In Vitro Assessment of the Antioxidant and Anticancer Properties of Flammulina velutipes Stipe Extracts.

BACKGROUND/AIM: Flammulina velutipes (FV), also known as the golden needle mushroom, is an edible and medicinal fungus that contains bioactive substances regulating various physiological functions. While the fruiting bodies of FV are commonly consumed, their stipes are often discarded despite containing polysaccharides. In this study, the biological functions of FV stipes (FV-S) were investigated to reduce waste and pollution while increasing their value. MATERIALS AND METHODS: The antioxidant activity of FV was evaluated using three methods: the DPPH radical-scavenging capacity assay, ferrous ion chelating assay, and reducing power analysis. The anti-cancer potential was assessed through MTT viability and immunoblotting analyses. RESULTS: Results showed that FV-S had higher polysaccharide and total phenolic contents and greater antioxidant abilities, particularly in ethanolic extracts. FV-S also exhibited significant anticancer properties, specifically in hot water extracts with high polysaccharide contents, and suppressed prostate cancer cell viability by inhibiting androgen receptor and PCa-specific antigen mRNA expression while inducing caspase-3/7 activation. CONCLUSION: FV-S is rich in bioactive components, possesses higher antioxidant and anticancer abilities, and has potential as an anticancer agent, which could enhance the value of FV.

The Biosynthetic Pathway of Ergothioneine in Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes).

Ergothioneine is a natural 2-thiol-amidazole amino acid that plays an important role in inflammation, depression, and cardiovascular disease. Flammulina velutipes is a common basidiomycete mushroom rich in ergothioneine (EGT). However, the biosynthetic pathway of EGT in F. velutipes is still unclear. In this study, the F. velutipes ergothioneine biosynthetic gene 1 (Fvegtl), F. velutipes ergothioneine biosynthetic gene 2 (Fvegt2), and F. velutipes ergothioneine biosynthetic gene 3 (Fvegt3) were cloned and expressed, and the activities of the proteins encoded by these three genes (FvEgt1, F. velutipes ergothioneine biosynthase 1; FvEgt2, F. velutipes ergothioneine biosynthase 2; and FvEgt3, F. velutipes ergothioneine biosynthase 3) were identified. The results showed that FvEgtl not only has the function of methyltransferase, but also has the function of hercynlcysteineteine sulfoxide (Hersul) synthase, which can catalyze the production of Hersul from histidine and cysteine in F. velutipes. FvEgt2 and FvEgt3 are two functionally different cysteine desulfurase enzymes. Among them, FvEgt2 is a cysteine-cysteine desulfurase-which catalyzes the activation of the S-H bond on cysteine, while FvEgt3 is a pyridoxal phosphate (PLP)-dependent cysteine desulfurase responsible for catalyzing the production of ketimine complex. Our results show that FvEgt1/FvEgt2/FvEgt3 can simultaneously catalyze the production of EGT by histidine, cysteine, and pyridoxal phosphate. Collectively, the in vitro synthesis of EGT in the edible fungus F. velutipes was first achieved, which laid the foundation for the biological production of EGT.

Combining transcriptomics and metabolomics to reveal the underlying molecular mechanism of ergosterol biosynthesis during the fruiting process of Flammulina velutipes.

