scholarly journals Accelerated discovery of novel glycoside hydrolases using targeted functional profiling and selective pressure on the rumen microbiome

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
André L. A. Neves ◽  
Jiangkun Yu ◽  
Yutaka Suzuki ◽  
Marisol Baez-Magana ◽  
Elena Arutyunova ◽  
...  
Keyword(s):  
Selective Pressure ◽  
De Novo ◽  
Structural Diversity ◽  
Glycoside Hydrolases ◽  
Reference Database ◽  
Xylanolytic Activity ◽  
Rumen Microbiome ◽  
Functional Profiling ◽  
Microbial Environment ◽  
Living Organisms

Abstract Background Carbohydrate-active enzymes (CAZymes) form the most widespread and structurally diverse set of enzymes involved in the breakdown, biosynthesis, or modification of lignocellulose that can be found in living organisms. However, the structural diversity of CAZymes has rendered the targeted discovery of novel enzymes extremely challenging, as these proteins catalyze many different chemical reactions and are sourced by a vast array of microbes. Consequently, many uncharacterized members of CAZyme families of interest have been overlooked by current methodologies (e.g., metagenomic screening) used to discover lignocellulolytic enzymes. Results In the present study, we combined phenotype-based selective pressure on the rumen microbiota with targeted functional profiling to guide the discovery of unknown CAZymes. In this study, we found 61 families of glycoside hydrolases (GH) (out of 182 CAZymes) from protein sequences deposited in the CAZy database—currently associated with more than 20,324 microbial genomes. Phenotype-based selective pressure on the rumen microbiome showed that lignocellulolytic bacteria (e.g., Fibrobacter succinogenes, Butyrivibrio proteoclasticus) and three GH families (e.g., GH11, GH13, GH45) exhibited an increased relative abundance in the rumen of feed efficient cattle when compared to their inefficient counterparts. These results paved the way for the application of targeted functional profiling to screen members of the GH11 and GH45 families against a de novo protein reference database comprised of 1184 uncharacterized enzymes, which led to the identification of 18 putative xylanases (GH11) and three putative endoglucanases (GH45). The biochemical proof of the xylanolytic activity of the newly discovered enzyme validated the computational simulations and demonstrated the stability of the most abundant xylanase. Conclusions These findings contribute to the discovery of novel enzymes for the breakdown, biosynthesis, or modification of lignocellulose and demonstrate that the rumen microbiome is a source of promising enzyme candidates for the biotechnology industry. The combined approaches conceptualized in this study can be adapted to any microbial environment, provided that the targeted microbiome is easy to manipulate and facilitates enrichment for the microbes of interest.

PSIX-3 Comparison of 16S rRNA gene profiles of rumen microbiome from Raramuri Criollo, European and Criollo x European lineages

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 441-442
Author(s):  
Adrian Maynez-Perez ◽  
Francisco Jahuey-Martinez ◽  
Jose A Martinez-Quintana ◽  
Michael E Hume ◽  
Robin C Anderson ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Beta Diversity ◽  
Chihuahuan Desert ◽  
Reference Database ◽  
Rrna Gene ◽  
Linear Discriminant ◽  
Microbiome Composition ◽  
Northern Mexico ◽  
Rumen Microbiome

