S-Adenosyl-L-methionine-dependent cinnamic acid carboxyl methyltransferase from the basidiomycete Tricholoma matsutake, which has low identity but similar overall structure to plant SABATH-family enzymes


A novel rapid artificial drying schedule for a Japanese cedar board retaining natural durability

スギ板材の迅速な新規人工乾燥法の検討

The objective of this study was to develop a novel and rapid artificial drying schedule for a boards of Japanese cedar (Cryptomeria japonica). Boards of green wood were dried to a moisture content of 15% under 9 artificial drying conditions with a combination of three dry-bulb temperatures (50°C, 70°C and 90°C) and three wet-bulb depressions (5°C, 10°C and 20°C). We found that the condition of 90°C dry-bulb temperature and 20°C wet-bulb depression produced dried boards in the shortest period. The boards indicated no significant color change for practical use. In addition, the dried boards showed durability against brown-rot fungi Fomitopsis palustris and subterranean termites Coptotermes formosanus comparable to naturally dried heartwood boards. The relative contents of cadinols demonstrated negative correlation with weight loss in decay tests. These results may lead to improvement of drying schedules for Japanese cedar boards while retaining natural durability.

スギ板材の，新しい迅速な人工乾燥条件の開発を目的に，スギ生材を製材した板材を乾球温度(DBT)50℃，70℃，90℃，乾湿球温度差(ΔT)5℃，10℃，20℃を組み合わせた，9種類の条件で，含水率15%まで乾燥させた．要した時間はDBT 90℃，ΔT 20℃が最短であった．本条件で乾燥された板材は天然乾燥や他の人工乾燥条件に対し強度性能の違いは見られず，材色変化も実用上問題は無かった．本条件で乾燥されたスギ板材は，木材腐朽菌オオウズラタケに対する耐腐朽性，イエシロアリに対する耐蟻性は，天然乾燥されたスギ板材と同等であった．cadinol類の相対含有濃度とオオウズラタケを用いた耐腐朽性試験における質量減少率とは，高い負の相関が認められた．以上の結果は，耐腐朽性，耐蟻性を保持したスギ板材の乾燥スケジュールの改善につながると思われる．


The ectomycorrhizal fungus Tricholoma matsutakebiosynthesizes benzoic acid and benzaldehyde independently


Downregulation of p-COUMAROYL ESTER 3-HYDROXYLASE in rice leads to altered cell wall structures and improves biomass saccharification.

The Plant Journal : for Cell and Molecular Biology


Regulation of CONIFERALDEHYDE 5-HYDROXYLASE expression to modulate cell wall lignin structure in rice

Regulation of a gene encoding coniferaldehyde 5-hydroxylase leads to substantial alterations in lignin structure in rice cell walls, identifying a promising genetic engineering target for improving grass biomass utilization. The aromatic composition of lignin greatly affects utilization characteristics of lignocellulosic biomass and, therefore, has been one of the primary targets of cell wall engineering studies. Limited information is, however, available regarding lignin modifications in monocotyledonous grasses, despite the fact that grass lignocelluloses have a great potential for feedstocks of biofuel production and various biorefinery applications. Here, we report that manipulation of a gene encoding coniferaldehyde 5-hydroxylase (CAld5H, or ferulate 5-hydroxylase, F5H) leads to substantial alterations in syringyl (S)/guaiacyl (G) lignin aromatic composition in rice (Oryza sativa), a major model grass and commercially important crop. Among three CAld5H genes identified in rice, OsCAld5H1 (CYP84A5) appeared to be predominantly expressed in lignin-producing rice vegetative tissues. Down-regulation of OsCAld5H1 produced altered lignins largely enriched in G units, whereas up-regulation of OsCAld5H1 resulted in lignins enriched in S units, as revealed by a series of wet-chemical and NMR structural analyses. Our data collectively demonstrate that OsCAld5H1 expression is a major factor controlling S/G lignin composition in rice cell walls. Given that S/G lignin composition affects various biomass properties, we contemplate that manipulation of CAld5H gene expression represents a promising strategy to upgrade grass biomass for biorefinery applications.


