Microbial and Carbohydrate Active Enzyme profile of buffalo rumen metagenome and their alteration in response to variation in the diet

Rumen microbiome represents rich source of enzymes degrading complex plant polysaccharides. We describe here analysis of Carbohydrate Active Enzymes (CAZymes) from 3.5 gigabase sequences of metagenomic data from rumen samples of Mehsani buffaloes fed on different proportions of green or dry roughages to concentrate ration. A total of 2597 contigs encoding putative CAZymes were identified by CAZyme Analysis Toolkit (CAT). The phylogenetic analysis of these contigs by MG-RAST revealed predominance of Bacteroidetes, followed by Firmicutes, Proteobacteria, and Actinobacteria phyla. Moreover, a higher abundance of oligosaccharide degrading and debranching enzymes in buffalo rumen metagenome and that of cellulases and hemicellulases in termite hindgut was observed when we compared glycoside hydrolase (GH) profile of buffalo rumen metagenome with cow rumen, termite hindgut and chicken caecum metagenome. Further, comparison of microbial profile of green or dry roughage fed animals showed significantly higher abundance (p-value < 0.05) of various polysaccharide degrading bacterial genera like Fibrobacter, Prevotella, Bacteroides, Clostridium and Ruminococcus in green roughage fed animals. In addition, we found a significantly higher abundance (p-value < 0.05) of enzymes associated with pectin digestion such as pectin lyase (PL) 1, PL10 and GH28 in green roughage fed animals. Our study outlines CAZyme profile of buffalo rumen metagenome and provides a scope to study the role of abundant enzyme families (oligosaccharide degrading and debranching enzymes) in digestion of coarse feed.     

瘤胃中木质纤维素降解菌及降解酶基因的研究进展

摘要：反刍动物瘤胃是公认的木质纤维素高效降解的天然反应器,对瘤胃微生物的研究成为开发生物能源的热点领域之一。其研究手段已经从传统的依赖分离培养从瘤胃中获得木质纤维素降解菌,并对降解菌中的木质纤维素降解酶逐一分析,发展到通过基因组/元基因组技术,直接从瘤胃中发现获得大量新的木质纤维素降解酶基因/基因簇,进而探讨其降解的分子机理。已有的研究结果表明,瘤胃微生物降解木质纤维素的过程非常复杂,其中涉及到大量不同种类的微生物、酶及基因/基因簇,随着新分析技术的建立和完善,对这些微生物、酶和基因的研究已取得了诸多进展。本论文综述报道了近期有关该方向的研究进展。     

基于16S rRNA高通量测序技术分析安格斯牛瘤胃微生物多样性和功能预测的研究

本研究旨在系统探索和分析安格斯牛瘤胃微生物多样性及其基因功能。选用体重550 kg左右的安格斯牛6头分成2组,利用基于16S rRNA高通量测序技术检测牛瘤胃液样本进行组学分析。结果表明,2组样本通过Illumina Miseq测序平台共获得86 298条高质量优质序列,聚类为346个操作分类单元(OUT),经分类学鉴定分属13个门,21个纲,24个目,40个科,123个属。拟杆菌门(Bacteroidetes)43.16%和厚壁菌门(Firmicutes)36.29%为优势菌群。基于属的组成,依次为普雷沃氏菌属_7 (Prevotella_7) 29.28%、琥珀酸弧菌科_UCG-001 (Succinivibrionaceae_UCG-001)11.30%、琥珀酸菌属(Succiniclasticum) 11.10%、普雷沃氏菌属_1 (Prevotella_1) 6.65%、瘤胃球菌属_1(Ruminococcus_1) 5.17%、琥珀酸弧菌属(Succinivibrio) 2.75%等。16S功能预测和COG、KEGG代谢通路数据库对比发现,功能集中在氨基酸运输和代谢、碳水化合物转运及代谢的相关基因上,可能含有丰富的蛋白分解、转运及代谢酶相关基因和大量的纤维素以及木质素降解酶基因。经碳水化合物活性酶(CAZymes)注释和KEGG代谢酶数据库比对显示,预测的功能基因糖苷水解酶和糖基转移酶所占比例较高;产乙酸和丁酸相关酶基因丰度较高。安格斯牛瘤胃内含有丰富的蛋白质分解、木质纤维素降解和挥发性脂肪酸生成菌群及酶系,为展示瘤胃微生物多样性,发现和筛选新的功能酶基因提供参考。     

