Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-β-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed β-1,3-galactan, but did not act on β-1,3-glucan, β-1,3:1,4-glucan, xyloglucan, and agarose. It released various β-1,3-galactooligosaccharides together with Gal from β-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on β-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-β-1,3-glucanase and endo-β-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-β-1,3-galactanase.     

An agglutinin with mitogenic and antiproliferative activities from the mushroom Flammulina velutipes

A hemagglutinin with a molecular mass of 12 kDa was isolated from the fruiting bodies of the mushroom Flammulina velutipes. Its molecular mass is similar to that of the fungal immunomodulatory protein isolated from F. velutipes (FIP-fve) with ice-cold 5% acetic acid and 50 mM 2-mercaptoethanol as extraction medium and to that of the larger 12 kDa subunit of F. velutipes lectin isolated with phosphate buffer as extraction medium. Its hemagglutinating activity cannot be inhibited by a variety of carbohydrates tested. The activity is stable between pH 4 and pH 11. Loss in activity occurred when the temperature is raised to 60 C and 70 C. Activity is indiscernible at and above 80 C. Its N-terminal sequence shows differences from that of FIP-fve. F. velutipes hemagglutinin stimulates [3H-methyl] thymidine uptake by mouse splenocytes. It inhibits proliferation of leukemia L1210 cells with an IC50 of 13 microM.     

Flammulin: a novel ribosome-inactivating protein from fruiting bodies of the winter mushroom Flammulina velutipes.

A protein with a molecular weight of 40 kDa, capable of inhibiting cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 0.25 nM, was isolated from fruiting bodies of the mushroom Flammulina velutipes. The protein, designated flammulin, was devoid of ribonuclease activity. Flammulin was unadsorbed on DEAE-cellulose at neutral pH and low ionic strength and adsorbed on CM-Sepharose and Affi-gel blue gel under similar conditions. Its N-terminal sequence demonstrates sites of similarity to those of plant ribosome-inactivating proteins (RIPs).     

Structural characterization of a polysaccharide and a beta-glucan isolated from the edible mushroom Flammulina velutipes

Two polysaccharides were isolated from the basidiomycete Flammulina velutipes, via successive hot extraction with water, 2% and 25% aq. KOH, and then submitted to freeze-drying. The precipitate formed by repeated freeze-thawing from the 2% aq. KOH extraction PK2 was analyzed by determination of its monosaccharide composition, as well as by methylation analyses using GC-MS, mono- ((13)C, (1)H NMR) and bidimensional ((1)H (obs.), (13)C HMQC) spectroscopy, and controlled Smith degradations. It was established to be a branched beta-glucan, with a main chain of (1-->3)-linked-Glcp residues, substituted at O-6 by single-unit beta-Glcp side chains. The precipitate formed by repeated freeze-thawing from the 25% KOH extraction PK25 contained Xyl, Man, and Glc and was heterogeneous by HSPEC and extraction with DMSO gave a soluble xylomannan (XM). It was homogeneous with a molar mass 30.8 x 10(4)g/mol (dn/dc=0.186). Using the above chemical analyses, it was a xylomannan with Man and Xyl in a 3:2 molar ratio. Its main chain consisted of (1-->3)-linked alpha-Manp units, mainly substituted at O-4 by beta-Xylp units or with some beta-Xylp-(1-->3)-beta-Xylp groups.     

Agrobacterium tumefaciens-mediated transformation of the vegetative dikaryotic mycelium of the cultivated mushroom Flammulina velutipes

Agrobacterium tumefaciens was used to transform the vegetative dikaryotic mycelium of Flammulina velutipes using a hygromycin B resistance gene as selectable marker. The gene coding for urogen III methyltransferase (cob) was introduced into F. velutipes dikaryotic cells. The resulting transformant cells generated a bright red fluorescence, indicating that cob is promising as a reporter gene in F. velutipes.     

Relationship between fruiting body development and extracellular laccase production in the edible mushroom Flammulina velutipes

The biochemical mechanism underlying the development of fruiting bodies in Flammulina velutipes, an edible mushroom, was investigated using the YBLB colorimetric assay to distinguish between the normal strain (FVN-1) and the degenerate strain (FVD-1). In this assay, the color of the YBLB medium (blue-green) inoculated with FVN-1 exhibiting normal fruiting body development changed to yellow, while the color of the medium inoculated with FVD-1 changed to blue. In this study, we found that this color difference originated from extracellular laccase produced by FVN-1. Moreover, FVN-1 exhibited considerably higher extracellular laccase activity than FVD-1, under conditions facilitating fruiting body formation. Overall, these findings suggest that extracellular laccase is involved in the fruiting body development process in F. velutipes.     