MYB-mediated upregulation of lignin biosynthesis in Oryza Sativa towards biomass refinery

Lignin encrusts lignocellulose polysaccharides, and has long been considered an obstacle for the efficient use of polysaccharides during processes such as pulping and bioethanol fermentation. However, lignin is also a potential feedstock for aromatic products and is an important by-product of polysaccharide utilization. Therefore, producing biomass plant species exhibiting enhanced lignin production is an important breeding objective. Herein, we describe the development of transgenic rice plants with increased lignin content. Five (Arabidopsis) and one (rice) MYB transcription factor genes that were implicated to be involved in lignin biosynthesis were transformed into rice ( L. ssp. cv. Nipponbare). Among them, three Arabidopsis MYBs (, , and ) in transgenic rice T lines resulted in culms with lignin content about 1.5-fold higher than that of control plants. Furthermore, lignin structures in -overexpressing rice plants were investigated by wet-chemistry and two-dimensional nuclear magnetic resonance spectroscopy approaches. Our data suggested that heterologous expression of in rice increased lignin content mainly by enriching syringyl units as well as -coumarate and tricin moieties in the lignin polymers. We contemplate that this strategy is also applicable to lignin upregulation in large-sized grass biomass plants, such as , switchgrass, and .


A biosynthetic pathway for (E)-methyl cinnamate formation in the ectomycorrhizal fungus Tricholoma matsutake


The expression of a rice secondary wall-specific cellulose synthase gene, OsCesA7, is directly regulated by a rice transcription factor, OsMYB58/63

A rice MYB transcription factor, OsMYB58/63, was found to directly upregulate the expression of a rice secondary wall-specific cellulose synthase gene, cellulose synthase A7 ( OsCesA7 ); in contrast, the Arabidopsis putative orthologs AtMYB58 and AtMYB63 have been shown to specifically activate lignin biosynthesis. Although indirect evidence has shown that grass plants are similar to but partially different from dicotyledonous ones in transcriptional regulation of lignocellulose biosynthesis, little is known about the differences. This study showed that a rice MYB transcription factor, OsMYB58/63, directly upregulated the expression of a rice secondary wall-specific cellulose synthase gene, cellulose synthase A7 (OsCesA7). Gene co-expression analysis showed that, in rice, OsMYB58/63 and several rice MYB genes were co-expressed with genes encoding lignocellulose biosynthetic enzymes. The expression levels of OsMYB55/61, OsMYB55/61-L, OsMYB58/63, and OsMYB42/85 were commonly found to be high in culm internodes and nodes. All four MYB transcription factors functioned as transcriptional activators in yeast cells. OsMYB58/63 most strongly transactivated the expression of OsCesA7 in rice protoplasts. Moreover, recombinant OsMYB58/63 protein was bound to two distinct cis-regulatory elements, AC-II and SMRE3, in the OsCesA7 promoter. This is in sharp contrast to the role of Arabidopsis orthologs, AtMYB58 and AtMYB63, which had been reported to specifically activate lignin biosynthesis. The promoter analysis revealed that AC elements, which are the binding sites for MYB58 and MYB63, were lacking in cellulose and xylan biosynthetic genes in Arabidopsis, but present in cellulose, xylan, and lignin biosynthetic genes in rice, implying that the difference of transcriptional regulation between rice and Arabidopsis is due to the distinct composition of promoters. Our results provide a new insight into transcriptional regulation in grass lignocellulose biosynthesis.


Rapid copper transfer and precipitation by wood-rotting fungi can effect copper removal from copper sulfate-treated wood blocks during solid-state fungal treatment

International Biodeterioration & Biodegradation


A new O-methyltransferase for monolignol synthesis in Carthamus tinctorius.


Substrate-enantiomer selectivity of matairesinol O-methyltransferases

Previously we reported a cDNA encoding an O-methyltransferase (OMT) responsible for the O-methylation of matairesinol to afford arctigenin in Carthamus tinctorius. However, the regioselectivity and stereoselectivity of its reaction are not yet well understood. In this paper, we report the characterization and comparison of three matairesinol OMTs (MROMTs) encoded by cDNAs isolated from C. tinctorius (CtMROMT), Anthriscus sylvestris (AsMROMT), and Forsythia koreana (FkMROMT). Although they shared matairesinol as a substrate, each recombinant MROMT showed different catalytic behavior. AsMROMT and CtMROMT methylated matairesinol's hydroxyl group at the C4' position giving rise to arctigenin, while FkMROMT methylated the C4 position hydroxyl group giving rise to isoarctigenin. Analysis of the enantiomeric composition of products from racemic matairesinol showed all OMT reactions to be highly selective in terms of the substrate enantiomers and only use the (-)-enantiomer as the substrate.


