Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising b-1,3-glucan with b-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited b-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.

Lentinula edodes is a popular, cultivated edible and medicinal mushroom. Lentinula edodes is susceptible to postharvest problems, such as gill browning, fruiting body softening, and lentinan degradation. We constructed a de novo assembly draft genome sequence and performed gene prediction for Lentinula edodes. De novo assembly was carried out using short reads from paired-end and mate-paired libraries and by using long reads by PacBio, resulting in a contig number of 1,951 and an N 50 of 1 Mb. Furthermore, we predicted genes by Augustus using transcriptome sequencing (RNA-seq) data from the whole life cycle of Lentinula edodes, resulting in 12,959 predicted genes. This analysis revealed that Lentinula edodes lacks lignin peroxidase. To reveal genes involved in the loss of quality of Lentinula edodes postharvest fruiting bodies, transcriptome analysis was carried out using serial analysis of gene expression (SuperSAGE). This analysis revealed that many cell wall-related enzymes are upregulated after harvest, such as ␤-1,3-1,6-glucan-degrading enzymes in glycoside hydrolase (GH) families GH5, GH16, GH30, GH55, and GH128, and thaumatin-like proteins. In addition, we found that several chitin-related genes are upregulated, such as putative chitinases in GH family 18, exochitinases in GH20, and a putative chitosanase in GH family 75. The results suggest that cell wall-degrading enzymes synergistically cooperate for rapid fruiting body autolysis. Many putative transcription factor genes were upregulated postharvest, such as genes containing high-mobility-group (HMG) domains and zinc finger domains. Several cell deathrelated proteins were also upregulated postharvest.IMPORTANCE Our data collectively suggest that there is a rapid fruiting body autolysis system in Lentinula edodes. The genes for the loss of postharvest quality newly found in this research will be targets for the future breeding of strains that keep fresh longer than present strains. De novo Lentinula edodes genome assembly data will be used for the construction of a complete Lentinula edodes chromosome map for future breeding.

We compared the gene expression patterns of Lentinula edodes fresh fruiting bodies and fruiting bodies 3 days after harvest, by suppression subtractive hybridization, to characterize the physiologic changes that occur after harvest, such as gill browning and cell wall lysis of the fruiting body, which are responsible for the loss of food quality and value. We found increase of transcription levels of several enzyme encoding genes, such as, two phenol oxidases encoding genes (tyr tyrosinase, lcc4 laccase), and several cell wall degradation-related enzyme-encoding genes, such as mixed-linked glucanase (mlg1), chitinases (chi1, chi2), chitin deacetylase (chd1), and chitosanase (cho1), after harvesting. We isolated a putative transcription factor-encoding gene (L. edodes exp1) with high similarity to exp1 from Coprinopsis cinerea, which is involved in autolysis of the cap during spore diffusion. Transcription of L. edodes exp1 increased post-harvest, which suggests that its target genes are up-regulated after harvesting. These enzymes and the transcription factor may be involved in L. edodes fruiting body senescence.

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as ␤-1,3-glucanase. In this study, a novel endo-type ␤-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited ␤-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of ␤-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).The cell walls of filamentous fungi have been investigated in several species of basidiomycota, including Schizophyllum commune (26), Agaricus bisporus (15), Coprinopsis cinerea (1), and Lentinula edodes (24a). These reports have indicated that the major components of the cell wall are chitin and ␤-1,3-glucan with ␤-1,6-linked branches. Several ␤-1,3-glucans from basidiomycetous mushrooms display antitumor activity; examples include lentinan from L. edodes (2) and schizophyllan from S. commune (17). Although lentinan can be purified from fresh shiitake mushrooms (L. edodes), it is degraded during storage as a result of ␤-1,3-glucanase activity (13,14). This suggests that ␤-1,3-glucanases are involved in cell wall lysis in the process of fruiting body senescence after harvesting.During the filamentous fungal life cycle, cell walls are synthesized, reoriented, and lysed (27,16). Cell wall lysis and changes in the constituent polysaccharides are essential for fruiting body development in basidiomycota (5, 6, 7). Autolysis of the pileus of fruiting bodies in C. cinerea also occurs following basidiospore formation (11). These observations suggest that cell wall-lytic enzymes, such as ␤-1,3-glucanase, are very important to the morphology of basidiomycetous fungi. There have been few reports on purified glucanases in basidiomycetes, such as endo-␤-1,3-glucanase in A. bisporus (3) and ␤-1,3(4)-glucanase in Phanerochaete chrysosporium (8); however, the physiological and biological functions of endo-␤-1,3-glucanase in basidiomycetous fungi are unclear.Previously, we reported that two exo-␤-1,3-glucanase-encoding genes (exg1 and exg2) are involved in morphogenesis of L. edodes (21,22) and that the enzyme encoded by exg2 is also involved in postharvest cell wall degradation (22). One endo-␤-1,3-glucanase, TLG1, which is a homolog of thaumatin-like protein, was purified from L. edodes fruiting bodies after harvest (4). The gene, tlg1, ...

