The Fvclp1 gene regulates mycelial growth and fruiting body development in edible mushroom Flammulina velutipes.

Fruiting body development in Agaricomycetes represents the most complex and unclear process in the fungi. Mating type pathways (A and B) and transcription factors are important regulators in the sexual development of mushrooms. It is known that clampless1 (clp1) is an additional gene that participate under the homeodomain (HD) genes in the matA pathway and clp1 inactivation blocks clamps formation in Coprinopsis cinerea. In this study we identified and analyzed a homologous Fvclp1 gene in the edible mushroom Flammulina velutipes. The coding sequence of the Fvclp1 was 1011 bp without intron interruption, encoding a protein of 336 amino acids. To exhibit the role of Fvclp1 in clamp development and fruiting body formation, knockdown and overexpression mutants were prepared. No significant difference was observed in the monokaryotic hyphal morphology of overexpression and knockdown transformants. In the dikaryotic hyphae from the compatible crossings between the wild-type L22 strain and Fvclp1 knockdown or overexpression mutants, clamp connections developed. However, knockdown mutants could generate fewer fruiting bodies than the wild-type strain. On the contrary, reduced mycelial growth rate but improved fruiting ability was observed in the dikaryotic Fvclp1 overexpression mutants as compared to the wild-type strain. These results indicate that Fvclp1 is necessary and actively involved in fruiting body development in F. velutipes. Overall, these findings suggest that further studies on the function of Fvclp1 would advance our understanding of sexual reproduction and fruiting body development in edible mushrooms.

Genome-wide analysis and prediction of genes involved in the biosynthesis of polysaccharides and bioactive secondary metabolites in high-temperature-tolerant wild Flammulina filiformis.

BACKGROUND: Flammulina filiformis (previously known as Asian F. velutipes) is a popular commercial edible mushroom. Many bioactive compounds with medicinal effects, such as polysaccharides and sesquiterpenoids, have been isolated and identified from F. filiformis, but their biosynthesis and regulation at the molecular level remains unclear. In this study, we sequenced the genome of the wild strain F. filiformis Liu355, predicted its biosynthetic gene clusters (BGCs) and profiled the expression of these genes in wild and cultivar strains and in different developmental stages of the wild F. filiformis strain by a comparative transcriptomic analysis. RESULTS: We found that the genome of the F. filiformis was 35.01 Mb in length and harbored 10,396 gene models. Thirteen putative terpenoid gene clusters were predicted and 12 sesquiterpene synthase genes belonging to four different groups and two type I polyketide synthase gene clusters were identified in the F. filiformis genome. The number of genes related to terpenoid biosynthesis was higher in the wild strain (119 genes) than in the cultivar strain (81 genes). Most terpenoid biosynthesis genes were upregulated in the primordium and fruiting body of the wild strain, while the polyketide synthase genes were generally upregulated in the mycelium of the wild strain. Moreover, genes encoding UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase, which are involved in polysaccharide biosynthesis, had relatively high transcript levels both in the mycelium and fruiting body of the wild F. filiformis strain. CONCLUSIONS: F. filiformis is enriched in a number of gene clusters involved in the biosynthesis of polysaccharides and terpenoid bioactive compounds and these genes usually display differential expression between wild and cultivar strains, even in different developmental stages. This study expands our knowledge of the biology of F. filiformis and provides valuable data for elucidating the regulation of secondary metabolites in this unique F. filiformis strain.

Characterization and expression pattern analysis of pheromone receptor-like genes in Winter Mushroom Flammulina filiformis.