ATL54, a ubiquitin ligase gene related to secondary cell wall formation, is transcriptionally regulated by MYB46

We previously characterized Arabidopsis Tóxicos en Levadura54 (ATL54), a ubiquitin ligase associated with secondary cell wall formation. The ATL54 gene is co-expressed with secondary wall-associated genes, and the knock-out of ATL54 up-regulates the expression of cellulose, lignin, and xylan biosynthetic genes in apical stem portions of four-week-old plants. Here, we report the tissue-level localization patterns and the regulation of ATL54 expression. The β-glucuronidase (GUS) reporter gene driven by the ATL54 promoter was significantly expressed in interfascicular fibers, xylary fibers, and vessels in inflorescence stems. A transient transfection assay using Arabidopsis T87 cells showed that the expression of the firefly luciferase gene driven by the ATL54 promoter was activated by MYB46, which is a key transcriptional activator of secondary wall formation. In addition, the electrophoretic mobility of ATL54 promoter fragments was shifted by a recombinant MYB46 protein. These results indicate that ATL54 expression is regulated by MYB46, and support the view that ATL54 has a role in secondary wall formation.


Characterization of three cinnamylalcohol dehydrogenases from Carthamus tinctorius


A novel O-methyltransferase involved in the first methylation step of yatein biosynthesis from matairesinol in Anthriscus sylvestris.


CAD2 deficiency causes both brown midrib and gold hull and internode phenotypes in Oryza sativa L. cv. Nipponbare


Characterization of 5-hydroxyconiferaldehyde O-methyltransferase in Oryza sativa


A lignan O-methyltransferase catalyzing the regioselective methylation of matairesinol in carthamus tinctorius

Lignans are a group of plant phenolic compounds with various biological activities, including antitumor and antioxidant properties. O-Methylation is a critical step in biosynthesis of these compounds. However, little is known about the O-methyltransferase (OMT) enzymes that catalyze lignan O-methylation. We discovered a highly regioselective OMT activity in safflower (Carthamus tinctorius) seeds that catalyzed the methylation of matairesinol, a dibenzylbutyrolactone lignan, into 4'-O-methylmatairesinol (arctigenin) but not 4-O-methylmatairesinol (isoarctigenin). By examining such OMT activity in correlation with OMT transcript abundances during seed development, we cloned a few putative OMT cDNAs and produced their recombinant proteins in Escherichia coli. Among them, one protein exhibited O-methylation activity for matairesinol with the regioselectivity identical to that of the plant protein, and was named C. tinctorius matairesinol OMT (CtMROMT). CtMROMT did not show any detectable OMT activities towards phenylpropanoid monomers under the reaction conditions tested, while it methylated flavonoid apigenin efficiently into 4'-O-methylapigenin (acacetin). However, quantitative real-time polymerase chain reaction analysis demonstrated that expression of the CtMROMT gene was synchronized with the CtMROMT activity profile and arctigenin accumulation in the plant. These results demonstrated that CtMROMT is a novel plant OMT for lignan methylation.


ATL54, a RING-H2 domain protein selected by a gene co-expression network analysis, is associated with secondary cell wall formation in Arabidopsis.

Biosynthesis of plant secondary cell walls is controlled by several master transcription factors. Ubiquitin ligases, which mediate ubiquitination of proteins, including transcription factors in the protein degradation pathway, are also believed to regulate secondary wall biosynthesis; however, the exact ubiquitin ligases involved in secondary wall formation have not yet been identified. We conducted a gene co-expression network analysis and found that ATL54, annotated as a RING-finger protein, was highly co-expressed with several transcription factor and enzyme genes involved in secondary wall formation. A recombinant ATL54 protein showed ubiquitin ligase activity. The expression of several biosynthetic genes of cellulose, lignin, and xylan in apical portions of inflorescence stems was up-regulated by ATL54 knock-out. The expression of Xylem Cysteine Peptidase1 (XCP1), which participates in the programmed cell death process of xylem tracheary elements, was down-regulated in middle stem portions of both ATL54-knock-out and ATL54-overexpressed mutants. Alteration of ATL54 expression levels did not, however, affect lignin and polysaccharide content and composition in whole mature stems. Our results suggest that ATL54 is an E3 ubiquitin ligase involved in secondary wall biosynthesis and programmed cell death during xylogenesis.


