Abstract
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.
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Abbreviations
- CMC-1:
-
Carboxyl methyl cellulase-1
- E. coli :
-
Escherichia coli
- CMC:
-
Carboxyl methyl cellulose
- GHF5:
-
Glycosyl hydrolase family 5
- aa:
-
Amino acids
- M w :
-
Molecular weights
- pI:
-
Isoelectric points
- ORF:
-
Open reading frame
- LB:
-
Luria broth
- DNS:
-
Dinitrosalicylic acid
- VAL:
-
Valine
- ALA:
-
Alanine
- GLY:
-
Glycine
- GLU:
-
Glutamate
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Acknowledgements
We thank the researchers at our laboratories for their dedication and hard work. We would like to thank everyone who made this thesis possible.
Funding
This research was supported by the Key Research and Development Plan Project of the Yunnan province (2018BB001), Yunnan Agricultural Foundation Projects (2017FG001-061), National Natural Science Foundation of China (31672452), and Key Projects of Natural Science Foundation of the Yunnan Province (2017FA012).
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Conceived and designed the experiments: MHM, YSL. Performed the experiments: WSM, ZYX, ZXY, GZB, LJ, GX, DMY, SLY. Analyzed the data: WDW, WSM. Wrote the paper: WDW, VP, YSL. All authors read and approved the final manuscript.
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All experiments performed in this study were approved by the Institutional Animal Care and Use Committee of the Yunnan Agricultural University (Contract 2007–0081), China. The study complied with the guidelines of the institutional administrative committee and ethics committee of laboratory animals.
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Communicated by Erko Stackebrandt.
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Wu, D., Wang, S., Vinitchaikul, P. et al. Directed modification of a ruminal cellulase gene (CMC-1) from a metagenomic library isolated from Yunnan gayal (Bos frontalis). Arch Microbiol 202, 1117–1126 (2020). https://doi.org/10.1007/s00203-020-01812-3
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DOI: https://doi.org/10.1007/s00203-020-01812-3