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

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.

Recent Developments in Microbial Biotransformation and Biodegradation of Dioxins

“…Shinkyo et al [2003] produced a novel P-450 enzyme capable of metabolizing 2,3,7,8-TCDD by modifying rat CYP1A1 through site-directed mutagenesis. Recently, Orihara et al [2005] produced a recombinant white-rot fungus Coriolus hirsutus by transforming it with rat P-450 (CYPA1). Three transformants (ChTF5-2 (CYP1A1), ChTF5-4 (CYP1A1) and ChTF5-6 (CYP1A1) produced similar amounts of rat CYPA1 enzyme and efficiently transformed 2,7-and 2,8-DCDDs.…”

Possibility of application of cytochrome P450 to bioremediation of dioxins

“…In addition, we successfully expressed N‐terminal truncated F240A mutant (ΔF240A) in Escherichia coli cells . Shishido's group succeeded in the expression of rat CYP1A1 in white‐rot basidiomycete fungus cells . These results suggest possible application of fungal or prokaryotic cells expressing F240A or ΔF240A to the bioremediation of PCDD‐contaminated soil .…”

Agrobacterium-mediated transformation of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces commercially valuable fruit bodies, matsutake

“…Agrobacterium-mediated transformation of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces commercially valuable fruit bodies, matsutake Received: October 18, 2005/ Accepted: March 27, 2006 Marmeisse et al 1992;Ogawa et al 1998;Orihara et al 2005). Such a method, if developed, will facilitate molecular genetic analysis of T. matsutake to elucidate the biological functions involved in symbiosis, the unique colonization in the rhizosphere forming a massive aggregate called shiro, and fruit-body production, information that may be useful in the development of artificial culture practice.…”