Characterization of lignocellulose of Erianthus arundinaceus in relation to enzymatic saccharification efficiency.


Microscale thioacidolysis method for the rapid analysis of β-O-4 substructures in lignin


Characterization of lignocellulose of Erianthus ravennae in relation to enzymatic saccharification efficiency

Lignin is a major component of the secondary cell walls of vascular plants, and an obstacle in the conversion of plant cell wall polysaccharides into biofuels. Erianthus spp. are large gramineous plants of interest as potential energy sources. However, lignocelluloses of Erianthus spp. have not been chemically characterized. In this study, we analysed lignins, related compounds, enzymatic saccharification efficiencies, and minerals in the ash of the inner and outer parts of the internode, leaf blade and leaf sheath of Erianthus ravennae. Lignins in four organs consisted of guaiacyl, syringyl, and p-hydroxyphenyl units. The ratios of syringyl to guaiacyl lignins and lignin contents ranged from 0.43 to 0.79 and 20 to 28%, respectively, with values highest in the outer part of the internode. The amounts of ferulic acid were similar (7.3-11.8 mg g-1 dry weight of cell-wall material) in all four organs, while there was more p-coumaric acid in the inner part of the internode (44.7 mg g-1 dry weight of cell-wall material) than in other organs (25.7-28.8 mg g-1 dry weight of cell-wall material). The enzymatic saccharification efficiency (24 h reaction time) of the leaf blade was 21.6%, while those of the other organs ranged from 10.0 to 15.2%. The leaf blade had the highest ash content (17.1%); the main inorganic element was silicon. This paper provides the first fundamental knowledge of E. ravennae lignins.


Characterization of Jatropha curcas lignins


Silicon deficiency promotes lignin accumulation in rice

Although both lignin and silica accumulate on cell walls and confer rigidity, mechanical strength, and resistance to pathogen invasion in rice, it remains unclear whether silicon deficiency affects lignin accumulation. We demonstrate that low silicon rice mutated in a silicon influx transporter, Lsi1, or a silicon efflux transporter, Lsi2, contained larger amounts of lignin in the rice straws. Furthermore, wild-type rice cultivated on low silicon media also accumulated larger amounts of lignin in shoots. Significant accumulation of guaiacyl lignin upon silicon deficiency was determined by nitrobenzene oxidation analysis. These data indicate a negative correlation between silicon accumulation and lignin deposition.


Rapid analysis of transgenic rice straw using near-infrared spectroscopy

A rapid near-infrared (NIR) spectroscopy method was established to predict the lignin and starch contents and enzymatic saccharification efficiency of transgenic rice (Oryza sativa cv. Nipponbare) straw wherein expression of genes encoding lignin synthetic enzymes are regulated. Strong correlations were obtained between laboratory wet chemistry values and the NIR-predicted values. This method is useful to develop transgenic rice where cell-wall formation is engineered.


Occurrence of guaiacyl/p-hydroxyphenyl lignin in Arabidopsis thaliana T87 cells


Oxalate efflux transporter from the brown rot fungus Fomitopsis palustris.

An oxalate-fermenting brown rot fungus, Fomitopsis palustris, secretes large amounts of oxalic acid during wood decay. Secretion of oxalic acid is indispensable for the degradation of wood cell walls, but almost nothing is known about the transport mechanism by which oxalic acid is secreted from F. palustris hyphal cells. We characterized the mechanism for oxalate transport using membrane vesicles of F. palustris. Oxalate transport in F. palustris was ATP dependent and was strongly inhibited by several inhibitors, such as valinomycin and NH(4)(+), suggesting the presence of a secondary oxalate transporter in this fungus. We then isolated a cDNA, FpOAR (Fomitopsis palustris oxalic acid resistance), from F. palustris by functional screening of yeast transformants with cDNAs grown on oxalic acid-containing plates. FpOAR is predicted to be a membrane protein that possesses six transmembrane domains but shows no similarity with known oxalate transporters. The yeast transformant possessing FpOAR (FpOAR-transformant) acquired resistance to oxalic acid and contained less oxalate than the control transformant. Biochemical analyses using membrane vesicles of the FpOAR-transformant showed that the oxalate transport property of FpOAR was consistent with that observed in membrane vesicles of F. palustris. The quantity of FpOAR transcripts was correlated with increasing oxalic acid accumulation in the culture medium and was induced when exogenous oxalate was added to the medium. These results strongly suggest that FpOAR plays an important role in wood decay by acting as a secondary transporter responsible for secretion of oxalate by F. palustris.


