Naotake Konno

Endo-β-1,3-Glucanase GLU1, from the Fruiting Body of Lentinula edodes, Belongs to a New Glycoside Hydrolase Family

ABSTRACT The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-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 β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-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).

Crystal structure of polysaccharide lyase family 20 endo-β-1,4-glucuronan lyase from the filamentous fungus Trichoderma reesei

The crystal structure of endo‐β‐(1→4)‐glucuronan lyase from Trichoderma reesei (TrGL) has been determined at 1.8 Å resolution as the first three‐dimensional structure of polysaccharide lyase (PL) family 20. TrGL has a typical β‐jelly roll fold, which is similar to glycoside hydrolase family 16 and PL7 enzymes. A calcium ion is bound to the site far from the cleft and appears to contribute to the stability. There are several completely conserved residues in the cleft. Possible catalytic residues are predicted based on structural comparison with PL7 alginate lyase A1–II′.

Cloning of the Trichoderma reesei cDNA Encoding a Glucuronan Lyase Belonging to a Novel Polysaccharide Lyase Family

ABSTRACT The filamentous fungus Trichoderma reesei produces glucuronan lyase (TrGL) when it is grown on β-(1→4)-polyglucuronate (cellouronate) as a sole carbon source. The cDNA encoding TrGL was cloned, and the recombinant enzyme was heterologously expressed in Pichia pastoris. The cDNA of TrGL includes a 777-bp open reading frame encoding a 20-amino-acid signal peptide and the 238-amino-acid mature protein. The amino acid sequence showed no similarity to the amino acid sequences of previously described functional proteins, indicating that the enzyme should be classified in a novel polysaccharide lyase (PL) family. Recombinant TrGL catalyzed depolymerization of cellouronate endolytically by β-elimination and was highly specific for cellouronate. The enzyme was most active at pH 6.5 and 50°C, and its activity and thermostability increased in the presence of Ca2+, suggesting that its calcium dependence is similar to that of other PLs, such as pectate lyases.

Characterization of β-N-acetylhexosaminidase (LeHex20A), a member of glycoside hydrolase family 20, from Lentinula edodes (shiitake mushroom)

We purified and cloned a β-N-acetylhexosaminidase, LeHex20A, with a molecular mass of 79 kDa from the fruiting body of Lentinula edodes (shiitake mushroom). The gene lehex20a gene had 1,659 nucleotides, encoding 553 amino acid residues. Sequence analysis indicated that LeHex20A belongs to glycoside hydrolase (GH) family 20, and homologues of lehex20a are broadly represented in the genomes of basidiomycetes. Purified LeHex20A hydrolyzed the terminal monosaccharide residues of β-N-acetylgalactosaminides and β-N-acetylglucosaminides, indicating that LeHex20A is a β-N-acetylhexosaminidase classified into EC 3.2.1.52. The maximum LeHex20A activity was observed at pH 4.0 and 50°C. The kinetic constants were estimated using chitooligosaccharides with degree of polymerization 2-6. GH20 β-N-acetylhexosaminidases generally prefer chitobiose among natural substrates. However, LeHex20A had the highest catalytic efficiency (kcat/Km) for chitotetraose, and the Km values for GlcNAc6 were 3.9-fold lower than for chitobiose. Furthermore, the enzyme partially hydrolyzed amorphous chitin polymers. These results indicate that LeHex20A can produce N-acetylglucosamine from long-chain chitomaterials.

Effective induction of pblac1 laccase by copper ion in Polyporus brumalis ibrc05015

Polyporus brumalis ibrc05015 is a strain capable of high laccase (Lac) production. Among several inducers, 0.25 mM copper was most effective for Lac production. One of the Lacs induced by copper was PbLac1, and its transcription was induced within 60 min after copper addition. The promoter region of pblac1 contained six putative metal response elements and one Ace1 consensus cis-element. We cloned the P. brumalis PbAce1 transcription factor, a homologue of Saccharomyces cerevisiae transcription factor Ace1, which regulates metallothionein genes in response to excess copper. PbAce1 complemented the function of Ace1 in an S. cerevisiae Δace strain. The conserved N-terminal copper-fist DNA binding domain of PbAce1 was required for complementation. In the PbAce1 complemented Δace1 strain, the pblac1 promoter was constitutively expressed at a high level, independent of copper concentration. PbAce1 has two Cys-rich repeat motifs (PbC1 and PbC2), which are similar to the Cys-rich repeat domain in metallothionein proteins, and are uniquely conserved in the C-terminal domain of basidiomycetous Ace1 sequences. These C-terminal domains could be involved in copper sensing and concentration-dependent Lac production in basidiomycetous fungi.

Lentinula edodes Genome Survey and Postharvest Transcriptome Analysis

ABSTRACT 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 N50 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 β-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 death-related 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 novoLentinula edodes genome assembly data will be used for the construction of a complete Lentinula edodes chromosome map for future breeding.

Polysaccharide-Inducible Endoglucanases from Lentinula edodes Exhibit a Preferential Hydrolysis of 1,3–1,4-β-Glucan and Xyloglucan

Three genes encoding glycoside hydrolase family 12 (GH12) enzymes from Lentinula edodes, namely Lecel12A, Lecel12B, and Lecel12C, were newly cloned by PCR using highly conserved sequence primers. To investigate enzymatic properties, recombinant enzymes encoded by L. edodes DNAs and GH12 genes from Postia placenta (PpCel12A and PpCel12B) and Schizophyllum commune (ScCel12A) were prepared in Brevibacillus choshinensis. Recombinant LeCel12A, PpCel12A, and PpCel12B, which were grouped in GH12 subfamily 1, preferentially hydrolyzed 1,3-1,4-β-glucan. By contrast, LeCel12B, LeCel12C, and ScCel12A, members of the subfamily 2, exhibited specific hydrolysis of xyloglucan. These results suggest that two subfamilies of GH12 are separated based on the substrate specificity. Transcript levels of L. edodes genes increased 72 h after growth of L. edodes mycelia cells in the presence of plant cell wall polymers such as xyloglucan, 1,3-1,4-β-glucan, and cellulose. These results suggest that L. edodes GH12 enzymes have evolved to hydrolyze 1,3-1,4-β-glucan and xyloglucan, which might enhance hyphal extension and nutrient acquisition.