Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)

doi:10.1016/S2095-3119(15)61144-3

Journal of Integrative Agriculture ›› 2016, Vol. 15 ›› Issue (4): 855-861.DOI: 10.1016/S2095-3119(15)61144-3

Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)

LI Bi-feng^1*, ZHU Ya-xin^2*, GU Zhao-bing^1*, CHEN Yuan³, LENG Jing¹, GOU Xiao¹, FENG Li¹, LI Qing¹, XI Dong-mei¹, MAO Hua-ming¹, YANG Shu-Li¹

¹ Yunnan Provincial Key Laboratory of Animal Nutrition and Feed Science, Yunnan Agricultural University, Kunming 650201, P.R.China
² Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R.China
³ Department of Biological Science, Huizhou University, Huizhou 560001, P.R.China

收稿日期:2015-03-31 出版日期:2016-04-01 发布日期:2016-04-07
通讯作者: YANG Shu-li, Mobile: +86-13759115322, E-mail: yangshuli77@163.com
作者简介:LI Bi-feng, E-mail: ynjsclbf@163.com
基金资助:
This work was supported by the National Natural Science Foundation of China (31160467, 31360562, 31160449 and 31260543).

Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)

LI Bi-feng^1*, ZHU Ya-xin^2*, GU Zhao-bing^1*, CHEN Yuan³, LENG Jing¹, GOU Xiao¹, FENG Li¹, LI Qing¹, XI Dong-mei¹, MAO Hua-ming¹, YANG Shu-Li¹

¹ Yunnan Provincial Key Laboratory of Animal Nutrition and Feed Science, Yunnan Agricultural University, Kunming 650201, P.R.China
² Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R.China
³ Department of Biological Science, Huizhou University, Huizhou 560001, P.R.China

Received:2015-03-31 Online:2016-04-01 Published:2016-04-07
Contact: YANG Shu-li, Mobile: +86-13759115322, E-mail: yangshuli77@163.com
About author:LI Bi-feng, E-mail: ynjsclbf@163.com
Supported by:
This work was supported by the National Natural Science Foundation of China (31160467, 31360562, 31160449 and 31260543).

摘要/Abstract

摘要： Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as well as reeds and other plant species, and grow to higher mature live weights than Yunnan Yellow cattle maintained in similar harsh environments. The aim of this study was to identify specific cellulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38 400 clones with an average insert size of 35.5 kb. The Umcel-1 gene was isolated from this library. Investigation of the cellulase activity of 24 random clones led to the identification of the Umcel-1 gene, which exhibited the most potent cellulase activity. Sequencing the Umcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putative gene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the cellulase (GenBank accession no. YP_004310852.1) from Clostridium lentocellum DSM 5427, with 44% identity and 62% similarity. The Umcel-1 gene was heterologously expressed in Escherichia coli BL21, and recombinant Umcel-1 was purified. The activity of purified recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl cellulose with optimal activity at pH 5.5 and 45°C. To our knowledge, this study provides the first evidence for a cellulase produced by bacteria in gayal rumen.

关键词: gayal , ruminal microorganism , fosmid library , Umcel-1 gene , gene expression , cellulase activity

Abstract: Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as well as reeds and other plant species, and grow to higher mature live weights than Yunnan Yellow cattle maintained in similar harsh environments. The aim of this study was to identify specific cellulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38 400 clones with an average insert size of 35.5 kb. The Umcel-1 gene was isolated from this library. Investigation of the cellulase activity of 24 random clones led to the identification of the Umcel-1 gene, which exhibited the most potent cellulase activity. Sequencing the Umcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putative gene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the cellulase (GenBank accession no. YP_004310852.1) from Clostridium lentocellum DSM 5427, with 44% identity and 62% similarity. The Umcel-1 gene was heterologously expressed in Escherichia coli BL21, and recombinant Umcel-1 was purified. The activity of purified recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl cellulose with optimal activity at pH 5.5 and 45°C. To our knowledge, this study provides the first evidence for a cellulase produced by bacteria in gayal rumen.

Key words: gayal , ruminal microorganism , fosmid library , Umcel-1 gene , gene expression , cellulase activity

LI Bi-feng, ZHU Ya-xin, GU Zhao-bing, CHEN Yuan, LENG Jing, GOU Xiao, FENG Li, LI Qing, XI Dong-mei, MAO Hua-ming, YANG Shu-Li. Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)[J]. Journal of Integrative Agriculture, 2016, 15(4): 855-861.

参考文献

Cai S, Li J, Hu F Z, Zhang K, Luo Y, Janto B, Boissy R, Ehrlich G, Dong X. 2010. Cellulosilyticum ruminicola, a newly described rumen bacterium that possesses redundant fibrolytic-protein-encoding genes and degrades lignocellulose with multiple carbohydrate-borne fibrolytic enzymes. Applied and Environmental Microbiology, 76, 3818–3824.

Chang L, Ding M, Bao L, Chen Y, Zhou J, Lu H. 2011. Characterization of a bifunctional xylanase/endoglucanase from yak rumen microorganisms. Applied Microbiology and Biotechnologyl, 90, 1933–1942.

Cheema T A, Jirajaroenrat K, Sirinarumitr T, Rakshit S K. 2012. Isolation of a gene encoding a cellulolytic enzyme from swamp buffalo rumen metagenomes and its cloning and expression in Escherichia coli. Animal Biotechnology, 23, 261–277.

Cheng F, Sheng J, Dong R, Men Y, Gan L, Shen L. 2012. Novel xylanase from a Holstein cattle rumen metagenomic library and its application in xylooligosaccharide and ferulic acid production from wheat straw. Journal of Agricultural and Food Chemistry, 60, 12516–12524.