Microscale alkaline nitrobenzene oxidation method for high-throughput determination of lignin aromatic components.

The nitrobenzene oxidation method is widely used for structural analysis of lignin. However, the conventional nitrobenzene oxidation method has several drawbacks including the requirement of a sizeable amount of sample material and the rather slow completion of the reaction process. In this paper, we describe a microscale nitrobenzene oxidation method using deuterium-labeled vanillin and syringaldehyde as internal standards. Using this method, we show that the microscale nitrobenzene oxidation method realized high-throughput determination of lignin components using a small amount of sample, i.e. 10 mg per reaction, and high reproducibility. Due to the small sample sizes, the oxidation reaction and extraction steps of a number of samples can be completed in parallel, thus enabling us to process more than 40 samples per day.


Subunit composition of hinokiresinol synthase controls enantiomeric selectivity in hinokiresinol formation

Asparagus officinalis hinokiresinol synthase (HRS) is composed of two subunits, HRSalpha and HRSbeta. Individually, each subunit forms (E)-hinokiresinol (EHR) from 4-coumaryl 4-coumarate, whereas a mixture of both subunits forms (Z)-hinokiresinol (ZHR) from the same substrate. In this study, we analyzed the enantiomeric compositions of ZHR and EHR formed after incubation of 4-coumaryl 4-coumarate with recombinant subunit proteins, recHRSalpha and/or recHRSbeta, and with naturally occurring A. officinalis ZHR. The enantiomeric composition of ZHR formed by the mixture of recHRSalpha and recHRSbeta was (+)-100% enantiomer excess (e.e.), identical to that of A. officinalis ZHR. In contrast, the enantiomeric compositions of EHR formed by recHRSalpha and recHRSbeta, individually, were (-)-20.6 and (-)-9.0% e.e., respectively. These results clearly demonstrate that the subunit composition of A. officinalis HRS controls not only cis/trans isomerism but also enantioselectivity in hinokiresinol formation.


Analysis of expressed sequence tags in developing secondary xylem and shoot of Acacia mangium


High-throughput determination of thioglycolic acid lignin from rice

For research and development of plants suitable for forage and biofuel production, the development of a high-throughput system of lignin determination, especially for herbaceous species, is important. However, currently available methods for lignin determination are not suitable for high-throughput analysis of herbaceous samples. In this paper, we describe a straightforward, high-throughput method for lignin determination as thioglycolic acid lignin from rice straw using disposable plastic microtubes, TissueLyser, and a UV microplate reader. We successfully plotted calibration curves derived from the readings of a spectrophotometer and a microplate reader using thioglycolic acid lignin prepared from bamboo milled wood lignin as a lignin standard. Based on the calibration curve, we could determine the lignin content in various organs from dried rice straw and rice seedlings. We confirmed that 20 mg of dried material from rice plants is adequate for stable determination of the lignin content. Using this method, 100 independent samples from rice straw can be analyzed in three days per person.


At5g54160 gene encodes Arabidopsis thaliana 5-hydroxyconiferaldehyde O-methyltransferase


Characterization of Arabidopsis thaliana Pinoresinol Reductase, a New Type of Enzyme Involved in Lignan Biosynthesis

