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Metatranscriptomics of the Hu sheep rumen microbiome reveals novel cellulases.

ABSTRACT: Background:Cellulosic biomass has great potential as a renewable biofuel resource. Robust, high-performance enzymes are needed to effectively utilize this valuable resource. In this study, metatranscriptomics was used to explore the carbohydrate-active enzymes (CAZymes), especially glycoside hydrolases (GHs), present in the rumen microbiome of Hu sheep. Select CAZymes were experimentally verified and characterized after cloning and expression in E. coli. Results:The metatranscriptomes of six Hu sheep rumen microbiomes yielded 42.3 Gbp of quality-checked sequence data that represented in total 2,380,783 unigenes after de novo assembling using Trinity and clustered with CD-HIT-EST. Annotation using the CAZy database revealed that 2.65% of the unigenes encoded GHs, which were assigned to 111 different CAZymes families. Firmicutes (18.7%) and Bacteroidetes (13.8%) were the major phyla to which the unigenes were taxonomically assigned. In total, 14,489 unigenes were annotated to 15 cellulase-containing GH families, with GH3, GH5 and GH9 being the predominant. From these putative cellulase-encoding unigenes, 4225 open reading frames (ORFs) were predicted to contain 2151 potential cellulase catalytic modules. Additionally, 147 ORFs were found to encode proteins that contain carbohydrate-binding modules (CBMs). Heterogeneous expression of 30 candidate cDNAs from the GH5 family in E. coli BL21 showed that 17 of the tested proteins had endoglucanase activity, while 7 exhibited exoglucanase activity. Interestingly, two of the GH5 proteins (Cel5A-h28 and Cel5A-h11) showed high specific activity against carboxymethylcellulose (CMC) and p-nitrophenyl-?-d-cellobioside (pNPC) (222.2 and 142.8 U/mg), respectively. The optimal pH value for activity of Cel5A-h11 and Cel5A-h28 was 6.0 for both enzymes, and optimal temperatures were 40 and 50 °C, respectively. Both enzymes retained over 70 and 60%, respectively, of their original activities after incubation at 40 °C for 60 min. However, their activities were rapidly diminished upon exposure to higher temperatures. Cel5A-h11 and Cel5A-h28 retained more than 80 and 60% of their maximal enzymatic activities after incubation for 16 h in buffered solutions in the pH range from 4.0 to 9.0. Conclusion:The metatranscriptomic results revealed that the rumen microbiome of Hu sheep encoded a repertoire of new enzymes capable of cellulose degradation and metatranscriptomics was an effective method to discover novel cellulases for biotechnological applications.

SUBMITTER: He B

PROVIDER: S-EPMC6587244 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Metatranscriptomics of the Hu sheep rumen microbiome reveals novel cellulases.

He Bo B Jin Shuwen S Cao Jiawen J Mi Lan L Wang Jiakun J

Biotechnology for biofuels 20190620

<h4>Background</h4>Cellulosic biomass has great potential as a renewable biofuel resource. Robust, high-performance enzymes are needed to effectively utilize this valuable resource. In this study, metatranscriptomics was used to explore the carbohydrate-active enzymes (CAZymes), especially glycoside hydrolases (GHs), present in the rumen microbiome of Hu sheep. Select CAZymes were experimentally verified and characterized after cloning and expression in <i>E. coli</i>.<h4>Results</h4>The metatra ...[more]

