58NISR Research GRANT
2005 Research Grant

Development of the Biosystem Useful for Degradation of Polychlorinated Aromatic Hydrocarbons by Using Molecular-genetically Improved Basidiomycete Mushroom
Kazuo SHISHIDO
Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology

Research aim
One of the most important problems to be solved in 21st
century is the development of the biosystem useful forpreservation of environment. Especially, chlorinateddibenzo-p-dioxins (CDDs) and pentachlorophenol (PCP)
have been of public concern because of their toxicity in animal tests. The aim of this research is the development ofthe biosystem useful for degradation of CDDs and PCP.
Methods
Extracellular lignin peroxidase (LiP) and manganese peroxidase (MnP) produced by white-rot basidiomycetous
mushrooms have been reported to be involved in degradation of various CDDs (and PCP). Mammalian (rat, etc.) cytochrome P450, CYP1A1 has been reported to play animportant role in the metabolism (detoxification) ofMono–TriCDDs. So the white-rot mushroom strains producing large amounts of LiP and rat CYP1A1 are producedby molecular-genetical method. The Coriolus hirsutus
monokaryotic strain is used as a target of molecular breeding.
Results1) Efficient degradation of 2,7-DCDD by using moleculargenetically bred C. hirsutus strains producing large
amounts of LiP
The chromosome-integrating vector MIp30 carrying theC. hirsutus gpd gene promoter—Lentinula edodes priA
gene terminator and the selectable marker of C. hirsutus
ARG1 gene was constructed. The C. hirsutus LiP gene (lip)
was fused between the promoter and terminator of MIp30
and the resulting recombinant plasmid MIp30-lip was introduced into protoplasts of monokaryotic C. hirsutus Arg
Leu strain (OJ1078), followed by selection of Arg Lipcolonies. Two Arg Lip transformants, namely ChTF61(Ch.LiP) and ChTF6-2(Ch.LiP), possessed about fivecopies of the promoter–lip–terminator cassette on theirchromosomes and the extracellular LiP activities of the twoArg Lip transformants were about five times as high asthat of the Arg control. The supernatants of ChTF61(Ch.LiP) and ChTF6-2(Ch.LiP) showed remarkably high2,7-DCDD degradation activities: at the time when only
33.5% of 2,7-DCDD was degraded by the control Argtransformant, 73.7% and 63.5% of 2,7-DCDD were degraded by ChTF6-1(Ch.LiP) and ChTF6-2(Ch.LiP), respectively (Fig. 1). These two transformants also showed significantly high PCP conversion activity.
2) Degradation/transformation of 2,7/2,8-DCDDs by using
molecular-genetically bred C. hirsutus strains producing
rat cytochrome P450, CYP1A1
The expression cassette [C. hirsutus gpd promoter—C.
hirsutus gpd 5-portion (224-bp sequences of 1st exon—8thbase of 4th exon)–rat cyp1a1 cDNA–L. edodes priA terminator] was constructed and then inserted into pUCR1 carrying C. hirsutus ARG1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1+arg1) was introduced into protoplastsof C. hirsutus OJ1078 strain, obtaining three good Argtransformants. These transformants, namely ChTF52(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)
were estimated to carry nine, six, and seven copies of theexpression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants
produce similar amounts of rat CYP1A1. After 5-day cultivations, ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), andChTF5-6(CYP1A1) degraded/transformed 71.7%, 69.8%,
and 69.4% of 2,7/2,8-DCDDs, respectively, while recipient
OJ1078 degraded/transformed only 11.8% of 2,7/2,8DCDDs (Fig. 2).
Conclusion
We have succeeded in production of the C. hirsutus
strains producing rat cytochrome P450, CYP1A1 and thoseFig. 1. 2,7-DCDD degradation by the culture supernatants of C. hirsutus
transformants.

59Fig. 2. Degradation (transformation) of 2,7/2,8(1 : 1)-DCDDs during
cultivations of the three rat CYP1A1-producing transformants of C. hirsutus.

producing large amounts of LiP. A much more efficientdegradation/transformation of chlorinated dioxins isthought to be achieved by co-cultivation of these strains.
The dioxins are efficiently degraded by both extracellular
LiP (and MnP) and intracellular CYP1A1 (refer to Fig. 3).
The author is very grateful to Noda Institute for Scientific Research for supporting this work by research grant.
References1) K. Shishido (Editor and Author): Kinoko to Kabi no Kisokagaku toBaiogijutsu, IPC Press (2002).

Fig. 3. Degradation of dioxins outside and/or inside mycelial cells ofmolecular-genetically bred basidiomycetous mushrooms (fungi).
2) K. Shishido: Reuse of plant biomass resources and environmental
preservation by using molecular-genetically improved basidiomycete
mushrooms, Bioscience and Industry, 60: 523–526 (2002).
3) K. Shishido: Genetic engineering, Biological Activities and Functions of Mushrooms, CMC Press, 2005, pp. 63–71.
4) T. Yamazaki, Y. Okajima, H. Kawashima, A. Tsukamoto, J. Sugiura,and K. Shishido: Intron-dependent accumulation of mRNA in Coriolus hirsutus of lignin peroxidase gene the product of which is involved in conversion/degradation of polychlorinated aromatic hydrocarbons, Biosci. Biotecnol. Biochem. 70: 1293–1299 (2006).
5) K. Orihara, T. Yamazaki, R. Shinkyo, T. Sakaki, K. Inouye, A.
Tsukamoto, J. Sugiura, and K. Shishido: Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant
white-rot basidiomycete Coriolus hirsutus, Appl. Microbiol. Biotechnol. 69: 22–28 (2005).