Overexpression and repression of the tyrosinase gene in Lentinula edodes using the pChG vector.

Tyrosinase is an industrially useful enzyme, however, it causes gill browning of Lentinula edodes fruiting bodies during preservation. In this study, we constructed two vectors, pChG-gTs and pChG-gTa, expressing sense and antisense tyrosinase gene of L. edodes, respectively, using promoters derived from the glyceraldehyde-3-phosphate dehydrogenase gene. The host strain SR-1 of L. edodes was selected because of its fast growth, high protoplast yield, and high regeneration rate. Upon transformation of the host strain SR-1 with the pChG-gTs vector, a clone with 3.6-fold and 14.5-fold higher tyrosinase activity in vegetative mycelia and in fresh gills, respectively, than that of the host strain was obtained from nine transformants. Similarly, two clones containing the pChG-gTa vector with effectively repressed tyrosinase gene expression in vegetative mycelia and gills during the late stage of post-harvest preservation of fruiting bodies were obtained from 10 transformants. However, it remained unclear whether repression of the tyrosinase gene prevented gill browning, as the host strain also showed less browning than a commercial strain. Thus, this study highlights the usefulness of the pChG vector in expressing homologous enzyme coding genes in the vegetative mycelia and fruiting bodies of L. edodes.

New composite thixotropic hydrogel composed of a polymer hydrogelator and a nanosheet.

A composite gel composed of a water-soluble aromatic polyamide hydrogelator and the nanosheet Laponite®, a synthetic layered silicate, was produced and found to exhibit thixotropic behaviour. Whereas the composite gel contains the gelator at the same concentration as the molecular gel made by the gelator only, the composite gel becomes a softer thixotropic gel compared to the molecular gel made by the gelator only. The reason for this could be that bundles of polymer gelator may be loosened and the density of the polymer network increased in the presence of Laponite.

Gene silencing of the Lentinula edodes lcc1 gene by expression of a homologous inverted repeat sequence.

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 tyrosinase-encoding gene of Lentinula edodes, Letyr, is abundantly expressed in the gills of the fruit-body during post-harvest preservation.

The gill browning of Lentinula edodes fruit-bodies during preservation is thought to be due to melanin biosynthesis catalyzed by tyrosinase. We isolated a genomic DNA sequence and cDNA encoding a putative tyrosinase from the white rot basidiomycete Lentinula edodes (shiitake mushroom). The gene, named Letyr, consists of a 1,854-bp open reading frame interrupted by eight introns, and encodes a putative protein of 618 amino acid residues with an estimated molecular mass of 68 kDa. Amino acid residues known to be involved in copper-binding domains were conserved in the deduced amino acid residues of LeTyr. Transcriptional and translational expression of Letyr in the gills of the fruit-body increased during preservation after harvest. This correlation between Letyr expression and fruit-body preservation suggests that tyrosinase gene expression contributes to gill browning.

Isolation of industrial strains of Aspergillus oryzae lacking ferrichrysin by disruption of the dffA gene.

Based on studies using laboratory strains, the efficiency of gene disruption in Aspergillus oryzae, commonly known as koji mold, is low; thus, gene disruption has rarely been applied to the breeding of koji mold. To evaluate the efficiency of gene disruption in industrial strains of A. oryzae, we produced ferrichrysin biosynthesis gene (dffA) disruptants using three different industrial strains as hosts. PCR analysis of 438 pyrithiamine-resistant transformants showed dffA gene disruption efficiency of 42.9%-64.1%, which is much higher than previously reported. Analysis of the physiological characteristics of the disruptants indicated that dffA gene disruption results in hypersensitivity to hydrogen peroxide. To investigate the industrial characteristics of dffA gene disruptants, two strains were used to make rice koji and their properties were compared to those of the host strains. No differences were found between the dffA gene disruptants and the host strains, except that the disruptants did not produce ferrichrysin. Thus, this gene disruption technique is much more effective than conventional mutagenesis for A. oryzae breeding.

Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase.

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.

Lentinula edodes tlg1 encodes a thaumatin-like protein that is involved in lentinan degradation and fruiting body senescence.

Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising beta-1,3-glucan with beta-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 beta-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.

Identification of enzyme responsible for erythritol utilization and reaction product in yeast Lipomyces starkeyi.