BACKGROUND: Flammulina velutipes has been recognized as a useful basidiomycete with nutritional and medicinal values. Ergosterol, one of the main sterols of F. velutipes is an important precursor of novel anticancer and anti-HIV drugs. Therefore, many studies have focused on the biosynthesis of ergosterol and have attempted to upregulate its content in multiple organisms. Great progress has been made in understanding the regulation of ergosterol biosynthesis in Saccharomyces cerevisiae. However, this molecular mechanism in F. velutipes remains largely uncharacterized. RESULTS: In this study, nine cDNA libraries, prepared from mycelia, young fruiting bodies and mature fruiting bodies of F. velutipes (three replicate sets for each stage), were sequenced using the Illumina HiSeq™ 4000 platform, resulting in at least 6.63 Gb of clean reads from each library. We studied the changes in genes and metabolites in the ergosterol biosynthesis pathway of F. velutipes during the development of fruiting bodies. A total of 13 genes (6 upregulated and 7 downregulated) were differentially expressed during the development from mycelia to young fruiting bodies (T1), while only 1 gene (1 downregulated) was differentially expressed during the development from young fruiting bodies to mature fruiting bodies (T2). A total of 7 metabolites (3 increased and 4 reduced) were found to have changed in content during T1, and 4 metabolites (4 increased) were found to be different during T2. A conjoint analysis of the genome-wide connection network revealed that the metabolites that were more likely to be regulated were primarily in the post-squalene pathway. CONCLUSIONS: This study provides useful information for understanding the regulation of ergosterol biosynthesis and the regulatory relationship between metabolites and genes in the ergosterol biosynthesis pathway during the development of fruiting bodies in F. velutipes.

Comparative Proteome Reveals Metabolic Changes during the Fruiting Process in Flammulina velutipes.

Understanding the molecular mechanisms regulating the fruiting process in macro-fungi, especially industrially cultivated mushrooms, has long been a goal in mycological research. To gain insights into the events accompanying the transformation of mycelia into fruit-bodies in Flammulina velutipes, proteins expressed characteristically and abundantly at primordium and fruit-body stages were investigated by using the iTRAQ labeling technique. Among the 171 differentially expressed proteins, a total of 68 displayed up-regulated expression levels that were associated with 84 specific KEGG pathways. Some up-regulated proteins, such as pyruvate carboxylase, aldehyde dehydrogenase, fatty acid synthase, aspartate aminotransferase, 2-cysteine peroxiredoxin, FDS protein, translation elongation factor 1-alpha, mitogen-activated protein kinases (MAPKs), and heat-shock protein 70 that are involved in carbohydrate metabolism, carotenoid formation, the TCA cycle, MAPK signaling pathway, and the biosynthesis of fatty acids and branched-chain amino acids, could serve as potential stage-specific biomarkers to study the fruiting process in F. velutipes. Knowledge of the proteins might provide valuable evidence to better understand the molecular mechanisms of fruit-body initiation and development in basidiomycete fungi. Furthermore, this study also offers valuable evidence for yield improvement and quality control of super golden-needle mushroom in practice.

Expression of enterovirus 71 virus-like particles in transgenic enoki (Flammulina velutipes).

No commercial vaccines are currently available for enterovirus 71 (EV71) infection. Oral virus-like particle (VLP) vaccines are regarded as a better choice for prevention from food-borne diseases compared with injected whole virus vaccines. Unfortunately, the application of oral VLP vaccines produced from transgenic plants was limited due to the concerns of gene contamination. Alternatively, using transgenic mushrooms retains the advantages of transgenic plants and tremendously reduce risks of gene contamination. Polycistronic expression vectors harboring the glyceraldehyde-3-phospho-dehydrogenase promoter to codrive EV71 structural protein P1 and protease 3C using the 2A peptide of porcine teschovirus-1 were constructed and introduced into Flammulina velutipes via Agrobacterium tumefaciens-mediated transformation. The analyses of the genomic PCR, Southern blotting, and RT-PCR showed that the genes of P1 and 3C were integrated into the chromosomal DNA through a single insertion, and their resulting mRNAs were transcribed. The Western blotting analysis combined with LC-MS/MS demonstrated that EV71 VLPs were composed of the four subunit proteins digested from P1 polyprotein by 3C protease. Through the use of a single particle electron microscope, images of 1705 particles with diameter similar to the EV71 viron were used for 3D reconstruction. Protrusions were observed on the surface in the 2D class averages, and a 3D reconstruction of the VLPs was obtained. In conclusion, EV71 VLPs were successfully produced in transgenic F. velutipes using a polycistronic expression strategy, which indicates that this approach is promising for the development of oral vaccines produced in mushrooms.