Pheromone receptor-like genes (PRLGs) belong to the G protein-coupled receptors (GPCRs) family that interacts with biotic and abiotic stimulants and transmits signals to intracellular downstream pathways in eukaryotic cells. In this study, we investigated the structure and expressions patterns of PRLGs in Winter Mushroom Flammulina filiformis. Based on the alignment analysis, the structure of PRLGs was found conserved in F. filiformis strains expect few single-nucleotide polymorphism (SNP) sites. Six PRLGs were found at five different unlinked loci, scattered in the genomes of F. filiformis strains. These genes contain 2-5 introns; however, the introns were not found in the same relative positions regarding the encoded protein sequences in tested strains of F. filiformis. Three conserved motifs were identified in peptides structures of PRLGs, however, FfSte3.s6 contained only two types, suggests its difference in evolution and function. We have further analyzed the expression patterns of each PRLGs in different developmental stages of the fruiting body in F. filiformis by quantitative real-time polymerase chain reaction (qRT-PCR). The results exhibited expression variation of PRLGs at different developmental stages of the F. filiformis. Especially, FfSte3.s1 and FfSte3.s2 exhibited maximum expression level in mycelia stage. Other PRLGs exhibited high expression level in fruiting body stages. This study suggests that PRLGs could be vital genes involving in fruiting body development in F. filiformis. However, further studies could be performed to reveal their specific functional pathways in the fruiting body development.

A putative transcription factor LFC1 negatively regulates development and yield of winter mushroom.

Basidioma is the fruiting body of mushroom species. The deep understanding on the mechanism of basidioma development is valuable for mushroom breeding and cultivation. From winter mushroom (Flammulina velutipes), one of the top five industrially cultivated mushrooms, a novel putative Zn(II)2Cys6 transcription factor LFC1 with negative regulatory function in basidioma development was identified. The transcript level of lfc1 was dramatically decreased during basidioma development. Neither overexpression nor knockdown of lfc1 affected hyphal vegetative growth. However, knockdown of lfc1 could promote basidioma development and shorten cultivation time by 2 days, while overexpression of lfc1 delayed the optimal harvest time by 3 days. In the lfc1 knockdown strain, in which the lfc1 expression was reduced by 72%, mushroom yield and biological efficiency could be increased at least by 24%. Knockdown of lfc1 did not affect the shape of caps but significantly increased basidioma length and number, while its overexpression did not affect basidioma length but dramatically reduced basidioma number. In addition, rather than producing basidiomata with round caps as in wild type, the caps of basidiomata in the lfc1 overexpression mutants were significantly larger and the cap edge was wrinkled. RNA-seq analysis revealed that 455 genes had opposite transcriptional responses to lfc1 overexpression and knockdown. Some of them were previously reported as genes involved in basidioma development, including 3 hydrophobin encoding genes, 2 lectin encoding genes, FVFD16, an Eln2 ortholog encoding gene, and 3 genes encoding membrane components. As LFC1 homologs are widely present in mushroom species, lfc1 can be useful in mushroom breeding.Key Points• A novel transcription factor LFC1 negatively regulates fruiting in winter mushroom• LFC1 regulated transcription of more than 400 genes.• Reduction of LFC1 expression could shorten cultivation time and increase yield.• lfc1 could be a potentially useful reference gene for mushroom breeding.

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.

A Single Transcription Factor (PDD1) Determines Development and Yield of Winter Mushroom (Flammulina velutipes).

