Prospecting Metagenomic Enzyme Subfamily Genes for DNA Family Shuffling by a Novel PCR-based Approach*
Prospecting Metagenomic Enzyme Subfamily Genes for DNA Family Shuffling by a Novel PCR-based Approach*
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DOI：
10.1074/jbc.m110.139659
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发表时间：
2010-10
期刊：
影响因子：
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通讯作者：
Qiuyan Wang;Huili Wu;Anming Wang;Pengfei Du;Xiaolin Pei;Haifeng Li;Xiaopu Yin;Lifeng Huang;Xiaolong Xiong
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文献类型：
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作者：
Qiuyan Wang;Huili Wu;Anming Wang;Pengfei Du;Xiaolin Pei;Haifeng Li;Xiaopu Yin;Lifeng Huang;Xiaolong Xiong
DNA 家族改组是酶工程的一种强大方法，它利用自然发生的功能多样性的重组来加速实验室指导的进化。然而，由于基因组数据库中缺乏具有所需序列同一性水平的家族基因，该技术的使用受到阻碍。我们在这里描述了一种通过截短的宏基因组基因特异性 PCR (TMGS-PCR) 从环境样本中收集用于 DNA 改组的宏基因组同源基因的策略。使用已鉴定的宏基因组基因特异性引物，从 60 个宏基因组样本中检索到 23 个 921 bp 截短的脂肪酶基因片段，它们彼此具有 64-99% 的同一性，并形成独特的脂肪酶亚家族。对这些脂肪酶基因进行改组，并对选定的活性克隆进行表征。正如功能表征和序列分析所证明的那样，嵌合克隆显示出广泛的功能和遗传多样性。我们的结果表明，TMGS-PCR 捕获的基因同源序列可用作 DNA 家族改组的合适遗传材料，在酶工程中具有广泛的应用。
DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64–99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering.