan breakdown, while the more distant homologs were found either in alginate-targeting PULs or else outside PUL contexts. Although the BD-FAE structure adopts a typical α/β-hydrolase fold with a catalytic triad (Ser-Asp-His), it is distinct from other biochemically characterized CEs. Conclusions The bifunctional CE, BD-FAE, represents a new candidate for biomass processing given its capacity to remove ferulic acid and acetic acid from natural corn and birchwood xylan substrates, respectively. Its detailed biochemical characterization and solved crystal structure add to the toolbox of enzymes for biomass valorization as well as structural information to inform the classification of new CEs.

scholarly journals dbCAN-PUL: a database of experimentally characterized CAZyme gene clusters and their substrates

2020 ◽  
Vol 49 (D1) ◽  
pp. D523-D528 ◽  
Author(s):  
Catherine Ausland ◽  
Jinfang Zheng ◽  
Haidong Yi ◽  
Bowen Yang ◽  
Tang Li ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Gene Clusters ◽  
Homologous Gene ◽  
Sequence Information ◽  
Experimental Characterization ◽  
Online Database ◽  
Carbohydrate Active Enzymes ◽  
Bacterial Phyla ◽  
Efficient Retrieval ◽  
Polysaccharide Utilization Loci

Abstract PULs (polysaccharide utilization loci) are discrete gene clusters of CAZymes (Carbohydrate Active EnZymes) and other genes that work together to digest and utilize carbohydrate substrates. While PULs have been extensively characterized in Bacteroidetes, there exist PULs from other bacterial phyla, as well as archaea and metagenomes, that remain to be catalogued in a database for efficient retrieval. We have developed an online database dbCAN-PUL (http://bcb.unl.edu/dbCAN_PUL/) to display experimentally verified CAZyme-containing PULs from literature with pertinent metadata, sequences, and annotation. Compared to other online CAZyme and PUL resources, dbCAN-PUL has the following new features: (i) Batch download of PUL data by target substrate, species/genome, genus, or experimental characterization method; (ii) Annotation for each PUL that displays associated metadata such as substrate(s), experimental characterization method(s) and protein sequence information, (iii) Links to external annotation pages for CAZymes (CAZy), transporters (UniProt) and other genes, (iv) Display of homologous gene clusters in GenBank sequences via integrated MultiGeneBlast tool and (v) An integrated BLASTX service available for users to query their sequences against PUL proteins in dbCAN-PUL. With these features, dbCAN-PUL will be an important repository for CAZyme and PUL research, complementing our other web servers and databases (dbCAN2, dbCAN-seq).

scholarly journals Evolution of Bacterial Gene Transfer Agents

2018 ◽  
Author(s):  
Rosemary J. Redfield ◽  
Shannon M. Soucy
Keyword(s):  
Gene Transfer ◽  
Gene Clusters ◽  
Bacterial Gene ◽  
Chromosomal Genes ◽  
Potential Benefits ◽  
Small Virus ◽  
Fitness Benefits ◽  
The Cost ◽  
Encoding Genes ◽  
Transfer Agents

AbstractBacterial gene transfer agents (GTAs) are small virus-like particles that package DNA fragments and inject them into cells. They are encoded by gene clusters resembling defective prophages, with genes for capsid head and tail components. These gene clusters are usually assumed to be maintained by selection for the benefits of GTA-mediated recombination, but this has never been tested. We rigorously examined the potential benefits of GTA-mediated recombination, considering separately transmission of GTA-encoding genes and recombination of all chromosomal genes. In principle GTA genes could be directly maintained if GTA particles spread them to GTA−cells often enough to compensate for the loss of GTA-producing cells. However careful bookkeeping showed that losses inevitably exceed gains for two reasons. First, cells must lyse to release particles to the environment. Second, GTA genes are not preferentially replicated before DNA is packaged. A simulation model was then used to search for conditions where recombination of chromosomal genes makes GTA+populations fitter than GTA−populations. Although the model showed that both synergistic epistasis and some modes of regulation could generate fitness benefits large enough to overcome the cost of lysis, these benefits neither allowed GTA+cells to invade GTA−populations, nor allowed GTA+populations to resist invasion by GTA−cells. Importantly, the benefits depended on highly improbable assumptions about the efficiencies of GTA production and recombination. Thus, the selective benefits that maintain GTA gene clusters over many millions of years must arise from consequences other than transfer of GTA genes or recombination of chromosomal genes.

