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.

中文翻译：

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来自云南gayal（Bos frontalis）的宏基因组文库中瘤胃纤维素酶基因（CMC-1）的定向修饰

云南地区的Gayal（Bos frontalis）很好地适应了恶劣的环境条件。它的饮食主要由竹子、芦苇和木本植物组成，这表明该物种的瘤胃含有许多降解纤维的细菌和纤维素酶。本研究的目的是鉴定和修饰在同性恋瘤胃中发现的特定纤维素酶。在本研究中，采用易错 PCR 的定向进化策略来提高从同性恋瘤胃中分离的纤维素酶基因 (CMC-1) 的活性或最佳温度。CMC-1基因在大肠杆菌(E.coli)BL21中异源表达，重组CMC-1蛋白水解羧甲基纤维素(CMC)，在pH 5.0和50°C时具有最佳活性。构建了突变瘤胃 CMC-1 基因文库并鉴定了突变 EP-15 基因。测序分析表明，EP-15 和 CMC-1 属于糖基水解酶家族 5 (GHF5)，与来自普氏菌科细菌 HUN156 的纤维素酶（登录号 WP_083429257.1）具有最高的同源性。在 EP-15 和 CMC-1 中有类似的预测 GH5 结构域。EP-15 基因在 pH 5.0 的大肠杆菌 BL21 中异源表达并表现出纤维素酶活性，但其活性的最适温度从 CMC-1（50 ℃）降低至 45 ℃，更接近于瘤胃的生理温度 (40 °C)。EP-15 的纤维素酶活性在 45 °C 或 PH 5.0 时比 CMC-1 高约两倍，并且与 CMC-1 相比，对温度和 pH 变化的响应也更稳定。本研究成功地从云南大白菜宏基因组文库中分离并修饰了一个瘤胃纤维素酶基因。