Yoshihito Shiono

Characterization of two distinct feruloyl esterases, AoFaeB and AoFaeC, from Aspergillus oryzae

Two hypothetical proteins XP_001818628 and XP_001819091 (designated AoFaeB and AoFaeC, respectively), showing sequence identity with known type-C feruloyl esterases, have been found in the genomic sequence of Aspergillus oryzae. We cloned the putative A. oryzae feruloyl esterase-encoding genes and expressed them in Pichia pastoris. Both purified recombinant AoFaeB (rAoFaeB) and AoFaeC (rAoFaeC) had apparent relative molecular masses of 61,000 and 75,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, both proteins had a relative molecular mass of 55,000. The optimum pH for rAoFaeB was 6.0, although it was stable at pH values ranging from 3.0 to 9.0; rAoFaeC had an optimum pH of 6.0 and was stable in the pH range of 7.0–10.0. Thermostability of rAoFaeC was greater than that of rAoFaeB. Whereas rAoFaeC displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, methyl ferulate, and methyl sinapate, rAoFaeB displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, and methyl ferulate but not toward methyl sinapate. Substrate specificity profiling of rAoFaeB and rAoFaeC revealed type-B and type-C feruloyl esterases, respectively. Ferulic acid was efficiently released from wheat arabinoxylan when both esterases were applied with xylanase from Thermomyces lanuginosus. Both recombinant proteins also exhibited hydrolytic activity toward chlorogenic acid.

Relative abundance of Δ5-sterols in plasma membrane lipids of root-tip cells correlates with aluminum tolerance of rice

We investigated variations in aluminum (Al) tolerance among rice plants, using ancestor cultivars from the family line of the Al-tolerant and widely cultivated Japonica cultivar, Sasanishiki. The cultivar Rikuu-20 was Al sensitive, whereas a closely related cultivar that is a descendant of Rikuu-20, Rikuu-132, was Al tolerant. These two cultivars were compared to determine mechanisms underlying variations in Al tolerance. The sensitive cultivar Rikuu-20 showed increased permeability of the plasma membrane (PM) and greater Al uptake within 1 h of Al treatment. This could not be explained by organic acid release. Lipid composition of the PM differed between these cultivars, and may account for the difference in Al tolerance. The tolerant cultivar Rikuu-132 had a lower ratio of phospholipids to Delta(5)-sterols than the sensitive cultivar Rikuu-20, suggesting that the PM of Rikuu-132 is less negatively charged and less permeabilized than that of Rikuu-20. We used inhibitors of Delta(5)-sterol synthesis to alter the ratio of phospholipids to Delta(5)-sterols in both cultivars. These inhibitors reduced Al tolerance in Rikuu-132 and its Al-tolerant ancestor cultivars Kamenoo and Kyoku. In addition, Rikuu-132 showed a similar level of Al sensitivity when the ratio of phospholipids to Delta(5)-sterols was increased to match that of Rikuu-20 after treatment with uniconazole-P, an inhibitor of obtusifoliol-14alpha-demethylase. These results indicate that PM lipid composition is a factor underlying variations in Al tolerance among rice cultivars.

Three Oxygenated Cyclohexenone Derivatives Produced by an Endophytic Fungus

Three cyclohexenone derivatives, (4S,5S,6S)-5,6-epoxy-4-hydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (1), (4R,5R)-4,5-dihydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (2), and (4R,5S,6R)-4,5,6-trihydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (3), were isolated from unpolished rice fermented with an xylariaceous endophytic fungus (strain YUA-026). The structures of three compounds were established on the basis of spectroscopic analyses and chemical conversion. The minimum inhibitory concentrations of 1 and 3 were 100 μg/ml and 400 μg/ml against Staphylococcus aureus, 100 μg/ml and 200 μg/ml against Pseudomonas aeruginosa, and 200 μg/ml and >400 μg/ml against Candida albicans, respectively. In addition, 1 and 3 exhibited phytotoxic activity against lettuce.

Pyrrospirones A and B, apoptosis inducers in HL-60 cells, from an endophytic fungus, Neonectria ramulariae Wollenw KS-246

Pyrrospirones A and B have been isolated from unpolished rice cultures of the endophytic fungus Neonectria ramulariae Wollenw KS-246. Their absolute stereostructures (1 and 2) were elucidated through spectroscopic methods using 1D and 2D NMR techniques and chemical transformations, including the modified Moshers method. The compounds exhibited cytotoxicity and induced apoptosis in human promyelocytic leukemia HL-60 cells.

