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Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus

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Abstract

Rat cytochrome P450, CYP1A1, has been reported to play an important role in the metabolism of mono-trichlorodibenzo-p-dioxins (M-TriCDDs). To breed lignin (and M-TetraCDDs)-degrading basidiomycete Coriolus hirsutus strains producing rat CYP1A1, an expression cassette [C. hirsutus gpd promoter-C. hirsutus gpd 5′ portion (224-bp of 1st exon–8th base of 4th exon)-rat cyp1a1 cDNA-Lentinula edodes priA terminator] was constructed and inserted into pUCR1 carrying the C. hirsutus arg1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1 + arg1) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078 (Arg−, Leu−), obtaining three good Arg+ transformants. These transformants [ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)] were estimated to carry nine, six, and seven copies of the expression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants produce similar amounts of rat CYP1A1 enzyme. ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), ChTF5-6(CYP1A1) and recipient OJ1078 were cultivated in a liquid medium containing 2,7/2,8(at a ratio of 1:1)-dichlorodibenzo-p-dioxins (2,7/2,8-DCDDs) and the amount of intra- and extracellular 2,7/2,8-DCDDs remaining was measured. The results showed that all three transformants efficiently transform 2,7/2,8-DCDDs through the action of the recombinant rat CYP1A1 enzyme.

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Acknowledgements

The authors are grateful to Mrs. M. Hayashi for her help in preparing the manuscript. This work was partly supported by research grants from Research Institute of Innovative Technology of Earth (RITE) and New Energy and Industrial Technology Development Organization (NEDO).

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  1. Toshiyuki Sakaki

    Present address: Faculty of Engineering, Biotechnology Research Center, Toyama Prefectural University, Kurokawa, Kosugi-machi, Imizu-gun, Toyama, 939-0398, Japan

Authors and Affiliations

  1. Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan

    Kanami Orihara, Takashi Yamazaki & Kazuo Shishido

  2. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan

    Raku Shinkyo, Toshiyuki Sakaki & Kuniyo Inouye

  3. Advanced Technology Research Laboratory, Oji Paper, Shinonome, Koto-ku, Tokyo, 135-8558, Japan

    Akira Tsukamoto & Jun Sugiura

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  1. Kanami Orihara
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  2. Takashi Yamazaki
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Correspondence to Kazuo Shishido.

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Orihara, K., Yamazaki, T., Shinkyo, R. et al. Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus . Appl Microbiol Biotechnol 69, 22–28 (2005). https://doi.org/10.1007/s00253-005-1943-0

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