We have identified the enzyme responsible for erythritol utilization and its reaction product in the yeast Lipomyces starkeyi CBS 1807. The enzyme, a polyol dehydrogenase requiring NAD+ as a coenzyme, was induced by erythritol in this yeast. We confirmed that the enzyme product was L-erythrulose by MS, NMR, and polarimeter analyses, meaning that we clarified the first step of erythritol utilization in yeasts for the first time. In the case of the oxidative reaction, D-threitol, (2R,3R)-2,3-butanediol, and erythritol were much better substrates than 21 other polyols tested. These three substrates are tetroses and have an R configuration at C-3, and whose third carbon results in easiest oxidation in this enzyme. The research of the substrate specificity in the reductive reaction demonstrated that L-erythrulose and dihydroxyacetone were better substrates, that D-acetoin was inactive and L-erythrose (aldose) was slightly active.

A novel two nucleotide deletion in the apolipoprotein A-I gene, apoA-I Shinbashi, associated with high density lipoprotein deficiency, corneal opacities, planar xanthomas, and premature coronary artery disease.

Familial HDL deficiency (FHD) is a rare autosomal dominant lipoprotein disorder. We describe a novel genetic variant of the apolipoprotein A-I (apoA-I) gene resulting in FHD. The proband is a 51-year-old woman who was hospitalized due to severe heart failure. Her plasma HDL-cholesterol (C) and apoA-I concentrations were 0.08mmol/l and 1mg/dl, respectively. She exhibited corneal opacities and planar xanthomas on eyelids and elbows. Coronary angiography demonstrated extensive obstructions in two major vessels. Genomic DNA sequencing of the patient's apoA-I gene revealed a homozygosity for a GC deletion between 5 GC repeats in exon 4, creating a frameshift and a stop codon at residue 178. We designated this mutation as apoA-I Shinbashi. The proband's father, son, and daughter were found to be heterozygous for this mutation and their HDL-C and apoA-I levels were about half of normal levels, demonstrating a gene dosage effect. The father underwent coronary bypass surgery at age of 70 years. Lecithin-cholesterol acyltransferase (LCAT) activity was decreased by 63% in the homozygote and 31% in heterozygotes, respectively. This new case of apoA-I deficiency, apoA-I Shinbashi, is the first case involving a single gene defect of the apoA-I gene to develop all the characteristics for apoA-I deficiency, including premature coronary heart disease.

Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies.

A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58.0 kDa. The enzyme had an isoelectric point of around pH 6.9. The optimum pH for enzyme activity was around 3.0 against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 degrees C and stable up to 50 degrees C. The enzyme contained 8.6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. Beta-(3,4-dihydroxyphenyl)alanine (L-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of L-dopa was identified as L-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain.

dffA gene from Aspergillus oryzae encodes L-ornithine N5-oxygenase and is indispensable for deferriferrichrysin biosynthesis.

We identified and analyzed the dffA gene from Aspergillus oryzae which encodes L-ornithine N5-oxygenase involved in the biosynthesis of deferriferrichrysin, a type of siderophore which is a low-molecular-weight iron chelating compound. From among more than 20,000 clones in an A. oryzae EST (expressed sequence tag) library, we found only one clone encoding a protein that exhibited homology to theUstilago maydis sid1 protein (Sid1) and Pseudomonas aeruginosa pvdA protein (PvdA), both known as the only examples of L-ornithine N5-oxygenase. The complete gene sequence shows that the dffA gene includes a 1575-bp open reading frame (ORF), one 66-bp intorn, which is a typical intorn length inA. oryzae, and encodes 502 amino acids with putative FAD-binding, NADP-binding, and 'FATGY' motifs, which are conserved inN-hydroxylating enzymes. As well as that of the U. maydis sid1 gene,dffA gene expression was induced under iron-limited conditions, and the promoter region has several GATA-type transcription regulator binding motifs. When the dffA gene was expressed under the control of the a-amylase promoter in A. oryzae, transformants revealed inducible high L-ornithine N5-oxygenase activities. In addition, a dffA gene disruptant showed no deferriferrichrysin production even under iron-limited conditions. These results clearly suggest that the dffA gene is indispensable for deferriferrichrysin biosynthesis in A. oryzae.

Development of a new chemically defined sporulation medium for the yeasts in the genus Lipomyces.

A chemically defined sporulation medium (AF medium) for the yeasts belonging to the genus Lipomyces was developed. The chemical composition was derived from chemical analyses of soybean extract. Some chemical modification of the AF medium indicated that the nitrogen sources (aspartic and glutamic acids) and zinc ion were essential for sporulation. The significance of medium pH was discussed.