PMID: 31249617

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Project description:Ruminant livestock is a major source of the potent greenhouse gas methane. The complex rumen microbiome, consisting of bacteria, archaea, and microbial eukaryotes, facilitates anaerobic plant biomass degradation in the cow rumen, leading to methane emissions. Using an integrated approach combining multidomain quantitative metatranscriptomics with gas and volatile fatty acid (VFA) profiling, we aimed at obtaining the most comprehensive picture of the active rumen microbiome during feed degradation to date. Bacterial, archaeal, and eukaryotic biomass, but also methane emissions and VFA concentrations, increased drastically within an hour after feed intake. mRNA profiling revealed a dynamic response of carbohydrate-active enzyme transcripts, transcripts involved in VFA production and methanogenesis. While the relative abundances of functional transcripts did not mirror observed processes, such as methane emissions, transformation to mRNA abundance per gram of rumen fluid echoed ruminant processes. The microbiome composition was highly individual, with, e.g., ciliate, Neocallimastigaceae, Prevotellaceae, Succinivibrionaceae, and Fibrobacteraceae abundances differing between cows. Microbiome individuality was accompanied by inter- and intradomain multifunctional redundancy among microbiome members during feed degradation. This likely enabled the robust performance of the anaerobic degradation process in each rumen. Neocallimastigaceae and ciliates contributed an unexpectedly large share of transcripts for cellulose- and hemicellulose-degrading enzymes, respectively. Methyl-reducing but not CO2-reducing methanogens were positively correlated with methane emissions. While Methanomassiliicoccales switched from methanol to methylamines as electron acceptors, Methanosphaera became the dominating methanol-reducing methanogen. This study for the first time linked rumen meta-omics with processes and enabled holistic insights into the contribution of all microbiome members to feed degradation. IMPORTANCE Ruminant animals, such as cows, live in a tight symbiotic association with microorganisms, allowing them to feed on otherwise indigestible plant biomass as food sources. Methane is produced as an end product of the anaerobic feed degradation in ruminants and is emitted to the atmosphere, making ruminant animals among the major anthropogenic sources of the potent greenhouse gas methane. Using newly developed quantitative metatranscriptomics for holistic microbiome analysis, we here identified bacterial, archaeal, and eukaryotic key players and the short-term dynamics of the rumen microbiome during anaerobic plant biomass degradation and subsequent methane emissions. These novel insights might pave the way for novel ecologically and economically sustainable methane mitigation strategies, much needed in times of global climate change.

Project description:Muskox (Ovibos moschatus), as the biggest herbivore in the High Arctic, has been enduring the austere arctic nutritional conditions and has evolved to ingest and digest scarce and high lignified forages to support the growth and reproduce, implying probably harbor a distinct microbial reservoir for the deconstruction of plant biomass. Therefore, metagenomics approach was applied to characterize the rumen microbial community and understand the alteration in rumen microbiome of muskoxen fed either triticale straw or brome hay. The difference in the structure of microbial communities including bacteria, archaea, fungi, and protozoa between the two forages was observed at the taxonomic level of genus. Further, although the highly abundant phylotypes in muskoxen rumen fed either triticale straw or brome hay were almost the same, the selective enrichment different phylotypes for fiber degrading, soluble substrates fermenting, electron and hydrogen scavenging through methanogenesis, acetogenesis, propionogenesis, and sulfur-reducing was also noticed. Specifically, triticale straw with higher content of fiber, cellulose selectively enriched more lignocellulolytic taxa and electron transferring taxa, while brome hay with higher nitrogen content selectively enriched more families and genera for degradable substrates-digesting. Intriguingly, the carbohydrate-active enzyme profile suggested an over representation and diversity of putative glycoside hydrolases (GHs) in the animals fed on triticale straw. The majority of the cellulases belonged to fiver GH families (i.e., GH5, GH6, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus, Piromyces, Neocallimastix, and Fibrobacter. Abundance of major genes coding for hemicellulose digestion was higher than cellulose mainly including GH8, GH10, GH16, GH26, and GH30, and these enzymes were produced by members of the genera Fibrobacter, Ruminococcus, and Clostridium. Oligosaccharides were mainly of the GH1, GH2, GH3, and GH31 types and were associated with the genera Prevotella and Piromyces. Our results strengthen metatranscriptomic evidence in support of the understanding of the microbial community and plant polysaccharide response to changes in the feed type and host animal. The study also establishes these specific microbial consortia procured from triticale straw group can be used further for efficient plant biomass hydrolysis.

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Metatranscriptomics of the Hu sheep rumen microbiome reveals novel cellulases.

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Metatranscriptomics of the Hu sheep rumen microbiome reveals novel cellulases.

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