Fusaristatins A and B, two new cyclic lipopeptides from an endophytic Fusarium sp.

Two new cyclic lipopeptides, fusaristatins A (1) and B (2) were isolated from rice cultures of a Fusarium sp. YG-45 in the course of a screening of endophytic fungi. Their structures of 1 and 2 were determined by spectroscopic methods. 2 showed a moderate inhibitory effect on topoisomerases I (IC50: 73 μM) and II (IC50: 98 μM) without cleavable complexes. Furthermore, 1 and 2 showed the growth-inhibitory activity toward lung cancer cells LU 65 with IC50 values of 23 and 7 μM, respectively.

Identification of Diterpene. Biosynthetic Gene Clusters and Functional Analysis of Labdane-Related Diterpene Cyclases in Phomopsis amygdali

Two diterpene biosynthesis gene clusters in the fusicoccin-producing fungus, Phomopsis amygdali, were identified by genome walking from PaGGS1 and PaGGS4 which encode the geranylgeranyl diphosphate (GGDP) synthases. The diterpene cyclase-like genes, PaDC1 and PaDC2, were respectively located proximal to PaGGS1 and PaGGS4. The amino acid sequences of these two enzymes were similar to those of fungal labdane-related diterpene cyclases. Recombinant PaDC1 converted GGDP mainly into phyllocladan-16α-ol via (+)-copalyl diphosphate (CDP) and trace amounts of several labdane-related hydrocarbons which had been identified from the P. amygdali F6 mycelia. Since phyllocladan-16α-ol had not been identified in P. amygdali F6 mycelia, we isolated phyllocladan-16α-ol from the mycelia. Recombinant PaDC2 converted GGDP into (+)-CDP. Furthermore, we isolated the novel diterpenoid, phyllocladan-11α,16α,18-triol, which is a possible metabolite of phyllocladan-16α-ol in the mycelia. We propose that genome walking offers a useful strategy for the discovery of novel natural products in fungi.

A novel Aspergillus oryzae esterase that hydrolyzes 4-hydroxybenzoic acid esters

In this study we report the biochemical characterization of a hypothetical protein from Aspergillus oryzae exhibiting sequence identity with feruloyl esterase and tannase from the genus Aspergillus. The purified recombinant protein showed a hydrolytic activity toward the ethyl, propyl, or butyl esters of 4‐hydroxybenzoic acid, but did not show feruloyl esterase or tannase activity. Finally, the enzyme decreased the antimicrobial activity of parabens against A. oryzae via hydrolysis of the ester bond present in butyl 4‐hydroxybenzoic acid.

Isopimarane diterpene glycosides, isolated from endophytic fungus Paraconiothyrium sp. MY-42.

Six isopimarane diterpenes, compounds 1-6, were isolated from the endophytic fungus Paraconiothyrium sp. MY-42. Compound 1 possesses a 19-glucopyranosyloxy group. Its structure was first elucidated by spectroscopic data analysis and finally confirmed by X-ray crystallography, whereas structures 2-6 were mainly elucidated based on the analysis of spectroscopic evidence. Compounds 2 and 3 showed moderate cytotoxicities against the human promyelocytic leukemia cell line HL60 (IC₅₀ 6.7 μM value for 2 and 9.8 μM for 3).

Isopimarane diterpene glycosides, apoptosis inducers, obtained from fruiting bodies of the ascomycete Xylaria polymorpha

The methanol extract of fruiting bodies of the ascomycete Xylaria polymorpha afforded three isopimarane diterpene glycosides, namely, 16-alpha-D-mannopyranosyloxyisopimar-7-en-19-oic acid (1), 15-hydroxy-16-alpha-D-mannopyranosyloxyisopimar-7-en-19-oic acid (2), and 16-alpha-D-glucopyranosyloxyisopimar-7-en-19-oic acid (3). Their structures were determined by spectroscopic methods and by single-crystal X-ray analysis. They showed cytotoxicity against human cancer cell lines and exhibited IC50 values ranging from 71 to 607 microM. Further studies on the cytotoxicity of these compounds against HL60 cells demonstrated that they induced apoptosis along with typical DNA fragmentation. It was observed that 2 was less active than 1 and 3.

A new benzoxepin metabolite isolated from endophytic fungus Phomopsis sp.

