Functional screening of a metagenomic library for genes involved in microbial degradation of aromatic compounds

Authors

  • Hikaru Suenaga,

    1. Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
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  • Tsutomu Ohnuki,

    1. Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
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  • Kentaro Miyazaki

    Corresponding author
    1. Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
    2. Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
      *E-mail miyazaki-kentaro@aist.go.jp; Tel. (+81) 29 861 6060; Fax (+81) 29 861 6413.
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*E-mail miyazaki-kentaro@aist.go.jp; Tel. (+81) 29 861 6060; Fax (+81) 29 861 6413.

Summary

A metagenomic approach was taken to retrieve catabolic operons for aromatic compounds from activated sludge used to treat coke plant wastewater. Metagenomic DNA extracted from the sludge was cloned into fosmids and the resulting Escherichia coli library was screened for extradiol dioxygenases (EDOs) using catechol as a substrate, yielding 91 EDO-positive clones. Based on their substrate specificity for various catecholic compounds, 38 clones were subjected to sequence analysis. Each insert contained at least one EDO gene, and a total of 43 EDO genes were identified. More than half of these belonged to new EDO subfamilies: I.1.C (2 clones), I.2.G (20 clones), I.3.M (2 clones) and I.3.N (1 clone). The fact that novel I.2.G family genes were over-represented in these clones suggested that these genes play a specific role in environmental aromatic degradation. The I.2.G clones were further classified into six groups based on single-nucleotide polymorphisms (SNPs). Based on the combination of the SNPs, the evolutionary lineage of the genes was reconstructed; further, taking the activities of the clones into account, potential adaptive mutations were identified. The metagenomic approach was thus used to retrieve novel EDO genes as well as to gain insights into the gene evolution of EDOs.

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