Genome-Wide Comparison of Carbohydrate-Active Enzymes (CAZymes) Repertoire of Flammulina ononidis

Whole-genome sequencing of Flammulina ononidis, a wood-rotting basidiomycete, was performed to identify genes associated with carbohydrate-active enzymes (CAZymes). A total of 12,586 gene structures with an average length of 2009 bp were predicted by the AUGUSTUS tool from a total 35,524,258 bp length of de novo genome assembly (49.76% GC). Orthologous analysis with other fungal species revealed that 7051 groups contained at least one F. ononidis gene. In addition, 11,252 (89.5%) of 12,586 genes for F. ononidis proteins had orthologs among the Dikarya, and F. ononidis contained 8 species-specific genes, of which 5 genes were paralogous. CAZyme prediction revealed 524 CAZyme genes, including 228 for glycoside hydrolases, 21 for polysaccharide lyases, 87 for glycosyltransferases, 61 for carbohydrate esterases, 87 with auxiliary activities, and 40 for carbohydrate-binding modules in the F. ononidis genome. This genome information including CAZyme repertoire will be useful to understand lignocellulolytic machinery of this white rot fungus F. ononidis.

Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes

Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi.

Genome-Wide Identification and Characterization of Novel Laccase Genes in the White-Rot Fungus Flammulina velutipes

The aim of this study was to identify and characterize new Flammulina velutipes laccases from its whole-genome sequence. Of the 15 putative laccase genes detected in the F. velutipes genome, four new laccase genes (fvLac-1, fvLac-2, fvLac3, and fvLac-4) were found to contain four complete copper-binding regions (ten histidine residues and one cysteine residue) and four cysteine residues involved in forming disulfide bridges, fvLac-1, fvLac-2, fvLac3, and fvLac-4, encoding proteins consisting of 516, 518, 515, and 533 amino acid residues, respectively. Potential N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified in the cDNA sequence of fvLac-1 (Asn-454), fvLac-2 (Asn-437 and Asn-455), fvLac-3 (Asn-111 and Asn-237), and fvLac4 (Asn-402 and Asn-457). In addition, the first 19~20 amino acid residues of these proteins were predicted to comprise signal peptides. Laccase activity assays and reverse transcription polymerase chain reaction analyses clearly reveal that CuSO4 affects the induction and the transcription level of these laccase genes.

Inducible and constitutive expression of fip-fve from Flammulina velutipes in Pichia pastoris GS115 / 生物工程学报

We transformed the fip-fve gene into Pichia pastoris GS115 for inducible and constitutive expression to obtain feasible bioactvie recombinant Fip-fve. The fip-fve gene was cloned from Flammulina velutipes fruting body by PCR and ligated to pPIC9 to construct inducible expression vector pPIC9-FIP-fve, and promotor pgap was used to replace the paox1 to construct constitutive expression vector pPIC9-PGAP-FIP-fve. These two vectors were used to transform P. pastoris by PEG method. The fip-fve was expressed after histamine-absence screening and yeast colony PCR. The inducible expression level reached 158.2 mg/L at the fourth day and the constitutive expression level was 46.3 mg/L and 29.5 mg/L using glucose and glycerol, respectively. The SDS-PAGE and Western blotting both proved the correctness of rFip-fve, and the hemagglutination test indicats the rFip-fve's bioactivity.

Use of P450 cytochrome inhibitors in studies of enokipodin biosynthesis

Enokipodins A, B, C, and D are antimicrobial sesquiterpenes isolated from the mycelial culture medium of Flammulina velutipes, an edible mushroom. The presence of a quaternary carbon stereocenter on the cyclopentane ring makes enokipodins A-D attractive synthetic targets. In this study, nine different cytochrome P450 inhibitors were used to trap the biosynthetic intermediates of highly oxygenated cuparene-type sesquiterpenes of F. velutipes. Of these, 1-aminobenzotriazole produced three less-highly oxygenated biosynthetic intermediates of enokipodins A-D; these were identified as (S)-(-)-cuparene-1,4-quinone and epimers at C-3 of 6-hydroxy-6-methyl-3-(1,2,2-trimethylcyclopentyl)-2-cyclohexen-1-one. One of the epimers was found to be a new compound.