Cloning and characterization of a novel O-methyltransferase from Flammulina velutipes that catalyzes methylation of pyrocatechol and pyrogallol structures in polyphenols.

A novel O-methyltransferase gene was isolated from Flammulina velutipes. The isolated full-length cDNA was composed of a 690-nucleotide open reading frame encoding 230 amino acids. A database search revealed that the deduced amino acid sequence was similar to those of other O-methyltransferases; the highest identity was only 61.8% with Laccaria bicolor. The recombinant enzyme was expressed by Escherichia coli. BL21 (DE3) was assessed for its ability to methylate (-)-epigallocatechin-3-O-gallate (EGCG). LC-TOF-MS and NMR revealed that the enzyme produced five kinds of O-methylated EGCGs: (-)-epigallocatechin-3-O-(3-O-methyl)gallate, (-)-epigallocatechin-3-O-(4-O-methyl)gallate, (-)-epigallocatechin-3-O-(3,4-O-dimethyl)gallate, (-)-epigallocatechin-3-O-(3,5-O-dimethyl)gallate, and (-)-4'-O-methylepigallocatechin-3-O-(3,5-O-dimethyl)gallate. The substrate specificity of the enzyme for 20 kinds of polyphenols was assessed using the crude recombinant enzyme of O-methyltransferase. This enzyme introduced methyl group(s) into polyphenols with pyrocatechol and pyrogallol structures.

Cloning and analysis of a functional promoter of fungal immunomodulatory protein from Flammulina velutipes.

Fugal immunomodulatory protein from Flammulina velutipes (FIP-fve) belongs to FIPs family, which has precious pharmaceutical value. To understand the regulatory mechanism of FIP-fve expression, we have cloned a 900 bp genomic DNA fragment from the transcriptional start site of the FIP-fve gene using genomic walker technology. Sequence analysis showed the presence of several eukaryotic transcription factor binding motifs in the 900 bp of upstream region of the FIP-fve gene, which contains one putative TATA-boxes, four possible CAAT-boxes, one ABRE, one ARE, three CGTCA-motifs, two TGA-elements and four Skn-1 motifs. The eukaryotic expression vector pfveP:: GUS-GFP was transferred into tobacco via an agrobacterium-mediated leaf disc transformation. The results showed that the FIP-fve promoter could induce the reporter gene GUS or GFP expression in different tissues of tobaccos. This study would lay a foundation for expression regulation of FIP-fve and development of genetic-modified plant products.

Bioproduction of baccatin III, an advanced precursor of paclitaxol, with transgenic Flammulina velutipes expressing the 10-deacetylbaccatin III-10-O-acetyl transferase gene.

BACKGROUND: 10-Deacetylbaccatin III (10-DAB) and baccatin III are intermediates in the biosynthesis of Taxol (an anti-cancer drug) and useful precursors for semi-synthesis of the drug. In this study, a bioconversion system was established for the production of baccatin III, an advanced precursor of paclitaxel, in the transgenic mushroom Flammulina velutipes expressing the 10-deacetylbaccatin III-10ß-O-acetyltransferase gene. The expression vector pgFvs-TcDBAT containing the 10-deacetylbaccatin III-10ß-O-acetyltransferase (DBAT) gene was constructed and transformed into the cells of F. velutipes by polyethylene glycol-mediated protoplast transformation. RESULTS: Polymerase chain reaction and Southern blotting analysis verified the successful integration of the exogenous DBAT gene into the genome of F. velutipes. Reverse transcription polymerase chain reaction and enzyme activity analyses confirmed that the DBAT gene was expressed in F. velutipes, and DBAT is able to convert substrate into baccatin III. CONCLUSION: The DBAT gene from the plant Taxus chinensis can be functionally expressed in F. velutipes. Transgenic F. velutipes expressing the DBAT gene is able to produce the target product, baccatin III. This is the first report about the transformation and expression of paclitaxel biosynthetic gene in the edible mushroom F. velutipes. This represents a significant step towards bio-production of paclitaxel and its advanced precursor baccatin III in an edible fungus.