Endophytes live symptomlessly and intracellularly inside host plants. The interaction of endophytes with host plants has, in particular, received considerable attention. Studies carried out in the last two decades have shown that endophytes are rich sources of structurally diverse natural products with interesting biological activities.1 These studies emphasize that chemical compounds help control the equilibrium between the endophytes and the host plants. In our previous research on novel bioactive compounds isolated from endophytic fungi, we reported eremoxylarins A and B isolated from endophytic fungus xylariaceous YUA-026 as novel calcineurin inhibitors. These compounds were obtained as potential inhibitors of Ca2+-signaling by the use of a yeast-based screening system for the activity that restores the growth of a Ca2+-sensitive, drug-sensitive strain of Saccharomyces cerevisiae (zds1D erg3D pdr1D pdr3D) on solid medium containing CaCl2. 2,3 Among the microbial compounds similarly screened, we found that benzophomopsin A (1) inhibited the Ca2+-signal transduction more strongly than did a known compound, xylarinol A (2) (Figure 1). Herein, we report the isolation and structure elucidation of a new benzoxepin derivative 1 together with 2 and their effects on Ca2+-signal transduction. The producing strain, Phomopsis sp. KS-37-2, was isolated from the stem of a cherry tree in Yamagata, Japan. Phomopsis sp. KS-37-2 was cultivated on sterilized, unpolished rice (20 g/Petri dish 50) at 25 1C for 3 weeks. The moldy, unpolished rice was extracted with MeOH, and the MeOH extract was concentrated. The resulting aqueous concentrate was extracted with n-hexane at first, and the water layer was subsequently partitioned with EtOAc. The purification of the EtOAc layer was guided by the intense blue characteristic coloration with vanillin–sulfuric acid solution on TLC plates. The EtOAc residue was purified on a silica gel column using a stepwise gradient of n-hexane–EtOAc (100:0–0:100). The 50–60% EtOAc fractions (85.0 mg) were combined and further purified by octa decyl silyl (ODS, Fuji Silysia Chemical Ltd., Aichi, Japan) column chromatography using 80% aqueous MeOH as the eluent to afford fractions 1–15 (100 ml each). Fraction 5 (35.0 mg) was rechromatographed on a silica gel column using CHCl3–MeOH (80:20) as the eluent to yield benzophomopsin A (1, 5.5 mg) and xylarinol A (2, 11.0 mg). The metabolite xylarinol A (2) was isolated as a white powder. Xylarinol A has recently been isolated from the fruiting bodies of Xylaria polymorpha as a radical scavenger.4 A comparison of our spectroscopic data with the literature values confirmed that 2 was xylarinol A. The molecular formula of 1 was C12H12O3, which required seven degrees of unsaturation, as revealed by HR–FAB–MS. The IR spectrum of 1 showed absorption bands at 3384, 1584 and 1465 cm 1, which are characteristic of the hydroxyl and aromatic groups. The formation of a monomethoxyl derivative (1a) [C13H14O3 (FAB–MS: m/z 241 [M+Na]+); dH 3.83 (3H, s, OMe)] after treating 1 with trimethylsilyldiazomethane confirmed the presence of a phenolic hydroxyl group. The UV spectrum of 1 revealed the presence of aromatic rings. The 13C NMR and DEPT spectra of 1 showed peaks corresponding to five sp2 methine (d 103.5, 114.8, 123.0, 128.0, 131.3), three sp2 quaternary carbons (d 126.6, 136.9, 152.3), one sp3 quaternary carbon (d 104.1), one sp3 methylene (d 56.8), one sp3 methine (d 66.9) and one methyl group (d 16.0). The seven unsaturation equivalents implied by the molecular formula indicated that 1 has three rings. The 1H-NMR spectrum showed signals attributable to a vicinal sp2 spin network [d 6.82 (1H, d, J1⁄47.6 Hz), 6.89 (1H, d, J1⁄47.6 Hz), 7.09 (1H, t, J1⁄47.6 Hz)] and two protons of a cis double bond [d 5.95 (1H, d, J1⁄412.7 Hz) and 6.82 (1H, d, J1⁄412.7 Hz)]. In addition, the 1H NMR spectrum revealed signals that were due to an isolated oxymethylene [d 4.63 (1H, d, J1⁄413.9 Hz), 5.12 (1H, d, J1⁄413.9 Hz)], a doublet methyl group [d 1.05 (3H, d, J1⁄46.5 Hz)] and an oxymethine