Comparative genomics of the mating-type loci of the mushroom Flammulina velutipes reveals widespread synteny and recent inversions

Background

Mating-type loci of mushroom fungi contain master regulatory genes that control recognition between compatible nuclei, maintenance of compatible nuclei as heterokaryons, and fruiting body development. Regions near mating-type loci in fungi often show adapted recombination, facilitating the generation of novel mating types and reducing the production of self-compatible mating types. Compared to other fungi, mushroom fungi have complex mating-type systems, showing both loci with redundant function (subloci) and subloci with many alleles. The genomic organization of mating-type loci has been solved in very few mushroom species, which complicates proper interpretation of mating-type evolution and use of those genes in breeding programs.

Methodology/Principal Findings

We report a complete genetic structure of the mating-type loci from the tetrapolar, edible mushroom Flammulina velutipes mating type A3B3. Two matB3 subloci, matB3a that contains a unique pheromone and matB3b, were mapped 177 Kb apart on scaffold 1. The matA locus of F. velutipes contains three homeodomain genes distributed over 73 Kb distant matA3a and matA3b subloci. The conserved matA region in Agaricales approaches 350 Kb and contains conserved recombination hotspots showing major rearrangements in F. velutipes and Schizophyllum commune. Important evolutionary differences were indicated; separation of the matA subloci in F. velutipes was diverged from the Coprinopsis cinerea arrangement via two large inversions whereas separation in S. commune emerged through transposition of gene clusters.

Conclusions/Significance

In our study we determined that the Agaricales have very large scale synteny at matA (∼350 Kb) and that this synteny is maintained even when parts of this region are separated through chromosomal rearrangements. Four conserved recombination hotspots allow reshuffling of large fragments of this region. Next to this, it was revealed that large distance subloci can exist in matB as well. Finally, the genes that were linked to specific mating types will serve as molecular markers in breeding.     

Enantioselective total synthesis of enokipodins A-D, antimicrobial sesquiterpenes produced by the mushroom, Flammulina velutipes

The first enantioselective total synthesis of enokipodins A, B, C and D, highly oxidized alpha-cuparenone-type sesquiterpenoids possessing antimicrobial activity, was accomplished in 8-28% overall yields from methyl (2,5-dimethoxy-4-methylphenyl)acetate by applying Meyers' diastereoselective alkylation protocol for the construction of their C7-quaternary asymmetric center. The present synthesis confirmed the absolute configuration of the enokipodins, and also constitutes a formal enantioselective synthesis of (S)-1,4-cuparenediol and (S)-cuparene-1,4-quinone.     

The first characterized asparaginase from a basidiomycete, Flammulina velutipes

Flammulina velutipes enjoys high popularity as an edible mushroom in Asian cuisines. Investigating the secretion of peptidases in nutrient media enriched with gluten, an enzyme was noticed that catalyzed the deamidation of L-asparagine and L-glutamine. The enzyme was purified to electrophoretic homogeneity by foaming and SEC. PAGE analysis revealed a protein of about 85 kDa with 13 kDa subunits indicating a hexameric protein. Degenerated primers were deduced from peptide fragments and the complete coding sequence of 372 bp was determined. The gene of Flammulina velutipes asparaginase (FvNase) over expressed in E. coli achieved an L-asparagine-hydrolyzing activity of 16 U/mL in crude extract, which was five times higher than its L-glutamine-hydrolyzing ability. The enzyme showed a pH-optimum at pH 7, remarkable tolerance towards elevated temperature and sodium chloride concentration in both the native and recombinant form, and no significant homology to any conserved domains of published asparaginases or glutaminases.     

Flammutoxin, a cytolysin from the edible mushroom Flammulina velutipes, forms two different types of voltage-gated channels in lipid bilayer membranes

Flammutoxin, a 31-kDa cardiotoxic and cytolytic protein from the edible mushroom Flammulina velutipes, has been shown to assemble into a pore-forming annular oligomer with outer and inner diameters of 10 and 5 nm on the target cells [Tomita et al., Biochem. J. 333 (1998) 129-137]. Here we studied electrophysiological properties of flammutoxin channels using planar lipid bilayer technique, and found that flammutoxin formed two types of moderately cation-selective, voltage-gated channels with smaller and larger current amplitudes (1-4.5 pA and 20-30 pA, respectively, at 20 mV) in the lipid bilayers composed of phospholipid and cholesterol. The larger-conductance single channel showed the properties of a wide water-filled pore such as a linear relationship between channel conductance and salt concentration of the bathing solution. The functional diameter of the larger-conductance channel was estimated to be 4-5 nm by measuring the current conductance in the presence of polyethylene glycols of various sizes. In contrast, the smaller-conductance single channels showed a non-linear current to voltage curve and a saturating conductance to increasing salt concentration. These results suggest that the larger-conductance channel of flammutoxin corresponds to the hemolytic pore complex, while the smaller-conductance channel may reflect the intermediate state(s) of the assembling toxin.     

