Identification of thebphC gene formeta-cleavage of aromatic pollutants from a metagenomic library derived from lake waters

Abstract

Useful genes can be screened from various environments by construction of metagenomic DNA libraries. In this study, water samples were collected from several lakes in mid Korea, and analyzed by T-RFLP to examine diversities of the microbial communities. The crude DNAs were extracted by the SDS-based freezing-thawing method, and then further purified using an UltraCleanTM kit (MoBio, USA). The metagenomic libraries were constructed with the DNAs partially digested withEcoR I,BamH I, andSac II inEscherichia coli DH 10B using the pBACe3.6 vector. About 44.0 Mb of metagenomic libraries were obtained with average inserts 13∼15 kb in size. ThebphC genes responsible for degradation of aromatic hydrocarbons viameta-cleavage were identified from the metagenomic libraries by colony hybridization using thebphC specific sequence as a probe. The 2,3-dihydroxybiphenyl (2,3-DHBP) dioxygenase gene (bphC), capable of degradation of 2,3-DHBP, was cloned and its nucleotide sequences analyzed. The genes consisted of 966 and 897 base pairs with an ATG initiation codon and a TGA termination codon. The activity of the 2,3-DHBP dioxygenase was highly expressed to 2,3-DHBP and showed a broad substrate range to 2,3-DHBP, catechol, 3-methylcatechol and 4-methylcatechol. These results indicated that thebphC gene identified from the metagenomes derived from lake water might be useful in the development of a potent strain for degradation of aromatic pollutants.