Most of the edible mushrooms cannot be cultivated or have low yield under industrial conditions, partially due to the lack of knowledge on how basidioma (fruiting body) development is regulated. From winter mushroom (Flammulina velutipes), one of the most popular industrially cultivated mushrooms, a transcription factor, PDD1, with a high-mobility group (HMG)-box domain was identified based on its increased transcription during basidioma development. pdd1 knockdown by RNA interference affected vegetative growth and dramatically impaired basidioma development. A strain with an 89.9% reduction in the level of pdd1 transcription failed to produce primordia, while overexpression of pdd1 promoted basidioma development. When the transcriptional level of pdd1 was increased to 5 times the base level, the mushroom cultivation time was shortened by 9.8% and the yield was increased by at least 33%. RNA sequencing (RNA-seq) analysis revealed that pdd1 knockdown downregulated 331 genes and upregulated 463 genes. PDD1 positively regulated several genes related to fruiting, including 6 pheromone receptor-encoding genes, 3 jacalin-related lectin-encoding genes, FVFD16, and 2 FVFD16 homolog-encoding genes. PDD1 is a novel transcription factor with regulatory function in basidioma development found in industrially cultivated mushrooms. Since its orthologs are widely present in fungal species of the Basidiomycota phylum, PDD1 might have important application prospects in mushroom breeding.IMPORTANCE Mushrooms are sources of food and medicine and provide abundant nutrients and bioactive compounds. However, most of the edible mushrooms cannot be cultivated commercially due to the limited understanding of basidioma development. From winter mushroom (Flammulina velutipes; also known as Enokitake), one of the most commonly cultivated mushrooms, we identified a novel transcription factor, PDD1, positively regulating basidioma development. PDD1 increases expression during basidioma development. Artificially increasing its expression promoted basidioma formation and dramatically increased mushroom yield, while reducing its expression dramatically impaired its development. In its PDD1 overexpression mutants, mushroom number, height, yield, and biological efficiency were significantly increased. PDD1 regulates the expression of some genes that are important in or related to basidioma development. PDD1 is the first identified transcription factor with defined functions in mushroom development among commercially cultivated mushroom species, and it might be useful in mushroom breeding.

FvC5SD overexpression enhances drought tolerance in soybean by reactive oxygen species scavenging and modulating stress-responsive gene expression.

KEY MESSAGE: Overexpression of FvC5SD improves drought tolerance in soybean. Drought stress is one of the most important abiotic stress factors that influence soybean crop quality and yield. Therefore, the creation of drought-tolerant soybean germplasm resources through genetic engineering technology is effective in alleviating drought stress. FvC5SD is a type of C-5 sterol desaturase gene that is obtained from the edible fungus Flammulina velutipes. This gene has good tolerance to the effects of stresses, including drought and low temperature, in yeast cells and tomato. In this study, we introduced the FvC5SD gene into the soybean variety Shennong9 through the Agrobacterium-mediated transformation of soybean to identify drought-tolerant transgenic soybean varieties. PCR, RT-PCR, and Southern blot analysis results showed that T-DNA was inserted into the soybean genome and stably inherited by the progeny. The ectopic expression of FvC5SD under the control of a CaMV 35S promoter in transgenic soybean plants enhanced the plant's tolerance to dehydration and drought. Under drought conditions, the transgenic plants accumulated lower levels of reactive oxygen species and exhibited higher activities and expression levels of enzymes and cell than wild-type soybean. iTRAQ analysis of the comparative proteomics showed that some exogenous genes coding either functional or regulatory proteins were induced in the transgenic lines under drought stress. FvC5SD overexpression can serve as a direct and efficient target in improving drought tolerance in soybean and may be an important biotechnological strategy for trait improvement in soybean and other crops.

Overexpression of the saccharopine dehydrogenase gene improves lysine biosynthesis in Flammulina velutipes.

Saccharopine dehydrogenase (EC 1.5.1.7) regulates the last step of fungal lysine biosynthesis. The gene (Fvsdh) encoding saccharopine dehydrogenase was identified and cloned from the whole genome of Flammulina velutipes. The genomic DNA of Fvsdh is 1257 bp, comprising three introns and four exons. The full-length complementary DNA of Fvsdh comprises 1107 bp with a deduced amino acid sequence of 368 residues. A 1,000-bp promoter sequence containing the TATA box, CAAT box, and several putative cis-acting elements was also identified. The results of tissue expression analysis showed that the expression level of the Fvsdh gene was higher in the pileus than in the stipe whether in the elongation or maturation stage. Further research showed that the lysine contents were 3.03 and 2.95 mg/g in maturation-pileus and elongation-pileus, respectively. In contrast, the lysine contents were 2.49 and 2.07 mg/g in elongation-stipe and maturation-stipe, respectively. To study the function of Fvsdh, we overexpressed Fvsdh in F. velutipes and found that Fvsdh gene expression was increased from 1.1- to 3-fold in randomly selected transgenic strains. The lysine contents were also increased from 1.12- to 1.3-fold in these five transformants, except for strain T3, in which the lysine contents were the same as the control. These results indicate that the expression of the Fvsdh gene can affect the lysine content of F. velutipes.

