Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences.

Pleurotus ostreatus, also known as the oyster mushroom, is a popular edible mushroom cultivated worldwide. This review aims to survey recent progress in the molecular genetics of this fungus and demonstrate its potential as a model mushroom for future research. The development of modern molecular genetic techniques and genome sequencing technologies has resulted in breakthroughs in mushroom science. With efficient transformation protocols and multiple selection markers, a powerful toolbox, including techniques such as gene knockout and genome editing, has been developed, and numerous new findings are accumulating in P. ostreatus. These include molecular mechanisms of wood component degradation, sexual development, protein secretion systems, and cell wall structure. Furthermore, these techniques enable the identification of new horizons in enzymology, biochemistry, cell biology, and material science through protein engineering, fluorescence microscopy, and molecular breeding. KEY POINTS: • Various genetic techniques are available in Pleurotus ostreatus. • P. ostreatus can be used as an alternative model mushroom in genetic analyses. • New frontiers in mushroom science are being developed using the fungus.

α-1,3-Glucanase from the gram-negative bacterium Flavobacterium sp. EK-14 hydrolyzes fungal cell wall α-1,3-glucan.

The glycoside hydrolase (GH) 87 α-1,3-glucanase (Agl-EK14) gene was cloned from the genomic DNA of the gram-negative bacterium Flavobacterium sp. EK14. The gene consisted of 2940 nucleotides and encoded 980 amino acid residues. The deduced amino acid sequence of Agl-EK14 included a signal peptide, a catalytic domain, a first immunoglobulin-like domain, a second immunoglobulin-like domain, a ricin B-like lectin domain, and a carboxyl-terminal domain (CTD) involved in extracellular secretion. Phylogenetic analysis of the catalytic domain of GH87 enzymes suggested that Agl-EK14 is distinct from known clusters, such as clusters composed of α-1,3-glucanases from bacilli and mycodextranases from actinomycetes. Agl-EK14 without the signal peptide and CTD hydrolyzed α-1,3-glucan, and the reaction residues from 1 and 2% substrates were almost negligible after 1440 min reaction. Agl-EK14 hydrolyzed the cell wall preparation of Aspergillus oryzae and released glucose, nigerose, and nigero-triose from the cell wall preparation. After treatment of A. oryzae live mycelia with Agl-EK14 (at least 0.5 nmol/ml), mycelia were no longer stained by red fluorescent protein-fused α-1,3-glucan binding domains of α-1,3-glucanase Agl-KA from Bacillus circulans KA-304. Results suggested that Agl-EK14 can be applied to a fungal cell wall lytic enzyme.

Nigero-oligosaccharide production by enzymatic hydrolysis from alkaline-pretreated α-1,3-glucan.

Nigero-oligosaccharides are α-1,3-linked oligomers of glucose. Glycoside hydrolase 87 type α-1,3-glucanase Agl-KA from Bacillus circulans KA304 is an endo-lytic enzyme that releases nigero-oligosaccharides (tetra-, tri-, and di-saccharide) from α-1,3-glucan. α-1,3-Glucan is insoluble under natural conditions, thus the efficiency of enzymatic hydrolysis is low and only 5 mM of reducing sugars were released from 1% glucan by Agl-KA. To improve hydrolytic efficiency, α-1,3-glucan was solubilized by 1 M NaOH and alkaline-solubilized glucan was adjusted to approximately pH 8. As a result, glucan maintained a solubilized state. This alkaline-pretreated α-1,3-glucan (1%) was hydrolyzed by Agl-KA (0.64 nmol/mL) and approximately 11.6 mM of reducing sugars were released at 240 min of reaction. When 0.016, 0.032, and 0.13 nmol/mL enzyme were added, reducing sugar reached approximately 5.1, 7.5, and 9.8 mM, respectively, and reaction mixtures containing 0.016 and 0.032 nmol/mL enzyme gradually became cloudy. Our findings suggest α-1,3-glucan cannot maintain its solubilized state and gradually becomes insoluble. For deletion enzyme of α-1,3-glucan binding domains from Agl-KA (AglΔDCD-UCD) on glucan hydrolysis (2%), reducing sugar concentrations released by AglΔDCD-UCD were almost the same as Agl-KA. These findings suggest that alkaline-pretreated α-1,3-glucan maintains a soluble state during a short time period and that glucan is efficiently hydrolyzed even by α-1,3-glucanase without α-1,3-glucan binding domains.

