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

Kanami Orihara, Takashi Yamazaki, Raku Shinkyo, Toshiyuki Sakaki, Kuniyo Inouye, Akira Tsukamoto, Jun Sugiura, Kazuo Shishido

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)22-28
Number of pages7
JournalApplied Microbiology and Biotechnology
Volume69
Issue number1
DOIs
Publication statusPublished - 2005 Nov
Externally publishedYes

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Basidiomycota
Cytochrome P-450 CYP1A1
Cytochrome P-450 Enzyme System
Rats
Enzymes
Chromosomes
Lignin
Metabolism
Exons
Genes
Shiitake Mushrooms
dichlorodibenzo-p-dioxin
Protoplasts
Liquids
Plasmids
Complementary DNA

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology
  • Bioengineering
  • Microbiology (medical)

Cite this

Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus. / Orihara, Kanami; Yamazaki, Takashi; Shinkyo, Raku; Sakaki, Toshiyuki; Inouye, Kuniyo; Tsukamoto, Akira; Sugiura, Jun; Shishido, Kazuo.

In: Applied Microbiology and Biotechnology, Vol. 69, No. 1, 11.2005, p. 22-28.

Research output: Contribution to journalArticle

Orihara, Kanami ; Yamazaki, Takashi ; Shinkyo, Raku ; Sakaki, Toshiyuki ; Inouye, Kuniyo ; Tsukamoto, Akira ; Sugiura, Jun ; Shishido, Kazuo. / Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus. In: Applied Microbiology and Biotechnology. 2005 ; Vol. 69, No. 1. pp. 22-28.
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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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AU - Shinkyo, Raku

AU - Sakaki, Toshiyuki

AU - Inouye, Kuniyo

AU - Tsukamoto, Akira

AU - Sugiura, Jun

AU - Shishido, Kazuo

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