Ribosomal ITS1 sequence-based diversity analysis of anaerobic rumen fungi in cattle fed on high fiber diet

Fiber degradation in the ruminant digestive process is a major activity accomplished by rumen microbes, a process in which the role of fungi is important. Therefore, the present study was conducted to establish the community structure of anaerobic rumen fungi in cattle fed on a high fiber diet using molecular approaches. Total community DNA was extracted, and the ribosomal internal transcribed spacer (ITS) 1 region was amplified, cloned, and sequenced. The resulting nucleotide sequences were used to construct a phylogenetic tree. A total of 52 clones were analyzed, revealing 31 different ITS1 gene phylotypes. Of these, 12 belonged to the genus Orpinomyces (48 % of clones), followed by uncultured Neocallimastigale clones (29 %), Cyllamyces spp. (9 %) and Anaeromyces spp. (8 %). Our results indicate that genus Orpinomyces dominates the rumen fungal community in Indian crossbred Karan Fries cattle.     

Carbohydrate active enzyme domains from extreme thermophiles: components of a modular toolbox for lignocellulose degradation

Lignocellulosic biomass is a promising feedstock for the manufacture of biodegradable and renewable bioproducts. However, the complex lignocellulosic polymeric structure of woody tissue is difficult to access without extensive industrial pre-treatment. Enzyme processing of partly depolymerised biomass is an established technology, and there is evidence that high temperature (extremely thermophilic) lignocellulose degrading enzymes [carbohydrate active enzymes (CAZymes)] may enhance processing efficiency. However, wild-type thermophilic CAZymes will not necessarily be functionally optimal under industrial pre-treatment conditions. With recent advances in synthetic biology, it is now potentially possible to build CAZyme constructs from individual protein domains, tailored to the conditions of specific industrial processes. In this review, we identify a ‘toolbox’ of thermostable CAZyme domains from extremely thermophilic organisms and highlight recent advances in CAZyme engineering which will allow for the rational design of CAZymes tailored to specific aspects of lignocellulose digestion.

     

Proteogenomic Analysis of a Thermophilic Bacterial Consortium Adapted to Deconstruct Switchgrass

Thermophilic bacteria are a potential source of enzymes for the deconstruction of lignocellulosic biomass. However, the complement of proteins used to deconstruct biomass and the specific roles of different microbial groups in thermophilic biomass deconstruction are not well-explored. Here we report on the metagenomic and proteogenomic analyses of a compost-derived bacterial consortium adapted to switchgrass at elevated temperature with high levels of glycoside hydrolase activities. Near-complete genomes were reconstructed for the most abundant populations, which included composite genomes for populations closely related to sequenced strains of Thermus thermophilus and Rhodothermus marinus, and for novel populations that are related to thermophilic Paenibacilli and an uncultivated subdivision of the little-studied Gemmatimonadetes phylum. Partial genomes were also reconstructed for a number of lower abundance thermophilic Chloroflexi populations. Identification of genes for lignocellulose processing and metabolic reconstructions suggested Rhodothermus, Paenibacillus and Gemmatimonadetes as key groups for deconstructing biomass, and Thermus as a group that may primarily metabolize low molecular weight compounds. Mass spectrometry-based proteomic analysis of the consortium was used to identify >3000 proteins in fractionated samples from the cultures, and confirmed the importance of Paenibacillus and Gemmatimonadetes to biomass deconstruction. These studies also indicate that there are unexplored proteins with important roles in bacterial lignocellulose deconstruction.     

Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library

Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose-degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.     

