• Korean Journal of Microbiology
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• v.42 no.1
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• pp.59-61
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• 2006

Genetic transformation of a mushroom-forming fungus Coprinellus congregatus to antibiotic resistance gene had been successfully carried out by electroporation to oidia instead of protoplasts. Since there was no protoplast generation step which required not only cell wall degrading enzymes but many skillful procedures, commercial herbicide (basta) could be used without any difficulty with simple procedure. The transformation yield was 10-20 $transformants/{\mu}g$ DNA, and the transformants were very stable even after 10 consecutive transfers through the non-selective medium.

• Journal of Microbiology
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• v.44 no.6
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• pp.617-621
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• 2006

Coprinellus congregatus secreted a laccase isozyme when the culture was transferred to an acidic liquid medium (pH 4.1). The laccase cDNA gene (clac2) was used as a probe for cloning of the genomic laccase gene (lac2) including the promoter (Plac2). The open reading frame (ORF) of lac2 had 526 deduced amino acids and four conserved copper binding domains as other fungal laccases. Recombinant plasmid (pRSlac2p-cDNA) of lac2 cDNA with its own promoter was transformed in Saccharomyces cerevisiae. Expression of the transformed lac2 gene was induced by oxidative stress ($H_2O_2$) in yeast and the survival rate of the transformed yeast strain was greatly increased when compared with that of the control strain transformed with pRS316 yeast vector.

• Korean Journal of Microbiology
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• v.41 no.4
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• pp.312-315
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• 2005

Coprinellus congregatus, known as an inky cap, is autolysed into ink soon after the maturation of the mushrooms. Electron microscopy was used to examine the ultrastructural changes associated with the autolysis as an initial step to understand the role of hydrolytic enzymes in this process. During the early stages of maturation of the mushrooms, most of cytoplasm of hymenial and subhymenial tissues seemed to be transported to the developing basidiospores. The depletion of cytoplasm within the tissues and the maturation of the basidiospores may initiate the degradation of the cell walls of the tissues. Both hymenial and subhymenial tissues seemed to degraded at the same time. This study suggested that the critical steps in the autolysis of mushrooms is not the degradation of the cytoplasm, but the degradation of the cell wall by hydrolytic enzymes such as chitinases.

• Korean Journal of Microbiology
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• v.48 no.4
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• pp.325-327
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• 2012

Experiments for growth inhibition of two different plant pathogenic fungal species, Alternaria alternata (KCTC26781) and Fusarium graminearum ZO3639 (type culture) and ASR1R1 (isolate from rice), by a chitinase (Chi2) expressed in the autolysing tissue of Coprinellus congregatus were carried out. In liquid media, Chi2 ($50{\mu}g/ml$) inhibited more than 90% of germination of A. alternata spore, and the growth of each fungal strain was totally inhibited by the addition of Chi2 at the concentrations of $70{\mu}g/ml$. When $6{\mu}g$ of Chi2 was added twice a day at the hyphal tip zone, both strains of F. graminearum showed growth inhibition as well as decreased hyphal branching.

• Korean Journal of Microbiology
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• v.49 no.4
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• pp.309-312
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• 2013

Fungal cell walls consist of various glucans and chitin. Fungi produce chitinases for their growth and development. The inky cap, Coprinellus congregatus, produces at least two different chitinases during its life cycle. Chitinase 1 (chi1) is expresses throughout its life cycle while chitinase 2 (chi2) is expressed at the mushroom autolysing phase. The cloned cDNA of chi1 is successfully expressed as a fusion protein with c-myc in Pichia pastoris, and purified by the affinity chromatography. The optimum pH and temperature of Chi1 was pH 8.0 and $35^{\circ}C$, respectively when 4-nitrophenyl N,N',N"-triacetyl-${\beta}$-D-chitotrioside was used as the substrate. The $K_m$ value and $V_{max}$ for the substrate was 0.780 mM and 0.10 OD $min^{-1}unit^{-1}$, respectively. The addition of purified Chi1 resulted in total growth inhibition against several plant pathogenic fungi such as Alternaria alternata, Fusarium graminearum and Trichoderma harzianum at the concentration of 60 ${\mu}g/ml$.

• Korean Journal of Microbiology
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• v.42 no.3
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• pp.235-237
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• 2006

When fruit bodies of Coprinellus congregatus were matured, they were autolysed to form black ink. During the developmental changes, cell walls of basidia were degraded. Laccase formed melanin which was the typical black pigment of fungi, and chitinase hydrolyzed the chitin which was a component of fungal cell wall. When laccase and chitinase genes were used as the probe for the Northern analysis to confirm their expression during the fruit body development, both gene expressions were increased as the mushroom was getting matured.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.249-253
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• 2016

The inky cap, Coprinellus congregatus, produces several laccase isozymes during its life cycle: both hyphal tip laccase and sclerotial laccase are involved in the fungal development. When this fungus was transferred to an acid liquid medium (pH 4.0-4.5), a new laccase was synthesized and secreted into the culture supernatant. In order to examine its regulation by external pH, green fluorescent protein gene was ligated at the downstream of the promoters having different lengths. These expression vectors having different promoter lengths were inserted into the fungal transformation vector, pBARGEM7-1. These expression vectors were introduced to the mating type a1 and a2 monokaryons, and the transformants were selected by the phosphinothricin resistance. Transformant a1 (a1TF) and transformant a2 (a2TF) were mated with each other to generate homozygotic dikaryon transformants. All these transformants were grown in neutral liquid medium for 5 days, and then the whole cell homogenates were transferred to the acidic liquid medium (pH 4.1). After 36 h incubation at $25^{\circ}C$, cells were harvested for the analysis of GFP expression. GFP expression was detected in the transformant having full-length promoter (2.0 kb), but other transformants having shorter length promoter (shorter than 1.29 kb) failed to show the fluorescence. Therefore, the acid-responsive element in the laccase promoter should be localized between -2.0 kb ~ -1.29 kb region.

• Proceedings of the Microbiological Society of Korea Conference
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• 2005.05a
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• pp.217.3-217
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• 2005

• Korean Journal of Microbiology
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• v.52 no.2
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• pp.226-229
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• 2016

The growth of two different fungal species, human pathogenic Cryptococcus neoformans and plant pathogenic Alternaria alternata were inhibited by a chitinase (Chi2) expressed in the autolysing tissue of Coprinellus congregatus. The cell wall thickness was reduced (up to 32%) in C. neoformans compared with that of normal cell, and polysaccharide fibers located outside of the cell wall were also severely removed. The hyphal growth of A. alternata on agar plate was stopped by the enzyme. The allergic inflammation induced by A. alternata was reduced by the enzyme reaction when compared with untreated control in a mouse model.