Abstract Raramuri Criollo cattle from the Chihuahuan desert in northern Mexico have been described as an ecological ecotype due to their enormous advantage in land grass utilization and their capacity to diversify their diet with cacti, forbs and woody plants. This diversification in diet utilization, could reflect upon their microbiome composition. The aim of this study was to characterize the rumen microbiome of Raramuri criollo cattle and to compare it to other lineages that graze in the same area. A total of 28 cows representing three linages [Criollo (n = 13), European (n = 9) and Criollo x European Crossbred (n = 6)] were grazed without supplementation for 45 days. DNA was extracted from ruminal samples and the V4 region of the 16S rRNA gene was sequenced on an Illumina platform. Data were analyzed with the QIIME2 software package and DADA2 plugin and the amplicon sequence variants were taxonomically classified with naïve Bayesian using the SILVA 16S rRNA gene reference database (version 132). Statistical analysis was performed by ANOVA and PERMANOVA for alpha and beta diversity indexes, respectively, and the non-strict version of linear discriminant analysis effect size (LEfSe) was used to determine significantly different taxa among lineages. Differences in beta diversity indexes (P < 0.05) were found in ruminal microbiome composition between Criollo and European groups, whereas the Crossbred showed intermediate values when compared to the pure breeds (Table 1). LEfSe analysis identified a total of 20 bacterial groups that explained differences between lineages, including one for Crossbreed, ten for European and nine for Criollo. These results show ruminal microbiome differences between Raramuri criollo cattle and the mainstream European breeds used in the northern Mexico Chihuahuan desert and reflect that those differences could be a consequence of dissimilar grazing behavior.

scholarly journals Draft genome assembly data of Anoxybacillus sp. strain MB8 isolated from Tattapani hot springs, India

2021 ◽  
Author(s):  
VISHNU PRASOODANAN P K ◽  
Shruti S. Menon ◽  
Rituja Saxena ◽  
Prashant Waiker ◽  
Vineet K Sharma
Keyword(s):  
Hot Springs ◽  
De Novo ◽  
Draft Genome ◽  
Gc Content ◽  
Central India ◽  
Glycoside Hydrolases ◽  
Rrna Gene ◽  
Aerobic Bacterium ◽  
Protein Coding ◽  
Protein Coding Genes

Discovery of novel thermophiles has shown promising applications in the field of biotechnology. Due to their thermal stability, they can survive the harsh processes in the industries, which make them important to be characterized and studied. Members of Anoxybacillus are alkaline tolerant thermophiles and have been extensively isolated from manure, dairy-processed plants, and geothermal hot springs. This article reports the assembled data of an aerobic bacterium Anoxybacillus sp. strain MB8, isolated from the Tattapani hot springs in Central India, where the 16S rRNA gene shares an identity of 97% (99% coverage) with Anoxybacillus kamchatkensis strain G10. The de novo assembly and annotation performed on the genome of Anoxybacillus sp. strain MB8 comprises of 2,898,780 bp (in 190 contigs) with a GC content of 41.8% and includes 2,976 protein-coding genes,1 rRNA operon, 73 tRNAs, 1 tm-RNA and 10 CRISPR arrays. The predicted protein-coding genes have been classified into 21 eggNOG categories. The KEGG Automated Annotation Server (KAAS) analysis indicated the presence of assimilatory sulfate reduction pathway, nitrate reducing pathway, and genes for glycoside hydrolases (GHs) and glycoside transferase (GTs). GHs and GTs hold widespread applications, in the baking and food industry for bread manufacturing, and in the paper, detergent and cosmetic industry. Hence, Anoxybacillus sp. strain MB8 holds the potential to be screened and characterized for such commercially relevant enzymes.

scholarly journals Engineering the substrate binding site of the hyperthermostable archaeal endo-β-1,4-galactanase from Ignisphaera aggregans

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Sebastian J. Muderspach ◽  
Folmer Fredslund ◽  
Verena Volf ◽  
Jens-Christian Navarro Poulsen ◽  
Thomas H. Blicher ◽  
...  
Keyword(s):  
Binding Site ◽  
Plant Cell Wall ◽  
Biological Diversity ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Plant Defence ◽  
Structural Diversity ◽  
Industrial Applications ◽  
Glycoside Hydrolases ◽  
Substrate Binding Site