Comparative Transcriptomics of Flammulina filiformis Suggests a High CO2 Concentration Inhibits Early Pileus Expansion by Decreasing Cell Division Control Pathways.

Carbon dioxide is commonly used as one of the significant environmental factors to control pileus expansion during mushroom cultivation. However, the pileus expansion mechanism related to CO2 is still unknown. In this study, the young fruiting bodies of a popular commercial mushroom Flammulina filiformis were cultivated under different CO2 concentrations. In comparison to the low CO2 concentration (0.05%), the pileus expansion rates were significantly lower under a high CO2 concentration (5%). Transcriptome data showed that the up-regulated genes enriched in high CO2 concentration treatments mainly associated with metabolism processes indicated that the cell metabolism processes were active under high CO2 conditions. However, the gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with cell division processes contained down-regulated genes at both 12 h and 36 h under a high concentration of CO2. Transcriptome and qRT-PCR analyses demonstrated that a high CO2 concentration had an adverse effect on gene expression of the ubiquitin-proteasome system and cell cycle-yeast pathway, which may decrease the cell division ability and exhibit an inhibitory effect on early pileus expansion. Our research reveals the molecular mechanism of inhibition effects on early pileus expansion by elevated CO2, which could provide a theoretical basis for a CO2 management strategy in mushroom cultivation.

Genetic diversity and structure of core collection of winter mushroom (Flammulina velutipes) developed by genomic SSR markers.

BACKGROUND: A core collection is a subset of an entire collection that represents as much of the genetic diversity of the entire collection as possible. The establishment of a core collection for crops is practical for efficient management and use of germplasm. However, the establishment of a core collection of mushrooms is still in its infancy, and no established core collection of the economically important species Flammulina velutipes has been reported. RESULTS: We established the first core collection of F. velutipes, containing 32 strains based on 81 genetically different F. veltuipes strains. The allele retention proportion of the core collection for the entire collection was 100%. Moreover, the genetic diversity parameters (the effective number of alleles, Nei's expected heterozygosity, the number of observed heterozygosity, and Shannon's information index) of the core collection showed no significant differences from the entire collection (p > 0.01). Thus, the core collection is representative of the genetic diversity of the entire collection. Genetic structure analyses of the core collection revealed that the 32 strains could be clustered into 6 groups, among which groups 1 to 3 were cultivars and groups 4 to 6 were wild strains. The wild strains from different locations harbor their own specific alleles, and were clustered stringently in accordance with their geographic origins. Genetic diversity analyses of the core collection revealed that the wild strains possessed greater genetic diversity than the cultivars. CONCLUSION: We established the first core collection of F. velutipes in China, which is an important platform for efficient breeding of this mushroom in the future. In addition, the wild strains in the core collection possess favorable agronomic characters and produce unique bioactive compounds, adding value to the platform. More attention should be paid to wild strains in further strain breeding.

De novo transcriptome sequencing of Flammulina velutipes uncover candidate genes associated with cold-induced fruiting.

