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

• Published in: Archives of microbiology 2020-07, Vol.202 (5), p.1117-1126
• Main Authors: Wu, Dongwang, Wang, Songming, Vinitchaikul, Paramintra, Zhu, Yaxin, Zhou, Xiongyan, Gu, Zhaobing, Leng, Jing, Gou, Xiao, Deng, Mingyue, Sun, Liyuan, Mao, Huaming, Yang, Shuli
• Format: Article
• Language: English
• Subjects: Bacteria; Bamboo; bamboos; Biochemistry; Biodegradation; Biomedical and Life Sciences; Biotechnology; Bos frontalis; Cell Biology; Cellulase; Cellulose; China; diet; Directed evolution; E coli; Ecology; endo-1,4-beta-glucanase; Environmental conditions; Escherichia coli; family; Gene sequencing; genes; genomic libraries; Glycosidases; Glycosyl hydrolase; Homology; Hydrolase; Libraries; Life Sciences; Metagenomics; Methylcellulose; Microbial Ecology; Microbiology; mithuns; mutants; Optimization; Original Paper; pH effects; Reeds; Rumen; Sequence analysis; species; temperature; Woody plants
• ISSN: 0302-8933; 1432-072X
• DOI: 10.1007/s00203-020-01812-3

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.