One new sterpurane sesquiterpene from cultures of the basidiomycete Pholiota nameko.

One new sterpurane sesquiterpene (1), named (3R,6S,7S,8R,10S)-3,7,14-trihydroxy-1-sterpurene was isolated from cultures of the basidiomycete Pholiota nameko. The structure of new compound was elucidated by extensive spectroscopic. Additionally, a single crystal X-ray diffraction not only confirmed the structure, but also determined the absolute configuration of the new compound. The compound was evaluated for cytotoxicity against five human cancer cell lines, but no significant cytotoxicity were found (IC50 values > 40 µM).

[New antibiotics produced by Bacillus subtilis strains].

Two Bacillus subtilis strains isolated from the fruiting body of a basidiomycete fungus Pholiota squarrosa exhibited a broad range of antibacterial activity, including those against methicillin-resistant Staphylococcus aureus INA 00761 (MRSA) and Leuconostoc mes6nteroides VKPM B-4177 resistant to glycopep-> tide antibiotics, as well as antifungal activity. The strains were identified as belonging to the "B. subtilis" com- plex based on their morphological and physiological characteristics, as well as by sequencing of the 16S rRNA gene fragments. Both strains (INA 01085 and INA 01086) produced insignificant amounts of polyene antibiotics (hexaen and pentaen, respectively). Strain INA 01086 produced also a cyclic polypeptide antibiotic containing Asp, Gly, Leu, Pro, Tyr, Thr, Trp, and Phe, while the antibiotic of strain INA 01085 contained, apart from these, two unidentified nonproteinaceous amino acids. Both polypeptide antibiotics were new compounds efficient against gram-positive bacteria and able to override the natural bacterial antibiotic resistance.

A-mating-type gene expression can drive clamp formation in the bipolar mushroom Pholiota microspora (Pholiota nameko).

In the bipolar basidiomycete Pholiota microspora, a pair of homeodomain protein genes located at the A-mating-type locus regulates mating compatibility. In the present study, we used a DNA-mediated transformation system in P. microspora to investigate the homeodomain proteins that control the clamp formation. When a single homeodomain protein gene (A3-hox1 or A3-hox2) from the A3 monokaryon strain was transformed into the A4 monokaryon strain, the transformants produced many pseudoclamps but very few clamps. When two homeodomain protein genes (A3-hox1 and A3-hox2) were transformed either separately or together into the A4 monokaryon, the ratio of clamps to the clamplike cells in the transformants was significantly increased to ca. 50%. We therefore concluded that the gene dosage of homeodomain protein genes is important for clamp formation. When the sip promoter was connected to the coding region of A3-hox1 and A3-hox2 and the fused fragments were introduced into NGW19-6 (A4), the transformants achieved more than 85% clamp formation and exhibited two nuclei per cell, similar to the dikaryon (NGW12-163 x NGW19-6). The results of real-time reverse transcription-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. microspora. Thus, we concluded that nearly 100% clamp formation requires high expression levels of homeodomain protein genes and that altered expression of the A-mating-type genes alone is sufficient to drive true clamp formation.

[Media optimization for exopolysaccharide by Pholiota squarrosa (Pers. ex Fr.) Quel. AS 5.245 on submerged fermentation].

Our previous work has indicated that mycelium growth and exopolysaccharide accumulation in submerged fermentation by Pholiota squarrosa (Pers. ex Fr.) Quel. AS 5.245 are strongly affected by many internal and external factors, including medium constituents and fermentation conditions. In this study, we use an effective two-phase statistical approach to enhance exopolysaccharide production. In the first phase, Plackett-Burman design was undertaken to evaluate the effects of the twenty factors, i.e., glucose, fructose, maltose, yeast extract, tryptone, K2HPO4, KH2PO4, (NH4)2SO4, NaNO3, FeSO4, MgSO4, MnCl2, ZnCl2, FeCl3, CuSO4.5H2O, vitamin B1, initial pH, the temperature, the medium volume and the duration, to the fermentation. By regression analysis, yeast extract, tryptone, fructose, MgSO4, MnCl2, initial pH and temperature were found to be important for exopolysaccharide production, while glucose, maltose, NaNO3, ZnCl2, vitamin B1, the duration and the volume are important to the mycelium biomass. In the second phase of the optimization process, a response surface methodology (RSM) was used to optimize the above critical internal factors, and to find out the optimal concentration levels and the relationships between these factors. Based on the results of the first phase, a five-level six-factor (yeast extract, fructose, MgSO4, maltose, ZnCl2 and initial pH) central composite rotatable design (CCRD) was employed. By solving the quadratic regression model equation using appropriate statistic methods, the optimal concentrations for obtaining 876.32 microg exopolysaccharide per milliliter of fermentation liquor were calculated as: 6.0g/L yeast extract, 11.5g/L fructose, 0.5g/L MgSO4, 9.6g/L maltose, 38.6mg/L ZnCl2 and with the initial pH 5.3. The experimental data under various conditions have validated the theoretical values.