Lentinula edodes is one of the most important edible mushrooms, but no method for analyzing its molecular genetics has yet been established. RNA interference (RNAi) is a mechanism that inhibits expression of specific genes at the post-transcriptional stage and has been used to analyze the genetics of several fungal species. RNAi was used to examine the expression of the laccase (EC 1.10.3.2) gene lcc1 of L. edodes, which encodes a lignin-degrading enzyme. Vector pChG'-ivrL1 that expressed a 40 bp homologous inverted repeat sequence from lcc1 was constructed. This was transformed into L. edodes using the restriction enzyme mediated integration method (REMI). Lcc1 protein was not detected in two of 57 transformants (ivrL1#26, ivrL1#32) where the lcc1 transcription levels were suppressed. Thus, a 40 bp inverted repeat sequence expression vector suppressed expression of the target gene in L. edodes. Lcc1 downregulated transformants (ivrL1#32) did not form a thick aerial mycelium mat on agar medium. Electron microscopy showed hyphae of ivrL1#32 had many short branches with low mycelial density, a thin cell wall, and few fibrous layers as compared to the host strain. These morphological phenotypes would be caused by the absence of Lcc1, and that provides some clue to resolve the biological function of Lcc1 in L. edodes. Our results show that RNAi can be used for gene silencing in L. edodes.

The edible white rot fungus Lentinula edodes possesses a variety of lignin degrading enzymes such as manganese peroxidases and laccases. Laccases belong to the multicopper oxidases, which have a wide range of catalytic activities including polyphenol degradation and synthesis, lignin degradation, and melanin formation. The exact number of laccases in L. edodes is unknown, as are their complete properties and biological functions. We analyzed the draft genome sequence of L. edodes D703PP-9 and identified 13 multicopper oxidase-encoding genes; 11 laccases in sensu stricto, of which three are new, and two ferroxidases. lcc8, a laccase previously reported in L. edodes, was not identified in D703PP-9 genome. Phylogenetic analysis showed that the 13 multicopper oxidases can be classified into laccase sensu stricto subfamily 1, laccase sensu stricto subfamily 2 and ferroxidases. From sequence similarities and expression patterns, laccase sensu stricto subfamily 1 can be divided into two subgroups. Laccase sensu stricto subfamily 1 group A members are mainly secreted from mycelia, while laccase sensu stricto subfamily 1 group B members are expressed mainly in fruiting bodies during growth or after harvesting but are lowly expressed in mycelia. Laccase sensu stricto subfamily 2 members are mainly expressed in mycelia, and two ferroxidases are mainly expressed in the fruiting body during growth or after harvesting, and are expressed at very low levels in mycelium. Our data suggests that L. edodes laccases in same group share expression patterns and would have common biological functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0151-2) contains supplementary material, which is available to authorized users.

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Laccase (Lcc) is a lignin-degrading enzyme produced by white-rot fungi and has been the subject of much interest in the field of bioremediation due to its ability to oxidize phenolic compounds. In this report, we describe the isolation and characterization of lcc1, a novel gene of Lentinula edodes that encodes Lcc1, and demonstrate that recombinant Lcc1 is expressed in an active, secreted form in tobacco BY-2 cells in culture. The open reading frame of lcc1 was 1,557 base pairs in length and encoded a putative protein of 518 amino acids. We introduced a chimeric form of lcc1 (CaMV35Sp:clcc1) into tobacco BY-2 cells and obtained several stable clcc1 transformants that expressed active Lcc1. Lcc1 activity in BY-2 culture media was higher than in cellular extracts, which indicated that recombinant Lcc1 was produced in a secreted form. Recombinant Lcc1 had a smaller apparent molecular weight and exhibited a different pattern of posttranslational modification than Lcc1 purified from L. edodes. The substrate specificity of purified recombinant Lcc1 was similar to L. edodes Lcc1, and both enzymes were able to decolorize the same set of dyes. These results suggest that heterologous expression of fungal Lcc1 in BY-2 cells will be a valuable tool for the production of sufficient quantities of active laccase for bioremediation.