A lignan, lariciresinol, was isolated from Arabidopsis thaliana, the most widely used model plant in plant bioscience sectors, for the first time. In the A. thaliana genome database, there are two genes (At1g32100 and At4g13660) that are annotated as pinoresinol/lariciresinol reductase (PLR). The recombinant AtPLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, which is in sharp contrast to conventional PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently to lariciresinol and secoisolariciresinol, respectively. Therefore, we renamed AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). The recombinant AtPrR2 encoded by At4g13660 reduced only (-)-pinoresinol to (-)-lariciresinol and not (+)-pinoresinol in the presence of NADPH. This enantiomeric selectivity accords with that of other PLRs of other plants so far reported, which can reduce one of the enantiomers selectively, whatever the preferential enantiomer. In sharp contrast, AtPrR1 encoded by At1g32100 reduced both (+)- and (-)-pinoresinols to (+)- and (-)-lariciresinols efficiently with comparative k(cat)/K(m) values. Analysis of lignans and spatiotemporal expression of AtPrR1 and AtPrR2 in their functionally deficient A. thaliana mutants and wild type indicated that both genes are involved in lariciresinol biosynthesis. In addition, the analysis of the enantiomeric compositions of lariciresinol isolated from the mutants and wild type showed that PrRs together with a dirigent protein(s) are involved in the enantiomeric control in lignan biosynthesis. Furthermore, it was demonstrated conclusively for the first time that differential expression of PrR isoforms that have distinct selectivities of substrate enantiomers can determine enantiomeric compositions of the product, lariciresinol.


Roles of 5-hydroxyconiferylaldehyde and caffeoyl-CoA O-methyltransferases in monolignol biosynthesis in Carthamus tinctorius


Subcellular localization of the oxalic acid-producing enzyme, cytochrome c-dependent glyoxylate dehydrogenase in brown-rot fungus Fomitopsis palustris


Cloning of a cDNA encoding a NAD-dependent formate dehydrogenase involved in oxalic acid metabolism from the white-rot fungus Ceriporiopsis subvermispora and its gene expression analysis

The authors have proposed previously that intracellular degradation of oxalic acid via formate to CO(2) occurs in the white-rot fungus Ceriporiopsis subvermispora. The formate degradation is catalyzed by NAD-dependent formate dehydrogenase (CsFDH). In this study, two cDNAs named CsFDH1 and CsFDH2 encoding CsFDH were cloned. Each cDNA consisting of 1077 bp encodes a mature protein composed of 358 amino acid residues. The amino acid sequences of the deduced CsFDH1 and CsFDH2 showed 99% identity to each other. The predicted molecular mass for each was 39.3 kDa, which was similar to that of CsFDH purified from the vegetative mycelia of Ceriporiopsis subvermispora (purified-CsFDH). The recombinant CsFDH1 and CsFDH2 expressed by Escherichia coli showed FDH activity with similar characteristics to the purified CsFDH. However, the amount of CsFDH1 transcript from the vegetative mycelia was 236-691 times greater than that of CsFDH2. Therefore, the results strongly suggest that CsFDH1, as compared with CsFDH2, predominantly contributes to the production of the purified CsFDH.


The subunit composition of hinokiresinol synthase controls geometrical selectivity in norlignan formation.

The selective formation of E- or Z-isomers is an important process in natural product metabolism. We show that the subunit composition of an enzyme can alter the geometrical composition of the enzymatic products. Hinokiresinol synthase, purified from Asparagus officinalis cell cultures, is responsible for the conversion of (7E,7'E)-4-coumaryl 4-coumarate to (Z)-hinokiresinol, the first step in norlignan formation. The protein is most likely a heterodimer composed of two distinct subunits, which share identity with members of the phloem protein 2 gene superfamily. Interestingly, each recombinant subunit of hinokiresinol synthase expressed in Escherichia coli solely converted (7E,7'E)-4-coumaryl 4-coumarate to the unnatural (E)-hinokiresinol, the E-isomer of (Z)-hinokiresinol. By contrast, a mixture of recombinant subunits catalyzed the formation of (Z)-hinokiresinol from the same substrate.

Proceedings of the National Academy of Sciences of the United States of America


Involvement of FpTRP26, a thioredoxin-related protein, in oxalic acid-resistance of the brown-rot fungus Fomitopsis palustris.

Brown-rot fungus Fomitopsis palustris grows vigorously at high concentrations of oxalic acid (OA), which is fungal metabolite during wood decay. We isolated a cDNA FpTRP26 from F. palustris by functional screening of yeast transformants with cDNAs grown on plates containing OA. FpTRP26 conferred a specific resistance to OA on the transformant. OA-content in transformants grown with 2mM OA decreased by 65% compared to that of the control. The amount of FpTRP26 transcript in F. palustris amplified with increasing OA-accumulation, and was maintained at high levels even in the stationary phase. Its transcription in F. palustris was inducible in response to exogenously added OA. These results suggest that FpTRP26 is involved in the OA-resistance in F. palustris.


