Directed modification of a ruminal cellulase gene (CMC-1) from a metagenomic library isolated from Yunnan gayal (Bos frontalis)

© 2020, Springer-Verlag GmbH Germany, part of Springer Nature. Gayal (Bos frontalis) of the Yunnan region is well adapted to harsh environmental conditions. Its diet consists predominantly of bamboo, reeds, and woody plants, suggesting that the rumen of this species contains many fiber-degrading bac...

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Main Authors: Dongwang Wu, Songming Wang, Paramintra Vinitchaikul, Yaxin Zhu, Xiongyan Zhou, Zhaobing Gu, Jing Leng, Xiao Gou, Mingyue Deng, Liyuan Sun, Huaming Mao, Shuli Yang
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Published: 2020
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spelling th-cmuir.6653943832-682672020-04-02T15:27:05Z Directed modification of a ruminal cellulase gene (CMC-1) from a metagenomic library isolated from Yunnan gayal (Bos frontalis) Dongwang Wu Songming Wang Paramintra Vinitchaikul Yaxin Zhu Xiongyan Zhou Zhaobing Gu Jing Leng Xiao Gou Mingyue Deng Liyuan Sun Huaming Mao Shuli Yang Biochemistry, Genetics and Molecular Biology Immunology and Microbiology © 2020, Springer-Verlag GmbH Germany, part of Springer Nature. Gayal (Bos frontalis) of the Yunnan region is well adapted to harsh environmental conditions. Its diet consists predominantly of bamboo, reeds, and woody plants, suggesting that the rumen of this species contains many fiber-degrading bacteria and cellulases. The aim of this study was to identify and modify specific cellulases found in the gayal rumen. In the present study, a directed evolution strategy of error-prone PCR was employed to improve the activity or optimal temperature of a cellulase gene (CMC-1) isolated from gayal rumen. The CMC-1 gene was heterologously expressed in Escherichia coli (E. coli) BL21, and the recombinant CMC-1 protein hydrolyzed carboxyl methyl cellulose (CMC) with an optimal activity at pH 5.0 and 50 °C. A library of mutated ruminal CMC-1 genes was constructed and a mutant EP-15 gene was identified. Sequencing analysis revealed that EP-15 and CMC-1 belonged to the glycosyl hydrolase family 5 (GHF5) and had the highest homology to a cellulase (Accession No. WP_083429257.1) from Prevotellaceae bacterium, HUN156. There were similar predicted GH5 domains in EP-15 and CMC-1. The EP-15 gene was heterologously expressed and exhibited cellulase activity in E. coli BL21 at pH 5.0, but the optimum temperature for its activity was reduced from that of CMC-1 (50 °C) to 45 °C, which was closer to the physiological temperature of the rumen (40 °C). The cellulase activity of EP-15 was about two times higher than CMC-1 at 45 °C or PH 5.0, and also was more stable in response to temperature and pH changes compared to CMC-1. This study successfully isolated and modified a ruminal cellulase gene from metagenomics library of Yunnan gayal. Our findings may obtain a useful cellulase in future applications and present the first evidence of modified cellulases in the gayal rumen. 2020-04-02T15:23:58Z 2020-04-02T15:23:58Z 2020-01-01 Journal 1432072X 03028933 2-s2.0-85079459176 10.1007/s00203-020-01812-3 https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85079459176&origin=inward http://cmuir.cmu.ac.th/jspui/handle/6653943832/68267
institution Chiang Mai University
building Chiang Mai University Library
country Thailand
collection CMU Intellectual Repository
topic Biochemistry, Genetics and Molecular Biology
Immunology and Microbiology
spellingShingle Biochemistry, Genetics and Molecular Biology
Immunology and Microbiology
Dongwang Wu
Songming Wang
Paramintra Vinitchaikul
Yaxin Zhu
Xiongyan Zhou
Zhaobing Gu
Jing Leng
Xiao Gou
Mingyue Deng
Liyuan Sun
Huaming Mao
Shuli Yang
Directed modification of a ruminal cellulase gene (CMC-1) from a metagenomic library isolated from Yunnan gayal (Bos frontalis)
description © 2020, Springer-Verlag GmbH Germany, part of Springer Nature. Gayal (Bos frontalis) of the Yunnan region is well adapted to harsh environmental conditions. Its diet consists predominantly of bamboo, reeds, and woody plants, suggesting that the rumen of this species contains many fiber-degrading bacteria and cellulases. The aim of this study was to identify and modify specific cellulases found in the gayal rumen. In the present study, a directed evolution strategy of error-prone PCR was employed to improve the activity or optimal temperature of a cellulase gene (CMC-1) isolated from gayal rumen. The CMC-1 gene was heterologously expressed in Escherichia coli (E. coli) BL21, and the recombinant CMC-1 protein hydrolyzed carboxyl methyl cellulose (CMC) with an optimal activity at pH 5.0 and 50 °C. A library of mutated ruminal CMC-1 genes was constructed and a mutant EP-15 gene was identified. Sequencing analysis revealed that EP-15 and CMC-1 belonged to the glycosyl hydrolase family 5 (GHF5) and had the highest homology to a cellulase (Accession No. WP_083429257.1) from Prevotellaceae bacterium, HUN156. There were similar predicted GH5 domains in EP-15 and CMC-1. The EP-15 gene was heterologously expressed and exhibited cellulase activity in E. coli BL21 at pH 5.0, but the optimum temperature for its activity was reduced from that of CMC-1 (50 °C) to 45 °C, which was closer to the physiological temperature of the rumen (40 °C). The cellulase activity of EP-15 was about two times higher than CMC-1 at 45 °C or PH 5.0, and also was more stable in response to temperature and pH changes compared to CMC-1. This study successfully isolated and modified a ruminal cellulase gene from metagenomics library of Yunnan gayal. Our findings may obtain a useful cellulase in future applications and present the first evidence of modified cellulases in the gayal rumen.
format Journal
author Dongwang Wu
Songming Wang
Paramintra Vinitchaikul
Yaxin Zhu
Xiongyan Zhou
Zhaobing Gu
Jing Leng
Xiao Gou
Mingyue Deng
Liyuan Sun
Huaming Mao
Shuli Yang
author_facet Dongwang Wu
Songming Wang
Paramintra Vinitchaikul
Yaxin Zhu
Xiongyan Zhou
Zhaobing Gu
Jing Leng
Xiao Gou
Mingyue Deng
Liyuan Sun
Huaming Mao
Shuli Yang
author_sort Dongwang Wu
title Directed modification of a ruminal cellulase gene (CMC-1) from a metagenomic library isolated from Yunnan gayal (Bos frontalis)
title_short Directed modification of a ruminal cellulase gene (CMC-1) from a metagenomic library isolated from Yunnan gayal (Bos frontalis)
title_full Directed modification of a ruminal cellulase gene (CMC-1) from a metagenomic library isolated from Yunnan gayal (Bos frontalis)
title_fullStr Directed modification of a ruminal cellulase gene (CMC-1) from a metagenomic library isolated from Yunnan gayal (Bos frontalis)
title_full_unstemmed Directed modification of a ruminal cellulase gene (CMC-1) from a metagenomic library isolated from Yunnan gayal (Bos frontalis)
title_sort directed modification of a ruminal cellulase gene (cmc-1) from a metagenomic library isolated from yunnan gayal (bos frontalis)
publishDate 2020
url https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85079459176&origin=inward
http://cmuir.cmu.ac.th/jspui/handle/6653943832/68267
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