Cloning and Characterization of a Novel Laccase Gene, fvlac7, Based on the Genomic Sequence of Flammulina velutipes

Laccases (EC 1.10.3.2) are copper-containing polyphenol oxidases found in white-rot fungi. Here, we report the cloning and analysis of the nucleotide sequence of a new laccase gene, fvlac7, based on the genomic sequence of Flammulina velutipes. A primer set was designed from the putative mRNA that was aligned to the genomic DNA of F. velutipes. A cDNA fragment approximately 1.6-kb long was then amplified by reverse transcriptase-PCR using total RNA, which was subsequently cloned and sequenced. The cDNA sequence of fvlac7 was then compared to that of the genomic DNA, and 16 introns were found in the genomic DNA sequence. The fvlac7 protein, which consists of 538 amino acids, showed only 42~51% identity with 12 different mushroom species containing two laccases of F. velutipes, suggesting the fvlac7 is a novel laccase gene. The first 25 amino acids of Fvlac7 correspond to a predicted signal sequence, four copper-binding sites, and four N-glycosylation sites. Fvlac7 cDNA was heterologously overexpressed in an Escherichia coli system with an approximate expected molecular weight of 60 kDa.

Characterization of Species of Cladobotryum which Cause Cobweb Disease in Edible Mushrooms Grown in Korea

Four Cladobotryum isolates were collected from four different commercially grown mushroom types infected with cobweb disease in Cheongdo-gun and Chilgok-gun of Gyeongbuk Province, Korea in 2010. The isolates were identified as C. mycophilum from Agaricus bisporus and Pleurotus eryngii, C. varium from Flammulina velutipes and Hypsizygus marmoreus. The cultural characteristics of the four isolates were investigated using potato dextrose agar (PDA) media under nine different temperatures ranging from 5~32degrees C. Rapid growth of the isolates to colony diameters of 47~82 mm was observed at conditions of 18~22degrees C. No growth was observed at 32degrees C. C. mycophilum produced a yellowish red pigment while C. varium produced a cream colored pigment after cultivation for 25 days on PDA. Phylogenetic analysis of the internal transcribed spacer region and partial 28S rDNA from the four isolates confirmed they were C. mycophilum and C. varium. Cross pathogenicity tests revealed that the two isolates of C. mycophilum were highly pathogenic toward three mushroom types, but not toward H. marmoreus. The two isolates of C. varium were less pathogenic than those of C. mycophilum, but were pathogenic toward all mushroom types evaluated.

Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/microg of DNA in 1 x 107 protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

Determination of Mineral Components in the Cultivation Substrates of Edible Mushrooms and Their Uptake into Fruiting Bodies

The mineral contents of the cultivation substrates, fruiting bodies of the mushrooms, and the postharvest cultivation substrates were determined in cultivated edible mushrooms Pleurotus eryngii, Flammulina velutipes, and Hypsizigus marmoreus. The major mineral elements both in the cultivation substrates and in the fruiting bodies were K, Mg, Ca, and Na. Potassium was particularly abundant ranging 10~13 g/kg in the cultivation substrates and 26~30 g/kg in the fruiting bodies. On the contrary, the calcium content in the fruiting bodies was very low despite high concentrations in the cultivation substrates, indicating Ca in the cultivation substrates is in a less bio-available form or the mushrooms do not have efficient Ca uptake channels. Among the minor mineral elements determined in this experiment, Cu, Zn, and Ni showed high percentage of transfer from the cultivation substrates to the fruiting bodies. It is noteworthy that the mineral contents in the postharvest cultivation substrates were not changed significantly which implies that the spent cultivation substrates are nutritionally intact in terms of mineral contents and thus can be recycled as mineral sources and animal feeds.

A Survey on Preferences for Vegetable Cooking Methods and Vegetable-aversion-related Factors among Elementary School Students in Kwangju and Chonnam Regions / 대한지역사회영양학회지