Metagenomic insights into the fibrolytic microbiome in yak rumen

The rumen hosts one of the most efficient microbial systems for degrading plant cell walls, yet the predominant cellulolytic proteins and fibrolytic mechanism(s) remain elusive. Here we investigated the cellulolytic microbiome of the yak rumen by using a combination of metagenome-based and bacterial artificial chromosome (BAC)-based functional screening approaches. Totally 223 fibrolytic BAC clones were pyrosequenced and 10,070 ORFs were identified. Among them 150 were annotated as the glycoside hydrolase (GH) genes for fibrolytic proteins, and the majority (69%) of them were clustered or linked with genes encoding related functions. Among the 35 fibrolytic contigs of >10 Kb in length, 25 were derived from Bacteroidetes and four from Firmicutes. Coverage analysis indicated that the fibrolytic genes on most Bacteroidetes-contigs were abundantly represented in the metagenomic sequences, and they were frequently linked with genes encoding SusC/SusD-type outer-membrane proteins. GH5, GH9, and GH10 cellulase/hemicellulase genes were predominant, but no GH48 exocellulase gene was found. Most (85%) of the cellulase and hemicellulase proteins possessed a signal peptide; only a few carried carbohydrate-binding modules, and no cellulosomal domains were detected. These findings suggest that the SucC/SucD-involving mechanism, instead of one based on cellulosomes or the free-enzyme system, serves a major role in lignocellulose degradation in yak rumen. Genes encoding an endoglucanase of a novel GH5 subfamily occurred frequently in the metagenome, and the recombinant proteins encoded by the genes displayed moderate Avicelase in addition to endoglucanase activities, suggesting their important contribution to lignocellulose degradation in the exocellulase-scarce rumen.     

Metagenomics of the Svalbard reindeer rumen microbiome reveals abundance of polysaccharide utilization loci

Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus). Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1). Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs) as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.     

Comprehensive Analysis of Carbohydrate-Active Enzymes from the Filamentous Fungus <Emphasis Type="Italic">Scytalidium candidum</Emphasis> 3C

Complete enzymatic degradation of plant polysaccharides is a result of combined action of various carbohydrate-active enzymes (CAZymes). In this paper, we demonstrate the potential of the filamentous fungus Scytalidium candidum 3C for processing of plant biomass. Structural annotation of the improved assembly of S. candidum 3C genome and functional annotation of CAZymes revealed putative gene sequences encoding such proteins. A total of 190 CAZyme-encoding genes were identified, including 104 glycoside hydrolases, 52 glycosyltransferases, 28 oxidative enzymes, and 6 carbohydrate esterases. In addition, 14 carbohydrate-binding modules were found. Glycoside hydrolases secreted during the growth of S. candidum 3C in three media were analyzed with a variety of substrates. Mass spectrometry analysis of the fungal culture liquid revealed the presence of peptides identical to 36 glycoside hydrolases, three proteins without known enzymatic function belonging to the same group of families, and 11 oxidative enzymes. The activity of endohemicellulases was determined using specially synthesized substrates in which the glycosidic bond between monosaccharide residues was replaced by a thiolinkage. During analysis of the CAZyme profile of S. candidum 3C, four β-xylanases from the GH10 family and two β-glucanases from the GH7 and GH55 families were detected, partially purified, and identified.     

Influence of Diet Composition on Cattle Rumen Methanogenesis: A Comparative Metagenomic Analysis in Indian and Exotic Cattle

Comparative metagenomics approach has been used in this study to discriminate colonization of methanogenic population in different breeds of cattle. We compared two Indian cattle breeds (Gir and Kankrej) and two exotic cattle (Holstein and Jersey) breeds. Using a defined dietary plan for selected Indian varieties, the diet dependent shifts in microbial community and abundance of the enzymes associated with methanogenesis were studied. This data has been compared with the available rumen metagenome data from Holstein and Jersey dairy cattle. The abundance of genes for methanogenesis in Holstein and Jersey cattle came from Methanobacteriales order whereas, majority of the enzymes for methanogenesis in Gir and Kankrej cattle came from Methanomicrobiales order. The study suggested that by using slow/less digestible feed, the propionate levels could be controlled in rumen; and in turn, this would also help in further reducing the hydrogenotrophic production of methane. The study proposes that with the designed diet plan the overall methanogenic microbial pool or the individual methanogens could be targeted for development of methane mitigation strategies.     