Functional expression of FIP-fve, a fungal immunomodulatory protein from the edible mushroom Flammulina velutipes in Pichia pastoris GS115.

FIP-fve is a bioactive protein isolated from the mushroom Flammulina velutipes, which belongs to the fungal immunomodulatory protein (FIP) family and demonstrates several kinds of biological activities including anti-allergy, anti-tumor and immunomodulation. In the current study, the FIP-fve gene was cloned and expressed in the yeast Pichia pastoris GS115, and its correctness was confirmed by SDS-PAGE and Western blot. Optimal expression of rFIP-fve was observed when the P. pastoris cells were cultured in 1% methanol for 9 6h, which resulted in a yield of 258.2 mg l(-1). The rFIP-fve protein was subsequently purified via ammonium sulfate precipitation and Sephadex G-100 gel chromatography. In vitro bioactivity examination showed that rFIP-fve could agglutinate human red blood cells and stimulate the cell viability of murine splenocytes. The immunomodulatory capacity and anti-tumor activity of rFIP-fve were demonstrated by enhanced interleukin-2 secretion and interferon-γ release from the murine lymphocytes, similar to the biological FIP-fve. In conclusion, the FIP-fve gene was functionally and effectively expressed in P. pastoris, and rFIP-fve displayed biological activities similar to those of native FIP-fve. These results indicated the potential use of rFIP-fve from P. pastoris as an effective and feasible source for therapeutic studies and medical applications.

Characterization of a gene coding for a putative adenosine deaminase-related growth factor by RNA interference in the basidiomycete Flammulina velutipes.

A full-length cDNA coding for a putative adenosine deaminase (Fv-ada) was isolated from the basidiomycete Flammulina velutipes. Fv-ada encodes a polypeptide consisting of 537 amino acid residues, which has a consensus sequence conserved among adenosine deaminase-related growth factors (ADGF) found in several metazoa, including chordates and insects. Fv-ada transcript was detected at all stages of growth in dikaryotic F. velutipes cells, with a peak at the primordial stage. Heterologous expression of Fv-ada in the yeast Pichia pastoris produced recombinant Fv-ADA that catalyzed the conversion of adenosine to inosine. Dikaryotic mycelia from F. velutipes were transformed with the binary plasmid pFungiway-Fv-ada, which was designed to suppress the expression of Fv-ada through RNA interference. The growth rates of the resulting transformants were retarded in response to the degree of suppression, indicating that Fv-ada plays an important role in the mycelial growth of F. velutipes. These results suggested that ADGF could function as growth factors in fungi, as is seen in other eukaryotes.

Expression of an oxalate decarboxylase impairs the necrotic effect induced by Nep1-like protein (NLP) of Moniliophthora perniciosa in transgenic tobacco.

Oxalic acid (OA) and Nep1-like proteins (NLP) are recognized as elicitors of programmed cell death (PCD) in plants, which is crucial for the pathogenic success of necrotrophic plant pathogens and involves reactive oxygen species (ROS). To determine the importance of oxalate as a source of ROS for OA- and NLP-induced cell death, a full-length cDNA coding for an oxalate decarboxylase (FvOXDC) from the basidiomycete Flammulina velutipes, which converts OA into CO(2) and formate, was overexpressed in tobacco plants. The transgenic plants contained less OA and more formic acid compared with the control plants and showed enhanced resistance to cell death induced by exogenous OA and MpNEP2, an NLP of the hemibiotrophic fungus Moniliophthora perniciosa. This resistance was correlated with the inhibition of ROS formation in the transgenic plants inoculated with OA, MpNEP2, or a combination of both PCD elicitors. Taken together, these results have established a pivotal function for oxalate as a source of ROS required for the PCD-inducing activity of OA and NLP. The results also indicate that FvOXDC represents a potentially novel source of resistance against OA- and NLP-producing pathogens such as M. perniciosa, the causal agent of witches' broom disease of cacao (Theobroma cacao L.).