This study was conducted to survey multiple factors of aversion to vegetables and preferences for vegetable-related recipes in school meal services among elementary school children in order to help develop various menus and recipes for school meal services. Questionnaire survey was carried out with the study subjects, who were 401 children in 6th grade attending elementary schools in Chonnam and Kwangju metropolitan regions. Results from the survey can be summarized as follows: 65.1% of respondents answered they try to eat vegetables and other namul side dishes served in school meal service for health. As for the frequency of taking vegetables and namul side dishes out of daily meals, 47.4% of respondents chose 'once or twice'. The reasons for aversion to vegetables in boys were taste and cooking method, while girls were taste and feeling between teeth. In boys there were no differences between regions of Kwangju and Chonnam but the tendency of aversion to vegetables was significantly high in girls. As for the aspects of vegetable aversion of subjects, 46.9% of respondents took up 'black & purple' in the unfavorable color of vegetables. 49.1% in 'bitterness' and 39.2% in 'greasiness''were in terms of the aversive taste of vegetables. The aversive vegetable recipes were 58.6% in 'raw &seasoned' and the unfavorable feeling of vegetables were 53.1% in 'squashiness'. There were differences between regions of Kwangju and Chonnam with boys in color and cooking method in girls. Results from the survey on their preferences for vegetable recipes showed that leafy vegetables like crown daisy (raw/slightly seasoned) and pak choi (broth/pot stew) fell to the most aversive category, while bean sprouts (broth/pot stew) were chosen as the most favorable one. Among root begetables bell-flowers were found to belong to the least preferred recipe, while potatoes were proven to be most preferable in terms of recipes. As for fruit vegetables and other vegetables, all respondents didn't like 'fatsia shoots' vegetable and it's cooking method and they preferred 'green pumpkins (broth/pot stew)'. In respect of mushrooms, enoki mushroom (broth/pot stew) was found most preferred and had high tendency of preferences in boys and girls in Kwangju compared with Chonnam region. The study results indicated that respondents did not show big differences in factors influencing them to be averse to vegetables and their preferences for vegetable recipes depending on regions. In order to have high preference and intake in children's diets, it needs to study in reform of menu about using namul or vegetables mixed with meats and fruits that children preferred or applying roasted and fried other less than namul.

Cytoprotective effect of polysaccharide isolated from different mushrooms against 7-ketocholesterol induced damage in mouse liver cell line (BNL CL. 2)

Cytoprotective ability of polysaccharides isolated from different edible mushrooms was investigated on the 7-ketocholesterol-induced damaged cell line. Polysaccharide extracts from six different edible mushrooms-Flammulina velutipes, Peurotus ostreatus, Lentinus edodes, Agrocybe aegerita, Agaricus blazei, and Cordyceps militaris- were prepared by hot water extraction and alcohol precipitation. Cytoprotective ability was evaluated by measuring the viable cells of the normal embryonic liver cell line (BNL CL. 2) in the presence of 7-ketocholesterol. At 80 microgram/mL of 7-ketocholesterol, cytotoxicity was very high with a loss of 98% of viable cells after 20 h of incubation. With the addition of 200 microgram/mL of each polysaccharide isolate to the cell line containing 80 microgram/mL of 7-ketocholesterol, polysaccharide isolates from both Flammulina velutipes and Peurotus ostreatus could significantly inhibit the 7-ketochoelsterol-induced cytotoxicity in the cells. But other polysaccharide isolates were not effective in inhibiting cell damage caused by the oxLDL-induced cytotoxicity.

Agrobacterium-mediated Transformation of the Winter Mushroom, Flammulina velutipes

Flammulina velutipes was transformed efficiently by Agrobacterium-mediated transformation system. The transformation frequency was about 16% with the gill tissues of the fungal fruiting body. Southern hybridization and genetic analysis suggest that the introduced DNA was inserted onto different locations of the fungal genome, and inherited stably to the next generation via basidiospores. Transformation or gene tagging with Agrobacterium T-DNA based vector should be useful for wide ranges of genetic or molecular biological studies of the mushroom.

Molecular Marker Related to Fruitbody Color of Flammulina velutipes

White and brown strains of Flammulina velutipes were inter-crossed. All F1 showed light-brown fruitbody, suggesting that a gene for the brown fruitbody was incompletely dominant against the white one. And backcross experiment showed that more than two genes were involved in color determination. To isolate a molecular marker linked to fruitbody color, a set of primers was designed from a sequence of clones derived by a bulked segregant analysis. These markers showed a specific band which co-segregated with brown fruitbody forming strains.

Breeding of Flammulina velutipes Strains Adaptable to Elevated-temperature

Winter mushroom, Flammulina velutipes, needs low temperature during its cultivation. To save on farm costs, especially during summer, a strain adaptable to a higher or elevated-temperature must be developed. At the start of breeding program, parental strains which could endure high temperature were obtained. Seuenty four dikaryotic strains were collected and divided into four groups according to the nature of temperature. They also had different fruiting temperature. Finally we selected three brown strains ASI 4048, 4057 and 4072, and collected their spores. These selected strains can germinate even at a high temperature of 32degrees C, which were dramatically higher than the other strains. Based on these results, the new white strain adapted to mid-temperature by backcross mating was developed. Molecular markers were applied to select white fruit-body producing strains without cultivation. They showed a specific band which co-segregated with brown fruitbody forming strains in BC1F1 progenies. Selected white strains were tested under several elevated temperature conditions.