厌氧真菌纤维降解酶及其应用潜力的研究进展

反刍动物的瘤胃是已知的纤维降解能力最强的天然发酵罐,其发酵粗纤维的能力很大程度上依赖于其中栖息的各类微生物的功能。厌氧真菌作为瘤胃内的一类低丰度菌群,最先定殖到宿主动物摄入的纤维质饲料上,并通过分泌大量高效的碳水化合物活性酶降解粗纤维。然而,由于缺乏足够的基因组信息和有效的厌氧真菌遗传操作系统,目前国内外对厌氧真菌分泌的纤维降解酶及其降解机制的研究进展有限。本文对厌氧真菌的分类及已发表的基因组信息进行概述,介绍了各类纤维降解酶及纤维小体的组成结构和催化机制特点,并对纤维降解酶在生物质能、饲料处理、纺织造纸及食品加工等方面的应用进行总结。研究厌氧真菌纤维降解酶的作用特性,将有助于完善其在瘤胃环境中有效竞争资源并降解粗纤维的知识体系,也将进一步了解其生物技术应用潜力,为工业生产中应用酶制剂提供新思路。     

Characterization of the rumen microbiome of Indian Kankrej cattle (Bos indicus) adapted to different forage diet

Present study described rumen microbiome of Indian cattle (Kankrej breed) to better understand the microbial diversity and largely unknown functional capacity of the rumen microbiome under different dietary treatments. Kankrej cattle were gradually adapted to a high-forage diet (four animals with dry forage and four with green forage) containing 50 % (K1), 75 % (K2) to 100 % (K3) forage, and remaining concentrate diet, each for 6 weeks followed by analysis of rumen fiber adherent and fiber-free metagenomic community by shotgun sequencing using ion torrent PGM platform and EBI-metagenomics annotation pipeline. Taxonomic analysis indicated that rumen microbiome was dominated by Bacteroidetes followed by Firmicutes, Fibrobacter, Proteobacteria, and Tenericutes. Functional analysis based on gene ontology classified all reads in total 157 categories based on their functional role in biological, molecular, and cellular component with abundance of genes associated with hydrolase activity, membrane, transport, transferase, and different metabolism (such as carbohydrate and protein). Statistical analysis using STAMP revealed significant differences (P?相似文献   

植物基因工程改善生物质能利用的研究进展

利用植物木质纤维资源发酵生产乙醇越来越受到人们的重视,但是要实现工业化生产仍然存在很多难题。最近,利用植物基因工程技术,改善植物自身性状,包括减少植物自身细胞壁中木质素含量、细胞中积累表达纤维素酶和木聚糖酶等方法,使自生产生的生物质更利于降解利用。目前,对这种新的能源转基因植物的研究取得了一定进展。     

Metagenomic analysis of virulence-associated and antibiotic resistance genes of microbes in rumen of Indian buffalo (Bubalus bubalis)

A major research goal in rumen microbial ecology is to understand the relationship between community composition and its function, particularly involved in fermentation process is of a potential interest. The buffalo rumen microbiota impacts human food safety as well as animal health. Although the bacteria of bovine rumen have been well characterized, techniques have been lacking to correlate total community structure with gene function. We applied 454 next generations sequencing technology to characterize general microbial diversity present in buffalo rumen metagenome and also identified the repertoire of microbial genes present, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that over six percent (6.44%) of the sequences from our buffalo rumen pool sample could be categorized as virulence genes and genes associated with resistance to antibiotic and toxic compounds (RATC), which is a higher proportion of virulence genes reported from metagenome samples of chicken cecum (5.39%), cow rumen (4.43%) and Sargasso sea (2.95%). However, it was lower than the proportion found in cow milk (11.33%) cattle faeces (8.4%), Antarctic marine derived lake (8.45%), human fecal (7.7%) and farm soil (7.79%). The dynamic nature of metagenomic data, together with the large number of RATC classes observed in samples from widely different ecologies indicates that metagenomic data can be used to track potential targets and relative amounts of antibiotic resistance genes in individual animals. In addition, these data can be also used to generate antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats.     

基因工程改善植物生物质能利用的研究进展

利用植物木质纤维资源发酵产乙醇越来越受到人们的重视,但是要达到工业生产仍然存在很多难题。最近在利用植物基因工程技术改善植物自身性状,以利于能源植物的研究方面取得了一定的进展,这些研究包括减少植物自身细胞壁中的木质素含量、细胞中积累表达纤维素酶和木聚耱酶等的方法,使产生的生物质更利于降解利用。