Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences
Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences
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DOI：
10.1371/journal.pone.0172545
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发表时间：
2017-03-08
期刊：
影响因子：
3.7
通讯作者：
Charles, Trevor C.
中科院分区：
文献类型：
--
作者：
Cheng, Jiujun;Romantsov, Tatyana;Charles, Trevor C.
宏基因组学技术使研究人员能够获得未培养微生物的基因组潜力，但仅基于DNA序列确定基因功能仍然存在重大障碍。功能宏基因组学，其中DNA克隆和表达在替代宿主中，可以克服这些障碍，并为发现新的酶做出重要贡献。在这项研究中，在IncP粘粒中携带的土壤宏基因组文库用于功能互补的β-半乳糖苷酶活性在苜蓿中华根瘤菌（α-变形菌）和大肠杆菌（γ-变形菌）的背景。一种α-半乳糖苷酶，由在两种宿主中选择的六个重叠克隆编码，被鉴定为糖苷水解酶家族2的成员。在其他19个测序的克隆中，我们不能鉴定出明显编码可能的β-半乳糖苷酶的ORF，这些克隆只能互补于S。苜蓿草。基于与其他已知糖苷水解酶而不是β-半乳糖苷酶的低序列同一性，进一步检查了这些ORF中的三个。生化分析证实，所有三个编码的α-半乳糖苷酶活性。Lac 36-W_ORF11和Lac161_ORF7具有保守的结构域，但与已知的糖苷水解酶缺乏相似性。Lac161_ORF10与已知的糖苷水解酶既没有保守的结构域，也没有相似性。生物信息学和结构建模表明Lac161_ORF10蛋白代表了一个新的酶家族，具有五叶螺旋桨糖苷水解酶结构域。通过发现三个新的α-半乳糖苷酶家族的创始成员，我们加强了功能宏基因组学的价值，分离新的基因，不能单独从DNA序列分析预测。
The techniques of metagenomics have allowed researchers to access the genomic potential of uncultivated microbes, but there remain significant barriers to determination of gene function based on DNA sequence alone. Functional metagenomics, in which DNA is cloned and expressed in surrogate hosts, can overcome these barriers, and make important contributions to the discovery of novel enzymes. In this study, a soil metagenomic library carried in an IncP cosmid was used for functional complementation for beta-galactosidase activity in both Sinorhizobium meliloti (alpha-Proteobacteria) and Escherichia coli (gamma-Proteobacteria) backgrounds. One a-galactosidase, encoded by six overlapping clones that were selected in both hosts, was identified as a member of glycoside hydrolase family 2. We could not identify ORFs obviously encoding possible beta-galactosidases in 19 other sequenced clones that were only able to complement S. meliloti. Based on low sequence identity to other known glycoside hydrolases, yet not beta-galactosidases, three of these ORFs were examined further. Biochemical analysis confirmed that all three encoded a-galactosidase activity. Lac36-W_ORF11 and Lac161_ORF7 had conserved domains, but lacked similarities to known glycoside hydrolases. Lac161_ORF10 had neither conserved domains nor similarity to known glycoside hydrolases. Bioinformatic and structural modeling implied that Lac161_ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain. By discovering founding members of three novel a-galactosidase families, we have reinforced the value of functional metagenomics for isolating novel genes that could not have been predicted from DNA sequence analysis alone.