Abstract Background Endo-β-1,4-galactanases are glycoside hydrolases (GH) from the GH53 family belonging to the largest clan of GHs, clan GH-A. GHs are ubiquitous and involved in a myriad of biological functions as well as being widely used industrially. Endo-β-1,4-galactanases, in particular hydrolyse galactan and arabinogalactan in pectin, a major component of the primary plant cell wall, with important functions in plant defence and application in the food and other industries. Here, we explore the family’s biological diversity by characterizing the first archaeal and hyperthermophilic GH53 galactanase, and utilize it as a scaffold for engineering enzymes with different product lengths. Results A galactanase gene was identified in the genome of the anaerobic hyperthermophilic archaeon Ignisphaera aggregans, and the isolated catalytic domain expressed and characterized (IaGal). IaGal presents the typical (βα)8 barrel structure of clan GH-A enzymes, with catalytic carboxylates at the end of the 4th and 7th barrel strands. Its activity optimum of at least 95 °C and melting point over 100 °C indicate extreme thermostability, a very advantageous property for industrial applications. If enzyme depletion is reduced, so is the need for re-addition, and thus costs. The main stabilizing features of IaGal compared to other structurally characterized members are π–π and cation–π interactions. The length of the substrate binding site—and thus produced oligosaccharide products—is intermediate compared to previously characterized galactanases. Variants inspired by the structural diversity in the GH53 family were rationally designed to shorten or extend the substrate binding groove, in order to modulate product length. Subsite-deleted variants produced shorter products than IaGal, as do the fungal galactanases inspiring the design. IaGal variants engineered with a longer binding site produced a less expected degradation pattern, though still different from that of wild-type IaGal. All variants remained extremely stable. Conclusions We have characterized in detail the most thermophilic endo-β-1,4-galactanase known to date and successfully engineered it to modify the degradation profile, while maintaining much of its desirable thermostability. This is an important achievement as oligosaccharide products length is an important property for industrial and natural GHs alike.

scholarly journals Chemoenzymatic elaboration of the Raper–Mason pathway unravels the structural diversity within eumelanin pigments

2020 ◽  
Vol 11 (30) ◽  
pp. 7836-7841 ◽  
Author(s):  
Qing Zhe Ni ◽  
Brianna N. Sierra ◽  
James J. La Clair ◽  
Michael D. Burkart
Keyword(s):  
Molecular Structure ◽  
Structural Diversity ◽  
Living Organisms

Melanin is a central polymer in living organisms, yet our understanding of its molecular structure remains unresolved.

scholarly journals Robust taxonomic classification of uncharted microbial sequences and bins with CAT and BAT

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
F. A. Bastiaan von Meijenfeldt ◽  
Ksenia Arkhipova ◽  
Diego D. Cambuy ◽  
Felipe H. Coutinho ◽  
Bas E. Dutilh
Keyword(s):  
Dna Sequences ◽  
De Novo ◽  
Taxonomic Classification ◽  
Classification Method ◽  
Reference Database ◽  
Annotation Tool ◽  
Multiple Signals

Abstract Current-day metagenomics analyses increasingly involve de novo taxonomic classification of long DNA sequences and metagenome-assembled genomes. Here, we show that the conventional best-hit approach often leads to classifications that are too specific, especially when the sequences represent novel deep lineages. We present a classification method that integrates multiple signals to classify sequences (Contig Annotation Tool, CAT) and metagenome-assembled genomes (Bin Annotation Tool, BAT). Classifications are automatically made at low taxonomic ranks if closely related organisms are present in the reference database and at higher ranks otherwise. The result is a high classification precision even for sequences from considerably unknown organisms.

scholarly journals Further Comments on the Causation of Malignant Disease

1928 ◽  
Vol 28 (1) ◽  
pp. 9-32
Author(s):  
Arthur Eastwood
Keyword(s):  
Latent Period ◽  
Malignant Disease ◽  
De Novo ◽  
Animal Cell ◽  
Systemic Resistance ◽  
Natural Immunity ◽  
Active Growth ◽  
Animal Body ◽  
Chronic Irritation ◽  
Living Organisms