金针菇谷氨酸脱氢酶基因的克隆及原核表达

利用简并引物和RT-PCR方法从金针菇(Flammulina velutipes)幼嫩子实体中克隆获得FvGDH全长cDNA序列.构建入门载体pGWC-FvGDH,利用Gateway克隆技术的LR反应构建原核重组表达载体pDESTl7-FvGDH,转化大肠杆菌BL21(DE3).通过IPTG法诱导表达融合蛋白并进行表达条件优化.SDS-PAGE蛋白电泳分析表明,融合蛋白相对分子质量约为53 kD,与预测的一致.最佳表达条件为温度30℃、IPTG浓度0.4mmol/L、诱导4 h.融合蛋白表达量较高,实现了FvGDH的高效表达,并利用Western blotting对其特异性进行鉴定.FvGDH基因高效原核表达体系的成功建立,为进一步研究FvGDH的酶促动力学奠定了基础.     

Highly oxidized cuparene-type sesquiterpenes from a mycelial culture of Flammulina velutipes

Cuparene-type sesquiterpenes were isolated from a culture broth of Flammulina velutipes (Curt.:Fr.) Sing. Using spectroscopic methods (HR-MS, 1H and 13C NMR, and 2D NMR, spectroscopy), their structures were determined to be 2,3,4,5-tetrahydro-2,7-dihydroxy-5,8,10,10-tetramethyl-2,5-methano-1- benzoxepin and 5-methyl-2-(3-oxo-1,2,2-trimethylcyclopentyl)benzoquinone. Both showed antimicrobial activity against Cladosporium herharum and Bacillus subtilis.     

Purification and characterization of a lectin from the mushroom, Flammulina veltipes

A lectin was purified to homogeneity from the mushroom, Flammulina veltipes, by zinc acetate treatment and CM-cellulose column chromatography. Its molecular weight was estimated to be 20,000 by gel filtration and polyacrylamide gel electrophoresis. The lectin does not contain carbohydrate, half-cystine, methionine, or histidine. On gel filtration sith Sepharose 6B in the presence of 6M guanidine-HCl, the purified lectin dissociated into two nonidentical subunits, FVA-L (molecular weight, 12,000) and FVA-S (8,000). The hemagglutinating activity was retained only in the FVA-L subunit. The lectin is mitogenic with respect to mouse spleen lymphocytes.     

金针菇液态发酵培养基的筛选

研究不同碳源、氮源及无机盐对金针菇菌丝体产量的影响,采用正交设计法进行金针菇液态发酵培养基配方筛选和优化,确定了最佳培养基配方为大米粉3%、酵母粉1%、KH2PO40.1%、MgSO4.7H2O 0.05%。同时利用此配方培养测定了发酵过程pH、还原糖和氨基氮含量的变化,结果表明:发酵过程中pH变化很小,培养末期略有上升;还原糖含量呈先增后降的变化,氨基氮含量呈上升趋势。     

Transformation of the mushroom species Hypsizigus marmoreus,Flammulina velutipes,and Grifola frondosa by an Agrobacterium-mediated method using a universal transformation plasmid

Agrobacterium tumefaciens-mediated transformation (AMT) was successfully applied to mycelia of the 3 economically important mushrooms Hypsizigus marmoreus, Flammulina velutipes, and Grifola frondosa. We used the hygromycin B resistance gene (hph) under the control of the Cryptococcus neoformans actin promoter. Eighty-six resistant strains of H. marmoreus, 4 of F. velutipes, and 2 of G. frondosa were obtained. All transformants were highly resistant to hygromycin B, suggesting that the C. neoformans actin promoter has a potential universal promoter activity in basidiomycetes. Southern analysis revealed random but single integration of the hph gene.     

一种金针菇核糖体失活蛋白的分离纯化研究

获得一种为研究其他菌类核糖体失活蛋白的对照品,并论述一种金针菇核糖体失活蛋白的分离纯化及其活性的研究结果。实验中采用了DEAE和CM-离子交换纤维素与Bio-Gel 100柱层析方法。从1 000 g新鲜金针菇中得到5.58 mg的核糖体失活蛋白-Velutin,并证明其具有明显的抑制蛋白质的翻译作用,同时简要介绍了它的应用及展望。分离纯化到具有活性的Velutin,分子量为13.8 ku。     

金针菇菌丝体的深层培养及多糖制取

从２０株金针菇菌株中筛选出适于液体发酵的９４Ｂ２株。该菌种适宜的摇瓶培养条件：培养温度２３～２４℃,ｐＨ６．５,接液体种量１０％,装液量≤５０ｍｌ／５００ｍｌ瓶;不同的间歇振荡形式对菌丝球产量和形态有显著影响。深层发酵工艺相似于抗生素发酵。发酵期间发酵液ｐＨ变化幅度很小,总糖、还原糖、氨基氮与菌丝生长量有一定的相关性。发酵酵的菌丝产量可达４７．５ｇ（湿重）／１００ｍｌ、胞外多糖达１７４．７ｋｇ／１００ｍｌ发酵液上清、胞内多糖达３３８．１ｍｇ／１００ｇ湿菌丝体。