Jun Sugiura

Purification and Characterization of Cellobiose Dehydrogenase from White-Rot Basidiomycete Trametes hirsuta

In order to save energy during the pulp making process, we tried to use white-rot basidiomycete, Trametes hirsuta, which degrades lignin efficiently. But a decrease in paper strength caused by cellulolytic activity ruled this out for practical application. Since the cellulolytic activity of the fungus must be decreased, we purified and characterized a cellobiose dehydrogenase (CDH) that was reported to damage pulp fiber. The CDH in the culture filtrate of C. hirsutus was purified by freeze-thawing and chromatographic methods. The pI of the enzyme was 4.2 and its molecular weight was 92 kDa. The optimal temperature and pH of the enzyme were 60–70 °C and 5.0 respectively. Since the purified CDH decreased the viscosity of pulp in the presence of Fe(III) and cellobiose, it was shown that the suppression of CDH should be an effective way to reduce cellulose damage.

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

The homobasidiomycete Coriolus hirsutus coding sequences of a lignin peroxidase (LiP) gene (lip, containing six (I–VI) introns), a lip cDNA (lipc), and three lipc derivatives containing one (I), three (I–III), or five (I–V) introns were inserted into chromosome-integrating expression vector. These recombinant plasmids were introduced into C. hirustus monokaryotic strain. The transformant carrying the promoter–lipc–terminator cassette did not contain enough mRNA molecules to be detectable by Northern-blot analysis. On the other hand, all the transformants carrying cassettes of genomic lip and intron(s)-containing lipc sequences contained sufficient amounts of mRNAs to be easily detected by Northern-blot analysis. LiP activities in the culture supernatants of these transformants were found to be about five times as high as those of transformants carrying the lipc cassette (or no cassette). The culture supernatants of the transformants with high LiP activity showed remarkably high conversion activity toward pentachlorophenol (PCP) and degradation activity toward 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). These results indicate that at least one intron (intron I) is required for accumulation of lip mRNA and its subsequent translational expression in C. hirsutus.

Transformation of the White-rot Basidiomycete Coriolus hirsutus Using the Ornithine Carbamoyltransferase Gene

An efficient transformation system for the basidiomycete Coriolus hirsutus was developed. A double-auxotrophic mutant of C. hirsutus, deficient both in ornithine carbamoyltransferase (OCTase) and 3-isopropylmalate dehydrogenase (3-IPM dehydrogenase), was transformed to Arg+ with each allelic type of the C. hirsutus genomic OCTase gene (arg1) newly cloned. The transformation frequency of 103-104 transformants per μg DNA per 106-107 oidial protoplasts was reached. Southern blots showed that the transforming DNA was integrated into chromosomal DNA with multi-copies. The Arg+ phenotype of the transformants was stably inherited through mitosis.

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.

Isolation of a ras gene from the basidiomycete Coriolus hirsutus and use of its promoter for the expression of Pleurotus ostreatus manganese(II) peroxidase cDNA in C. hirsutus

A ras gene homologue (named Ch.ras) was cloned from the basidiomycete Coriolus hirsutus. Ch.ras has a coding capacity of 215 amino acids (aa) interrupted by six small introns. The deduced Ch.Ras protein exhibited significant homology (86.5% or 86.0% identical) to the basidiomycete Coprinus cinereus Ras (215 aa) and Lentinula edodes Ras (217 aa) proteins. The 5′-upstream region of Ch.ras contains two GC boxlike sequences, one TATA boxlike sequence, one CCAAT box, and three CT-rich sequences. Primer extension analysis showed the presence of three transcriptional initiation sites: one is located in the most upstream CT-sequence and the other two just after it. By using the 1.4-kb fragment containing the promoter elements and transcriptional initiation sites, we have constructed the chromosome-integrating vector pHRP, which is useful for the expression of foreign genes in C. hirsutus. The Pleurotus ostreatus manganese(II) peroxidase (MnP) cDNA (designated mnpc) was inserted into the downstream of the Ch.ras promoter elements of pHRP, yielding pHRP-mnp. We obtained, with pHRP-mnp, C. hirsutus strains that show high levels of enzymatic activity of MnP and efficiently degrade pentachlorophenol (PCP), a chlorinated aromatic toxic compound.