Collins M D, Lawson P A, Willems A, Cordoba J J, Fernandez-Garayzabal J, Garcia P, Cai J, Hippe H, Farrow J A. 1994. The phylogeny of the genus Clostridium: Proposal of five new genera and eleven new species combinations. International Journal of Systematic Bacteriology, 44, 812–826.

Deng W D, Wang L P, Ma S C, Jin B, He T B, Yang Z F, Mao H M, Wanapat M. 2007. Comparison of gayal (Bos frontalis) and Yunnan Yellow cattle (Bos taurus): Rumen function, digestibilities and nitrogen balance during feeding of pelleted lucerne (Medicago sativum). Asian-Australasian Journal of Animal Sciences, 20, 900–907.

Duan C J, Xian L, Zhao G C, Feng Y, Pang H, Bai X L, Tang J L, Ma Q S, Feng J X. 2009. Isolation and partial characterization of novel genes encoding acidic cellulases from metagenomes of buffalo rumens. Journal of Applied Microbiology, 107, 245–256.

Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39, 783–791.

Feng Y, Duan C J, Pang H, Mo X C, Wu C F, Yu Y, Hu Y L, Wei J, Tang J L, Feng J X. 2007. Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Applied Microbiology and Biotechnology, 75, 319–328.

Ferrer M, Golyshina O V, Chemikova T N, Khachane A N, Reyes-Duarte D, Santos V A, Strompl C, Elborough K, Jarvis G, Neef A, Yakimov M M, Timmis K N, Golyshin P N. 2005. Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora. Environmental Microbiology, 7, 1996–2010.

Gong X, Gruninger R J, Qi M, Paterson L, Forster R J, Teather R M, McAllister T A. 2012. Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene. BMC Research Notes, 5, 566–576.

Handelsman J, Rondon M R, Brady S F, Clardy J, Goodman R M. 1998. Molecular biological access to the chemistry of unknown soil microbes: A new frontier for natural products. Chemistry & Biology, 5, 245–249.

Leng J, Xie L J, Zhu R J, Yang S L, Gou X, Li S, Mao H M. 2011. Dominant bacterial communities in the rumen of gayals (Bos frontalis), yaks (Bos grunniens) and Yunnan Yellow cattle (Bos taurus) revealed by denaturing gradient gel electrophoresis. Molecular Biology Reports, 38, 4863–4872.

Miller D A, Suen G, Bruce D, Copeland A, Cheng J F, Detter C, Goodwin L A, Han C S, Hauser L J, Land M L, Lapidus A, Lucas S, Meincke L, Pitluck S, Tapia R, Teshima H, Woyke T, Fox B G, Angert E R, Currie C R. 2011. Complete genome sequence of the cellulose-degrading bacterium Cellulosilyticum lentocellum. Journal of Bacteriology, 193, 2357–2358.

Miller G L. 1959. Use of dinitrosalicyclic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428.

Murray W D, Hoffman L, Campbell N L, Madden R H. 1986. Clostridium lentocellum sp. nov., a cellulolytic species from river sediment containing paper-mill waste. Systematic and Applied Microbiology, 8, 181–184.

Nelson K E, Zinder S H, Hance I, Burr P, Odongo D, Wasawo D, Odenyo A, Bishop R. 2003. Phylogenetic analysis of the microbial populations in the wild herbivore gastrointestinal tract: Insights into an unexplored niche. Environmental Microbiology, 1, 1212–1220.

Nguyen H, Maruset L, Uengwetwanit T, Mhuantong W, Harnpicharnchai P, Champreda V, Tanapongpipat S, Jirajaroenrat K, Rakshit S K, Eurwilaichitr L, Pongpattanakitshote S. 2012. Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library. Bioscience Biotechnology and Biochemistry, 76, 1075–1084.

Palackal N, Lyon C S, Zaidi S, Luginbuhl P, Dupree P, Goubet F, Macomber J L, Short J M, Hazlewood G P, Robertson D E, Steer B A. 2007. A multifunctional hybrid glycosyl hydrolase discovered in an uncultured microbial consortium from ruminant gut. Applied Microbiology and Biotechnology, 74, 113–124.

Rashamuse K J, Visser D F, Hennessy F, Kem J, Roux-van der Merwe M P, Badenhorst J, Ronneburg T, Francis-Pope R, Brady D. 2013. Characterization of two bifunctional cellulase-xylanase enzymes isolated from a bovine rumen metagenome library. Current Microbiology, 66, 145–51.

Saitou N, Nei M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406–425.

Streit W R, Schmitz R A. 2004. Metagenomics - the key to the uncultured microbes. Current Opinion in Microbiology, 7, 492–498.

Thompson J D, Higgins D G, Gibson T J. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673–4680.

Walter J, Mangold M, Tannock G W. 2005. Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice. Applied and Environmental Microbiology, 71, 2347–2354.

Wang F, Li F, Chen G, Liu W. 2009. Isolation and characterization of novel cellulase genes from uncultured microorganisms in different environmental niches. Microbiological Research, 164, 650–657.

Yang S L, Bu D P, Wang J Q, Hu Z Y, Li D, Wei H Y, Zhou L Y, Loor J J. 2009. Soybean oil and linseed oil supplementation affect profiles of ruminal microorganisms in dairy cows. Animal, 3, 1562–1569.

Yang S L, Ma S C, Chen J, Mao H M, He Y D, Xi D M, Yang L Y, He T B, Deng W D. 2010. Bacterial diversity in the rumen of gayals (Bos frontalis), swamp buffaloes (Bubalus bubalis) and Holstein cow as revealed by cloned 16S rRNA gene sequences. Molecular Biology Reports, 37, 2063–2073.

Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)

Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)

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