Development of metagenomic DNA shuffling for the construction of a xenobiotic gene

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. (c) 2006 Elsevier B.V. All rights reserved.