Antimicrobial Activity of Polysorbate 80-iodine Complex: Polysorbate 80 Firmly Retains Iodine.

Considering that iodine is highly volatile and has low solubility in water, it is utilized as an antiseptic in its complex form (iodophor) with a carrier material. Herein, we prepared the polysorbate 80-iodine complex and investigated its properties. In the presence of 0%, 0.01%, 0.1%, and 1% polysorbate, Pseudomonas putida NBRC 100650 growth was inhibited at 75, 75, 50, and 25 ppm iodine, respectively, indicating that high concentrations of polysorbate 80 enhanced the antibacterial activity of iodine. Absorption spectra of the mixtures of polysorbate 80 and iodine were analyzed; we observed that two peaks at 287 and 350 nm, derived from triiodide ions, shifted to the longer wavelength side in the presence of 0.1% and 1% polysorbate 80. Further, when 1% polysorbate 80 was added to the mixture of soluble starch and iodine, the peak around 580 nm arising from the amylose-iodine complex disappeared, indicating that polysorbate 80 captured iodine from the starch-iodine complex. We also found that polysorbate 80 retained iodine for approximately 4 months and prevented its volatilization; moreover, the mixture did not lose its growth inhibitory activity upon storage for approximately 4 months. Collectively, our data indicated that polysorbate 80 firmly retains low concentrations of iodine and that the polysorbate 80-iodine complex can serve as an antiseptic that can be stably stored for a long time.

GH-16 Type ß-1,3-Glucanase from Lysobacter sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase, and Its Glucan-binding Domain Binds to Fungal Cell-wall.

The GH-16 type ß-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type ß-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with ß-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using ß-glucans.

Construction of a fusion protein consisting of α-1,3-glucan-binding domains and tetrameric red fluorescent protein, which is involved in the aggregation of α-1,3-glucan and inhibition of fungal biofilm formation.

Agl-KA, an α-1,3-glucan-hydrolyzing enzyme from Bacillus circulans KA-304, has three α-1,3-glucan-binding domains DS1, CB6, and DS2 (DCD). While their individual binding activities toward insoluble α-1,3-glucan and fungal cell-wall are weak, the three domains in combination bind strongly to the α-1,3-glucan and the cell-wall. In this study, we constructed DCD-tetraRFP by fusing DCD with DsRed-Express2, a tetrameric red fluorescent protein. DCD-tetraRFP forms a tetramer in an aqueous solution and contains twelve substrate-binding domains in one complex. We also constructed DCD-monoGFP by fusing DCD with AcGFP1, a monomeric green fluorescent protein. The molecular weight of DCD-tetraRFP and DCD-monoGFP were compared. The results of gel filtration chromatography and dynamic light scattering indicated that DCD-tetraRFP was larger than DCD-monoGFP, suggesting that DCD-tetraRFP had a tetrameric structure. In addition, DCD-tetraRFP bound to insoluble α-1,3-glucan strongly, and the amount of DCD-tetraRFP binding to 0.01% α-1,3-glucan was about twice of DCD-monoGFP. The Kd values of DCD-tetraRFP (measurements per subunit) and DCD-monoGFP were 0.16 and 0.84 µM, respectively. Adding DCD-tetraRFP to a suspension of α-1,3-glucan caused glucan aggregation; however, adding DCD-monoGFP did not. These data suggested that DCD-tetraRFP had four DCDs sterically arranged in different directions so that DCD-tetraRFP cross-linked with the substrate, causing aggregation. Lastly, the aggregates of DCD-tetraRFP and α-1,3-glucan captured Aspergillus oryzae conidia and decreased their biofilm formation by 80% in a 24-well dish.