Invisible infective agents may be divided into: (1) true, ultramicroscopic, living viruses, which do not arise de novo and, so far as is known, are not ubiquitous; (2) transmissible infective agents which arise de novo and are propagated through living cells, but are not themselves living organisms; (3) stimulants to variation which arise de novo, are not transmissible, and are not living organisms.Class (1) is not represented in malignant disease. “Bacteriophage” is a representative of class (2); very probably the infective agent of fowl sarcoma comes under the same category, and possibly some important human diseases of doubtful aetiology. There is no satisfactory evidence that mammalian malignant disease is related to class (2); its causation, according to the “chronic irritation” theory, must be attributed to influences comprised within class (3).The stimulants to variation in class (3) depend for their effectiveness upon the unstable energy of living matter. The changes which they produce are “biological” in the sense that they are changes of chemical constitution which could not be obtained without the aid of vital processes.Regulation of normal growth in the animal body means regulation of the cell's facilities for obtaining energy. I think it is misleading to regard it as a forceful restraint (or stimulus) upon the cell's inherent capacity for unlimited growth.The assumption, borrowed from “natural immunity” towards bacteria, that there is in the animal body a natural principle which destroys frequently occurring foci of incipient malignancy is also unsubstantiated and misleading.During the latent period, certain cells, which subsequently grow into a neoplasm, lose their capacity to respond to inhibitory systemic influences. This change is brought about by local and not by systemic causes.As regards the special class of tumour derived from cells which have been displaced in foetal life, long residence in an abnormal situation does not appear to be equivalent to the ordinary latent period; but it may have had the effect of increasing their susceptibility, so that, if exposed to chronic irritation, the cells would more readily lapse into the latent period predisposing to malignancy.It is known that the various tissues of the animal body differ in their degree of susceptibility to the precancerous change. This is a cellular characteristic; so also is the difference in the susceptibility of one animal as compared with another. It is not a question of difference in a hypothetical humoral property of “systemic resistance.”On the termination of the latent period by a fresh stimulus to proliferation, certain cells commence active growth and are incapable of responding to systemic inhibitory influences. These conditions seem sufficient for the origin of an innocent neoplasm. But something more is required to explain malignancy, because the malignant cell is essentially different from the cells in a benign tumour.About the actual cause of the change to malignancy one can only offer conjectures. I have suggested a way in which the change may possibly be produced through the agency of the local endothelium and the autogenous formation of antibodies.On taking a broad view, the change into the malignant variant is not something unique; equally remarkable changes are to be found in the properties of bacteria. In both cases the facts have to be accepted, at present, without satisfactory explanation of the conditions which gave rise to them. One finds with bacteria that degradation or “roughness” may be a phase preparatory to the acquirement of new properties, just as the degradation of cells in the latent period seems to be a requisite preparation for acquiring the new property of malignancy. But the actual steps involved in the change from a bacterial saprophyte to an invasive parasite are as difficult to understand as are the processes involved in the conversion of a normal animal cell into its malignant variant.Throughout the study of cancer it is very desirable to maintain a clear distinction between cause and effect. For example, the enzymes peculiar to cancer are not the cause of cancer but the effect of the biological change which produced the cancerous cell.

scholarly journals Genotyping and De Novo Discovery of Allelic Variants at the Brassicaceae Self-Incompatibility Locus from Short-Read Sequencing Data

2019 ◽  
Vol 37 (4) ◽  
pp. 1193-1201 ◽  
Author(s):  
Mathieu Genete ◽  
Vincent Castric ◽  
Xavier Vekemans
Keyword(s):  
De Novo ◽  
Balancing Selection ◽  
Methodological Approach ◽  
Natural Populations ◽  
Sequence Divergence ◽  
Reference Database ◽  
Self Incompatibility ◽  
Sequencing Data ◽  
Allelic Series ◽  
S Alleles

