Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus.
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.
Orihara
Kanami
K
Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Yamazaki
Takashi
T
Shinkyo
Raku
R
Sakaki
Toshiyuki
T
Inouye
Kuniyo
K
Tsukamoto
Akira
A
Sugiura
Jun
J
Shishido
Kazuo
K
D000818:Animals; D001711:Biotransformation; D015153:Blotting, Western; D002849:Chromatography, Gas; D015825:Chromosomes, Fungal / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004147:Dioxins / Q000378:metabolism*; D018628:Gene Dosage; D015870:Gene Expression; D005822:Genetic Vectors; D010957:Plasmids; D020072:Polyporales / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D020942:Shiitake Mushrooms; D014170:Transformation, Genetic
D004147:Dioxins; D011994:Recombinant Proteins; C000925:2,7-dichlorodibenzo-4-dioxin; C503656:dichlorodibenzo-p-dioxin; D003577:Cytochrome P-450 Enzyme System; C068993:2,8-dichlorodibenzo-4-dioxin