Cloning of a gene encoding flavin reductase coupling with dibenzothiophene monooxygenase through coexpression screening using indigo production as selective indication

Toshiki Furuya, Shusuke Takahashi, Yoshitaka Ishii, Kuniki Kino, Kotaro Kirimura

    Research output: Contribution to journalArticle

    30 Citations (Scopus)

    Abstract

    The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50°C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E. coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55°C. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases.

    Original languageEnglish
    Pages (from-to)570-575
    Number of pages6
    JournalBiochemical and Biophysical Research Communications
    Volume313
    Issue number3
    DOIs
    Publication statusPublished - 2004 Jan 16

    Fingerprint

    Indigo Carmine
    Gene encoding
    Cloning
    Organism Cloning
    Screening
    Oxidoreductases
    Mixed Function Oxygenases
    Bacillus subtilis
    Genes
    Desulfurization
    Bacilli
    Sulfur
    Escherichia coli
    Nitroreductases
    Amino Acids
    Temperature
    4,6-dinitro-o-cresol
    dibenzothiophene monooxygenase
    Pigments
    Nutrients

    Keywords

    • Bacillus subtilis
    • Desulfurization
    • Dibenzothiophene
    • Flavin reductase
    • Flavin-dependent monooxygenase
    • Indigo production

    ASJC Scopus subject areas

    • Biochemistry
    • Biophysics
    • Molecular Biology

    Cite this

    @article{92bd7ee5f9734bb28172437a0b2106b9,
    title = "Cloning of a gene encoding flavin reductase coupling with dibenzothiophene monooxygenase through coexpression screening using indigo production as selective indication",
    abstract = "The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50°C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E. coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61{\%} identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55°C. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases.",
    keywords = "Bacillus subtilis, Desulfurization, Dibenzothiophene, Flavin reductase, Flavin-dependent monooxygenase, Indigo production",
    author = "Toshiki Furuya and Shusuke Takahashi and Yoshitaka Ishii and Kuniki Kino and Kotaro Kirimura",
    year = "2004",
    month = "1",
    day = "16",
    doi = "10.1016/j.bbrc.2003.11.157",
    language = "English",
    volume = "313",
    pages = "570--575",
    journal = "Biochemical and Biophysical Research Communications",
    issn = "0006-291X",
    publisher = "Academic Press Inc.",
    number = "3",

    }

    TY - JOUR

    T1 - Cloning of a gene encoding flavin reductase coupling with dibenzothiophene monooxygenase through coexpression screening using indigo production as selective indication

    AU - Furuya, Toshiki

    AU - Takahashi, Shusuke

    AU - Ishii, Yoshitaka

    AU - Kino, Kuniki

    AU - Kirimura, Kotaro

    PY - 2004/1/16

    Y1 - 2004/1/16

    N2 - The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50°C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E. coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55°C. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases.

    AB - The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50°C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E. coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55°C. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases.

    KW - Bacillus subtilis

    KW - Desulfurization

    KW - Dibenzothiophene

    KW - Flavin reductase

    KW - Flavin-dependent monooxygenase

    KW - Indigo production

    UR - http://www.scopus.com/inward/record.url?scp=0345802687&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=0345802687&partnerID=8YFLogxK

    U2 - 10.1016/j.bbrc.2003.11.157

    DO - 10.1016/j.bbrc.2003.11.157

    M3 - Article

    C2 - 14697229

    AN - SCOPUS:0345802687

    VL - 313

    SP - 570

    EP - 575

    JO - Biochemical and Biophysical Research Communications

    JF - Biochemical and Biophysical Research Communications

    SN - 0006-291X

    IS - 3

    ER -