Abstract Plant self-incompatibility (SI) is a genetic system that prevents selfing and enforces outcrossing. Because of strong balancing selection, the genes encoding SI are predicted to maintain extraordinarily high levels of polymorphism, both in terms of the number of functionally distinct S-alleles that segregate in SI species and in terms of their nucleotide sequence divergence. However, because of these two combined features, documenting polymorphism of these genes also presents important methodological challenges that have so far largely prevented the comprehensive analysis of complete allelic series in natural populations, and also precluded the obtention of complete genic sequences for many S-alleles. Here, we develop a powerful methodological approach based on a computationally optimized comparison of short Illumina sequencing reads from genomic DNA to a database of known nucleotide sequences of the extracellular domain of SRK (eSRK). By examining mapping patterns along the reference sequences, we obtain highly reliable predictions of S-genotypes from individuals collected from natural populations of Arabidopsis halleri. Furthermore, using a de novo assembly approach of the filtered short reads, we obtain full-length sequences of eSRK even when the initial sequence in the database was only partial, and we discover putative new SRK alleles that were not initially present in the database. When including those new alleles in the reference database, we were able to resolve the complete diploid SI genotypes of all individuals. Beyond the specific case of Brassicaceae S-alleles, our approach can be readily applied to other polymorphic loci, given reference allelic sequences are available.

scholarly journals Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases

2009 ◽  
Vol 106 (6) ◽  
pp. 1948-1953 ◽  
Author(s):  
Jennifer M. Brulc ◽  
Dionysios A. Antonopoulos ◽  
Margret E. Berg Miller ◽  
Melissa K. Wilson ◽  
Anthony C. Yannarell ◽  
...  
Keyword(s):  
Glycoside Hydrolases ◽  
Bovine Rumen ◽  
Rumen Microbiome

scholarly journals CD-HIT-OTU-MiSeq, an Improved Approach for Clustering and Analyzing Paired End MiSeq 16S rRNA Sequences

2017 ◽  
Author(s):  
Weizhong Li ◽  
Yuanyuan Chang
Keyword(s):  
16S Rrna ◽  
High Speed ◽  
De Novo ◽  
Sequence Data ◽  
Illumina Miseq ◽  
Poor Quality ◽  
Reference Database ◽  
Rrna Gene ◽  
Variable Regions ◽  
Novel Approach

AbstractIn recent years, Illumina MiSeq sequencers replaced pyrosequencing platforms and became dominant in 16S rRNA sequencing. One unique feature of MiSeq technology, compared with Pyrosequencing, is the Paired End (PE) reads, with each read can be sequenced to 250-300 bases to cover multiple variable regions on the 16S rRNA gene. However, the PE reads need to be assembled into a single contig at the beginning of the analysis. Although there are many methods capable of assembling PE reads into contigs, a big portion of PE reads can not be accurately assembled because the poor quality at the 3’ ends of both PE reads in the overlapping region. This causes that many sequences are discarded in the analysis. In this study, we developed a novel approach for clustering and annotation MiSeq-based 16S sequence data, CD-HIT-OTU-MiSeq. This new approach has four distinct novel features. (1) The package can clustering PE reads without joining them into contigs. (2) Users can choose a high quality portion of the PE reads for analysis (e.g. first 200 / 150 bases from forward / reverse reads), according to base quality profile. (3) We implemented a tool that can splice out the target region (e.g. V3-V4) from a full-length 16S reference database into the PE sequences. CD-HIT-OTU-MiSeq can cluster the spliced PE reference database together with samples, so we can derive Operational Taxonomic Units (OTUs) and annotate these OTUs concurrently. (4) Chimeric sequences are effectively identified through de novo approach. The package offers high speed and high accuracy. The software package is freely available as open source package and is distributed along with CD-HIT from http://cd-hit.org. Within the CD-HIT package, CD-HIT-OTU-MiSeq is within the usecase folder.

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