scholarly journals Identification and characterization of cytochrome P450 1232A24 and 1232F1 from Arthrobacter sp. and their role in the metabolic pathway of papaverine

2019 ◽  
Vol 166 (1) ◽  
pp. 51-66 ◽  
Author(s):  
Jan M Klenk ◽  
Max-Philipp Fischer ◽  
Paulina Dubiel ◽  
Mahima Sharma ◽  
Benjamin Rowlinson ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Metabolic Pathway ◽  
Biochemical Characterization ◽  
Gene Clusters ◽  
Cytochrome P450 Monooxygenases ◽  
Dihydroxyphenylacetic Acid ◽  
Meta Position ◽  
Redox Partners

AbstractCytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s’ role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

scholarly journals Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Cathleen Kmezik ◽  
Daniel Krska ◽  
Scott Mazurkewich ◽  
Johan Larsbrink
Keyword(s):  
Biochemical Characterization ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Active Enzymes ◽  
Human Gut ◽  
Glucuronoyl Esterase ◽  
Cob Biomass ◽  
Polysaccharide Utilization Loci ◽  
Hydrolase Family ◽  
Discrete Functions

AbstractBacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions—multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii. The enzyme, BeCE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length BeCE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs.

Genetic analysis of gliadin-encoding genes reveals gene clusters as well as single remote genes

1986 ◽  
Vol 73 (2) ◽  
pp. 278-285 ◽  
Author(s):  
E. V. Metakovsky ◽  
M. G. Akhmedov ◽  
A. A. Sozinov
Keyword(s):  
Genetic Analysis ◽  
Gene Clusters ◽  
Encoding Genes

scholarly journals Methanosarcina acetivorans contains a functional ISC system for iron-sulfur cluster biogenesis

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Thomas M. Deere ◽  
Divya Prakash ◽  
Faith H. Lessner ◽  
Evert C. Duin ◽  
Daniel J. Lessner
Keyword(s):  
Parent Strain ◽  
Biochemical Characterization ◽  
Sulfur Metabolism ◽  
Gene Clusters ◽  
Sulfur Source ◽  
Methanosarcina Acetivorans ◽  
Cysteine Desulfurase ◽  
The Core ◽  
Iron Sulfur ◽  
Core Components

Abstract Background The production of methane by methanogens is dependent on numerous iron-sulfur (Fe-S) cluster proteins; yet, the machinery involved in Fe-S cluster biogenesis in methanogens remains largely unknown. Methanogen genomes encode uncharacterized homologs of the core components of the ISC (IscS and IscU) and SUF (SufBC) Fe-S cluster biogenesis systems found in bacteria and eukaryotes. Methanosarcina acetivorans contains three iscSU and two sufCB gene clusters. Here, we report genetic and biochemical characterization of M. acetivorans iscSU2. Results Purified IscS2 exhibited pyridoxal 5′- phosphate-dependent release of sulfur from L-cysteine. Incubation of purified IscU2 with IscS2, cysteine, and iron (Fe2+) resulted in the formation of [4Fe-4S] clusters in IscU2. IscU2 transferred a [4Fe-4S] cluster to purified M. acetivorans apo-aconitase. IscU2 also restored the aconitase activity in air-exposed M. acetivorans cell lysate. These biochemical results demonstrate that IscS2 is a cysteine desulfurase and that IscU2 is a Fe-S cluster scaffold. M. acetivorans strain DJL60 deleted of iscSU2 was generated to ascertain the in vivo importance of IscSU2. Strain DJL60 had Fe-S cluster content and growth similar to the parent strain but lower cysteine desulfurase activity. Strain DJL60 also had lower intracellular persulfide content compared to the parent strain when cysteine was an exogenous sulfur source, linking IscSU2 to sulfur metabolism. Conclusions This study establishes that M. acetivorans contains functional IscS and IscU, the core components of the ISC Fe-S cluster biogenesis system and provides the first evidence that ISC operates in methanogens.

scholarly journals Expanding magnetic organelle biogenesis in the domain Bacteria

Microbiome ◽  
2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Wei Lin ◽  
Wensi Zhang ◽  
Greig A. Paterson ◽  
Qiyun Zhu ◽  
Xiang Zhao ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Biological Significance ◽  
Gene Clusters ◽  
Magnetic Sensors ◽  
Evolutionary Origin ◽  
Phylogenomic Analysis ◽  
Organelle Biogenesis ◽  
Taxonomic Distribution ◽  
Origin And Evolution ◽  
Bacterial Phyla

