Isolation of oxygenase genes for indigo-forming activity from an artificially polluted soil metagenome by functional screening using Pseudomonas putida strains as hosts

Hirofumi Nagayama, Tomonori Sugawara, Ryo Endo, Akira Ono, Hiromi Kato, Yoshiyuki Ohtsubo, Yuji Nagata, Masataka Tsuda

Research output: Contribution to journalArticlepeer-review

29 Citations (Scopus)

Abstract

Metagenomes contain the DNA from many microorganisms, both culturable and non-culturable, and are a potential resource of novel genes. In this study, a 5.2-Gb metagenomic DNA library was constructed from a soil sample (artificially polluted with four aromatic compounds, i.e., biphenyl, phenanthrene, carbazole, and 3-chlorobenzoate) in Escherichia coli by using a broad-host-range cosmid vector. The resultant library was introduced into naphthalene-degrading Pseudomonas putida-derived strains having deficiencies in their naphthalene dioxygenase components, and indigo-forming clones on the indole-containing agar plates were screened. Cosmids isolated from 29 positive clones were classified by their various properties (original screening hosts, hosts showing indigo-forming activity, and digestion patterns with restriction enzymes), and six representative cosmids were chosen. Sequencing and in vitro transposon mutagenesis of the six cosmids resulted in the identification of genes encoding putative class B and D flavoprotein monooxygenases, a multicomponent hydroxylase, and a reductase that were responsible for the indigo-forming activity in the host cells. Among them, the genes encoding the multicomponent hydroxylase were demonstrated to be involved in phenol degradation. Furthermore, two genes encoding ring-cleavage dioxygenases were also found adjacent to the genes responsible for the indigo formation, and their functions were experimentally confirmed.

Original languageEnglish
Pages (from-to)4453-4470
Number of pages18
JournalApplied Microbiology and Biotechnology
Volume99
Issue number10
DOIs
Publication statusPublished - 2015 May 1

Keywords

  • Aromatic compound
  • Biodegradation
  • Indigo
  • Metagenome
  • Oxygenase

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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