To understand molecular mechanism of cold-induced fruiting in Flammulina velutipes, which is one of most popular edible fungi in east Asia, de novo assembly of the F. velutipes transcriptome was carried out. There were 26,888,494 and 26,275,146 clean reads obtained from mycelium and primordia of F. velutipes, respectively. A total of 20,157 unigenes were de novo assembled and 15,058 of them were annotated. Moreover, 7935 unigenes were differentially expressed between mycelium and primordia, 4025 of them were up-regulated and 3910 were down-regulated. GO and KEGG pathway analysis of the differentially expressed unigenes indicated that functional groups associated with two-component signaling pathway, calcium signaling, mitogen-actived protein kinase pathway, molecular chaperones, cell wall and membrane system, play an important role in cold-induced fruiting of F. velutipes. In this work 643 EST-SSRs were identified in 20,157 unigenes and 1560 EST-SSRs primers pairs were designed. Moreover, 5548 and 5955 SNPs were detected in mycelium and primordia, respectively. Consequently, results of this work can serve as a valuable resource for functional genomics study of cold-induced fruiting in F. velutipes.

Genome Sequencing and Carbohydrate-Active Enzyme (CAZyme) Repertoire of the White Rot Fungus Flammulina elastica.

Next-generation sequencing (NGS) of the Flammulina elastica (wood-rotting basidiomycete) genome was performed to identify carbohydrate-active enzymes (CAZymes). The resulting assembly (31 kmer) revealed a total length of 35,045,521 bp (49.7% GC content). Using the AUGUSTUS tool, 12,536 total gene structures were predicted by ab initio gene prediction. An analysis of orthologs revealed that 6806 groups contained at least one F. elastica protein. Among the 12,536 predicted genes, F. elastica contained 24 species-specific genes, of which 17 genes were paralogous. CAZymes are divided into five classes: glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycosyltransferases (GTs), and auxiliary activities (AA). In the present study, annotation of the predicted amino acid sequences from F. elastica genes using the dbCAN CAZyme database revealed 508 CAZymes, including 82 AAs, 218 GHs, 89 GTs, 18 PLs, 59 CEs, and 42 carbohydrate binding modules in the F. elastica genome. Although the CAZyme repertoire of F. elastica was similar to those of other fungal species, the total number of GTs in F. elastica was larger than those of other basidiomycetes. This genome information elucidates newly identified wood-degrading machinery in F. elastica, offers opportunities to better understand this fungus, and presents possibilities for more detailed studies on lignocellulosic biomass degradation that may lead to future biotechnological and industrial applications.

iTRAQ-Based Comparative Proteomics Analysis of the Fruiting Dikaryon and the Non-fruiting Monokaryon of Flammulina velutipes.

Flammulina velutipes is a potentially excellent fungus to study basic mechanisms of basidiomycete mycelium biology. To provide a better understanding of the mechanism of hyphae growth and fruit-body formation, the biological functions of the differentially abundant proteins between the fruiting dikaryon and the non-fruiting monokaryon of F. velutipes were investigated at the proteomic level using iTRAQ-coupled two-dimensional liquid chromatography tandem mass spectrometry technique. Among the 1198 proteins identified with high confidence, a total of 472 proteins were detected differentially abundant at least one of the mycelium development stages. In-depth data analysis revealed that differentially expressed proteins were influenced a variety of cellular processes, particularly metabolic processes. Functional pathway analysis indicated that 63 up-regulated proteins at only the fruiting dikaryon (Fv13) stage were mainly distributed in 51 specific Kyoto Encyclopedia of Genes and Genome pathways, such as amino acids biosynthesis and metabolism, signaling pathway, and central carbon metabolism. These up-regulated proteins could possibly serve as potential biomarkers to study the mycelium development pathways as well as provide new insights on the mycelium heterogenic compatibility and fruit-body formation mechanisms of basidiomycetes.

Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase.

Soya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed-specific expression of an oxalate-degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, ß-ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDC-expressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate-degrading enzyme.

A Jacalin-Related Lectin Regulated the Formation of Aerial Mycelium and Fruiting Body in Flammulina velutipes.