Abstract Background The discovery of membrane-enclosed, metabolically functional organelles in Bacteria has transformed our understanding of the subcellular complexity of prokaryotic cells. Biomineralization of magnetic nanoparticles within magnetosomes by magnetotactic bacteria (MTB) is a fascinating example of prokaryotic organelles. Magnetosomes, as nano-sized magnetic sensors in MTB, facilitate cell navigation along the local geomagnetic field, a behaviour referred to as magnetotaxis or microbial magnetoreception. Recent discovery of novel MTB outside the traditionally recognized taxonomic lineages suggests that MTB diversity across the domain Bacteria are considerably underestimated, which limits understanding of the taxonomic distribution and evolutionary origin of magnetosome organelle biogenesis. Results Here, we perform the most comprehensive metagenomic analysis available of MTB communities and reconstruct metagenome-assembled MTB genomes from diverse ecosystems. Discovery of MTB in acidic peatland soils suggests widespread MTB occurrence in waterlogged soils in addition to subaqueous sediments and water bodies. A total of 168 MTB draft genomes have been reconstructed, which represent nearly a 3-fold increase over the number currently available and more than double the known MTB species at the genome level. Phylogenomic analysis reveals that these genomes belong to 13 Bacterial phyla, six of which were previously not known to include MTB. These findings indicate a much wider taxonomic distribution of magnetosome organelle biogenesis across the domain Bacteria than previously thought. Comparative genome analysis reveals a vast diversity of magnetosome gene clusters involved in magnetosomal biogenesis in terms of gene content and synteny residing in distinct taxonomic lineages. Phylogenetic analyses of core magnetosome proteins in this largest available and taxonomically diverse dataset support an unexpectedly early evolutionary origin of magnetosome biomineralization, likely ancestral to the origin of the domain Bacteria. Conclusions These findings expand the taxonomic and phylogenetic diversity of MTB across the domain Bacteria and shed new light on the origin and evolution of microbial magnetoreception. Potential biogenesis of the magnetosome organelle in the close descendants of the last bacterial common ancestor has important implications for our understanding of the evolutionary history of bacterial cellular complexity and emphasizes the biological significance of the magnetosome organelle.

scholarly journals Absynte: a web tool to analyze the evolution of orthologous archaeal and bacterial gene clusters

2011 ◽  
Vol 27 (20) ◽  
pp. 2905-2906 ◽  
Author(s):  
A. Despalins ◽  
S. Marsit ◽  
J. Oberto
Keyword(s):  
Gene Clusters ◽  
Web Tool ◽  
Bacterial Gene

scholarly journals The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans: Analysis and Expression of the Encoding Genes

2008 ◽  
Vol 191 (2) ◽  
pp. 588-599 ◽  
Author(s):  
Anke Schilhabel ◽  
Sandra Studenik ◽  
Martin Vödisch ◽  
Sandra Kreher ◽  
Bernhard Schlott ◽  
...  
Keyword(s):  
Amino Acid Sequence Identity ◽  
Protein Complexes ◽  
Gene Clusters ◽  
Single Copy ◽  
Anaerobic Microorganisms ◽  
Sequence Identity ◽  
Copy Gene ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Genetic Context

ABSTRACT Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A. dehalogenans, the genes for methyltransferase I, CP, and methyltransferase II of both O-demethylases are clustered. The single-copy gene for AE is not included in the O-demethylase gene clusters. It was found that AE grouped with COG3894 proteins, the function of which was unknown so far. Genes encoding COG3894 proteins with 20 to 41% amino acid sequence identity with AE are present in numerous genomes of anaerobic microorganisms. Inspection of the domain structure and genetic context of these orthologs predicts that these are also reductive activases for corrinoid enzymes (RACEs), such as carbon monoxide dehydrogenase/acetyl coenzyme A synthases or anaerobic methyltransferases. The genes encoding the O-demethylase components were heterologously expressed with a C-terminal Strep-tag in Escherichia coli, and the recombinant proteins methyltransferase I, CP, and AE were characterized. Gel shift experiments showed that the AE comigrated with the CP. The formation of other protein complexes with the O-demethylase components was not observed under the conditions used. The results point to a strong interaction of the AE with the CP. This is the first report on the functional heterologous expression of acetogenic phenyl methyl ether-cleaving O-demethylases.

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