Boubakri Hasna, Beuf Mélanie, Simonet Pascal, Vogel Timothy M
Ecologie Microbienne, UMR CNRS 5557, Université Claude Bernard Lyon I, 16 rue Dubois, F-69622 Villeurbanne Cedex, France.
Gene. 2006 Jun 21;375:87-94. doi: 10.1016/j.gene.2006.02.027. Epub 2006 Apr 1.
We describe a metagenomic DNA shuffling process by combining protein engineering process mutation generator and the high potential diversity of metagenomic DNA derived from the environment. Numerous previous shuffling processes attempted to recombine more or less related parental sequences. At the same time, metagenomic approaches unveiled a huge diversity of DNA sequences and genomes, which have not yet been identified to date. In this study, we attempted to combine these two approaches in order to regenerate a novel gene. Here, we present the possibility that DNA fragments from an entire microbial community (metagenome) might be available for the creation of novel genes capable of degrading pollutants. Metagenomic DNA extracted from non-polluted soil was shuffled in vitro to recreate the linA gene responsible for the first steps of lindane degradation. In this work, 74% of the ORF came from separate subsets of the metagenomic pool from a lindane-free and linA-free soil. Our results demonstrate that microbial community genetic diversity can serve as a source for novel gene construction during in vitro manipulation. This in vitro gene construction might also simulate the mosaic nature of novel genes. This demonstration might lead to other attempts to mimic bacterial adaptation and to construct degradative genes for novel compounds not yet released into the environment.
我们通过结合蛋白质工程过程突变生成器和源自环境的宏基因组DNA的高潜在多样性，描述了一种宏基因组DNA改组过程。此前众多的改组过程试图重组或多或少相关的亲本序列。与此同时，宏基因组学方法揭示了大量尚未被鉴定的DNA序列和基因组。在本研究中，我们试图将这两种方法结合起来以再生一个新基因。在此，我们提出来自整个微生物群落（宏基因组）的DNA片段可能可用于创建能够降解污染物的新基因。从未受污染土壤中提取的宏基因组DNA在体外进行改组，以重建负责林丹降解第一步的linA基因。在这项工作中，74%的开放阅读框来自无林丹且无linA基因的土壤中宏基因组库的不同子集。我们的结果表明，微生物群落遗传多样性可作为体外操作过程中新型基因构建的来源。这种体外基因构建也可能模拟新基因的镶嵌性质。这一证明可能会引发其他模仿细菌适应性以及构建针对尚未释放到环境中的新型化合物的降解基因的尝试。