Flammulina velutipes, one of the most popular mushroom species in the world, has been recognized as a useful model system to study the biochemical and physiological aspects of the formation and elongation of fruit body. However, few reports have been published on the regulation of fruiting body formation in F. velutipes at the molecular level. In this study, a jacalin-related lectin gene from F. velutipes was characterized. The phylogenetic tree revealed that Fv-JRL1 clustered with other basidiomycete jacalin-like lectins. Moreover, the transcriptional pattern of the Fv-JRL1 gene in different developmental stages of F. velutipes implied that Fv-JRL1 could be important for formation of fruit body. Additionally, RNA interference (RNAi) and overexpression analyses provided powerful evidence that the lectin gene Fv-JRL1 from F. velutipes plays important roles in fruiting body formation.

Complex Biochemical Analysis of Fruiting Bodies from Newly Isolated Polish Flammulina velutipes Strains.

The present study examined Polish strains of Flamulina velutipes as a potential source of nutraceuticals and found that their nutritional value is dependent on the fruiting bodies gathering time. To prove the above hypothesis protein, carbohydrate and phenolic substances concentration were determined. Moreover, catalase, superoxide dismutase, cellobiose dehydrogenase activities were assayed. In order to prove the healing properties of Enoki fruiting bodies the obtained extracts were tested for antioxidant and bacteriostatic abilities. We have proved that Polish F. velutipes fruiting bodies may be a rich source of antioxidants and that they are capable of inhibiting Staphylococcus aureus growth.

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.

Homocitrate synthase expression and lysine content in fruiting body of different developmental stages in Flammulina velutipes.

Homocitrate synthase (EC 2.3.3.14) regulates the first step of fungal lysine biosynthesis. The gene encoding homocitrate synthase was identified in whole genomic sequencing of Flammulina velutipes and contains seven introns. The homocitrate synthase gene of F. velutipes strain W23 (Fvhcs) is 1780 bp in length and encodes a 464 amino acid protein with a predicted molecular weight 50.7 kDa. Phylogenetic analysis of Fvhcs and other homocitrate synthase proteins from diverse fungi produced a topology congruent with the current best estimate of organismal phylogeny. Analysis of protein domains by InterProScan and a motif search found that Fvhcs gene encodes homocitrate synthase protein conserved across Agaricomycotina. In addition, we sequenced the transcriptome of different developmental stages and structures of the fruiting body to analyze the expression levels of the Fvhcs gene. The data showed a correlation between Fvhcs gene expression and lysine values in different developmental stages and structures of F. velutipes.

Reduction of oxalate levels in tomato fruit and consequent metabolic remodeling following overexpression of a fungal oxalate decarboxylase.

The plant metabolite oxalic acid is increasingly recognized as a food toxin with negative effects on human nutrition. Decarboxylative degradation of oxalic acid is catalyzed, in a substrate-specific reaction, by oxalate decarboxylase (OXDC), forming formic acid and carbon dioxide. Attempts to date to reduce oxalic acid levels and to understand the biological significance of OXDC in crop plants have met with little success. To investigate the role of OXDC and the metabolic consequences of oxalate down-regulation in a heterotrophic, oxalic acid-accumulating fruit, we generated transgenic tomato (Solanum lycopersicum) plants expressing an OXDC (FvOXDC) from the fungus Flammulina velutipes specifically in the fruit. These E8.2-OXDC fruit showed up to a 90% reduction in oxalate content, which correlated with concomitant increases in calcium, iron, and citrate. Expression of OXDC affected neither carbon dioxide assimilation rates nor resulted in any detectable morphological differences in the transgenic plants. Comparative proteomic analysis suggested that metabolic remodeling was associated with the decrease in oxalate content in transgenic fruit. Examination of the E8.2-OXDC fruit proteome revealed that OXDC-responsive proteins involved in metabolism and stress responses represented the most substantially up- and down-regulated categories, respectively, in the transgenic fruit, compared with those of wild-type plants. Collectively, our study provides insights into OXDC-regulated metabolic networks and may provide a widely applicable strategy for enhancing crop nutritional value.