Molecular Cloning, Characterization, and Differential Expression of a Glucoamylase Gene from the Basidiomycetous Fungus Lentinula edodes

Abstract

The complete nucleotide sequence of putative glucoamylase gene gla1 from the basidiomycetous fungus Lentinula edodes strain L54 is reported. The coding region of the genomic glucoamylase sequence, which is preceded by eukaryotic promoter elements CAAT and TATA, spans 2,076 bp. The gla1 gene sequence codes for a putative polypeptide of 571 amino acids and is interrupted by seven introns. The open reading frame sequence of the gla1 gene shows strong homology with those of other fungal glucoamylase genes and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The similarity between the Gla1 protein and other fungal glucoamylases is from 45 to 61%, with the region of highest conservation found in catalytic domains and starch-binding domains. We compared the kinetics of glucoamylase activity and levels of gene expression in L. edodes strain L54 grown on different carbon sources (glucose, starch, cellulose, and potato extract) and in various developmental stages (mycelium growth, primordium appearance, and fruiting body formation). Quantitative reverse transcription PCR utilizing pairs of primers specific for gla1 gene expression shows that expression of gla1 was induced by starch and increased during the process of fruiting body formation, which indicates that glucoamylases may play an important role in the morphogenesis of the basidiomycetous fungus.Basidiomycetous fungus Lentinula edodes (Berk.) Pegler is the second most widely cultivated mushroom in the world. The cultivation of this fungus makes use of significant amounts of woody polysaccharides, and utilization of the complex polysaccharides is dependent on its ability to synthesize hydrolytic and oxidative enzymes which convert woody polysaccharides into low-molecular-weight compounds that can be absorbed and assimilated for nutrition. Starch is a polymer of glucose and is perhaps, next to cellulose, the most widely available polymeric glucoside made by plants (16,18). Starch is, therefore, available to fungi growing on plants or plant residues. Digestion of starch requires a complex of enzymes. Glucoamylases (1,4-␣-D-glucan glucohydrolases; EC 3.2.1.3) are enzymes that, among others, are believed to be important in the utilization of starch by the basidiomycetous fungus. Glucoamylases are exohydrolases, which catalyze the release of ␤-D-glucose units from the nonreducing ends of amylose, amylopectin, and other polysaccharides (18). Glucoamylase-encoding genes have been cloned from several fungi including Aspergillus awamori (6, 15), Aspergillus niger (1, 3), Aspergillus oryzae (8), Aspergillus terreus (5, 23), and Neurospora crassa (22).Although there have been many reports on glucoamylases in fungi, few studies on the production and regulation of glucoamylases in basidiomycetous fungi have been carried out. El-Zalaki and Hamza (2) studied five basidiomycetous fungus species for their ability to hydrolyze starch. L. edodes was found to be the most promising strain for amylase production. It was also reported that the ...

Le.MAPK and its interacting partner, Le.DRMIP, in fruiting body development in Lentinula edodes

“…Studies of the initiation and development of the fruiting body will further improve cultivation of L. edodes and other mushrooms, since the details of the molecular mechanism are still unclear. Previous studies of fruiting body development in L. edodes include the isolation and differential profiling of the expression of various enzymes (Zhao and Kwan, 1999;Zhao et al, 2000;Ohga and Royse, 2001), isolation of differentially expressed signal transduction genes such as ras (Hori et al, 1991), and identification of developmentally expressed genes, including a MAP kinase homolog Le.MAPK, from the primordium of L. edodes (Leung et al, 2000).…”

Characterization of the glycoside hydrolase family 15 glucoamylase gene from the ectomycorrhizal basidiomycete Tricholoma matsutake

“…Orton ) and 67% identity and 81% similarity with the glucoamylase protein of L. edodes (Zhao et al 2000). The phylogenetic tree of fungal glucoamylases constructed by the unweighted pair group method with arithmetic mean (UPGMA) clustering method is shown in Fig.…”

“…The genes encoding GH6 or GH7 enzymes have been identified and characterized in the saprophytic fungi Coprinopsis cinerea (Schaeff) (Yoshida et al 2009;Stajich et al 2010) and L. edodes (Lee et al 2001). Moreover, glucoamylase genes have also been cloned and characterized in these two saprophytic fungal species (Stajich et al 2010;Zhao et al 2000). In the ectomycorrhizal fungus Laccaria bicolor, a gene for glucoamylase, but not cellobiohydrolase, has been identified .…”

“…The F15-GP2-AF and F15-GP2-BR primers were designed based on the amino acid sequences GLGEPKF and FDLWEEI, respectively, which are conserved in the glucoamylase proteins of Lentinula edodes and several other basidiomycetes (Zhao et al 2000). PCR was performed in a 50-ll reaction containing 19 Ex Taq buffer (Takara Bio, Shiga, Japan), 50 ng genomic DNA, 50 pmol each primer, 0.2 mM each dNTP, and 1.25 U Ex Taq polymerase (Takara Bio).…”

Purification and some properties of α-amylase from an ectomycorrhizal fungus, Tricholoma matsutake

“…: Fr. (Shimazaki et al 1984) and Lentinula edodes (Berkeley) Pegler (Yamasaki and Suzuki 1978;Zhao et al 2000). However, no characterization of highly purified α-amylase from a mushroom origin involving a mycorrhizal fungus has been reported to date.…”