Thermal Behavior of β-1 Subunits in Lignin: Pyrolysis of 1,2-Diarylpropane-1,3-diol-type Lignin Model Compounds

1,2-Diarylpropane-1,3-diol-type lignin model compounds, 1,2-bis(4-hydroxy-3-methoxyphenyl)propane-1,3-diol (1) and 1-(3,4-diethoxyphenyl)-2-(4-methoxyphenyl)propane-1,3-diol (2), were pyrolyzed at 500 degrees C for 4 s to clarify the thermal behavior of beta-1 subunits in lignin. Products were monitored by gas chromatography/mass spectrometry. The cleavage of the Calpha-Cbeta bond to produce benzaldehydes such as 4-hydroxy-3-methoxybenzaldehyde (9) and phenylethanals as the counterparts such as 4-hydroxy-3-methoxyphenylethanal (10) occurred in pyrolyses of both 1 and 2. In pyrolysis of 1, an oxetane pathway leading to the formation of Z/E-stilbenes without the gamma-CH2OH group such as Z/E-4,4'-dihydroxy-3,3'-dimethoxystilbene (3) was predominant. In pyrolysis of 2, the oxetane pathway was minor, while pathways producing a dimer with a =CgammaH2 group by loss of water and a dimer with an alpha-carbonyl group were predominant. Pyrolysis of Japanese cedar wood provided 3 and 10 in approximately 0.8% and 0.6% yields, respectively, based on the Klason lignin content, while pyrolysis of a guaiacyl bulk dehydrogenation polymer gave them in a very small amount.

Journal of Agricultural and Food Chemistry


Studies on the Dehydrogenative Polymerizations of Monolignol β Glycosides. Part 1. Syntheses of Monolignol β glycosides, (E) Isoconiferin, (E) Isosyringin, and (E) Triandrin

Journal of Wood Chemistry and Technology


Subcellular localization of glyoxylate cycle key enzymes involved in oxalate biosynthesis of wood-destroying basidiomycete Fomitopsis palustris grown on glucose.

This study investigated the subcellular localization of key enzymes of the glyoxylate cycle, i.e. isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (EC 2.3.3.9), that function constitutively in coordination with oxalate biosynthesis of glucose-grown Fomitopsis palustris. The ICL purified previously from F. palustris is termed FPICL1. Subcellular fractionation analysis of the cell homogenate by the sucrose density-gradient method showed that both key enzymes were present in peroxisomes, whereas acetyl-CoA synthase (EC 6.2.1.1) and oxalate-producing oxaloacetate acetylhydrolase (EC 3.7.1.1) were cytosolic. The peroxisomal localization of FPICL1 was further confirmed by electron microscopic and immunocytochemical analysis with anti-FPICL1 antibody. In addition, the peroxisomal target signal, composed of SKL at the C terminus of the cDNA encoding FPICL1, was found, which also suggests that FPICL1 is peroxisomal. Accordingly, it is postulated that transportation of succinate from peroxisomes to mitochondria, and vice versa, for the transportation of isocitrate or citrate, occurs in glucose-grown F. palustris for the constitutive metabolic coordination of the TCA and glyoxylate cycles with oxalate biosynthesis.


Purification and characterization of NAD-dependent formate dehydrogenase from the white-rot fungus Ceriporiopsis subvermispora and a possible role of the enzyme in oxalate metabolism


The ability of ectomycorrhizal fungi to utilize fatty acids and a lipid as a carbon source for mycelial growth


Purification and characterization of NADP-isocitrate dehydrogenase from the copper-tolerant wood-rotting basidiomycete Fomitopsis palustris

NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), a key enzyme of the tricarboxylic acid cycle, was purified 672-fold as a nearly homogeneous protein from the copper-tolerant wood-rotting basidiomycete Fomitopsis palustris. The purified enzyme, with a molecular mass of 115 kDa, consisted of two 55-kDa subunits, and had the Km of 12.7, 2.9, and 23.9 microM for isocitrate, NADP, and Mg2+, respectively, at the optimal pH of 9.0. The enzyme had maximum activity in the presence of Mg2+, which also helped to prevent enzyme inactivation during the purification procedures and storage. The enzyme activity was competitively inhibited by 2-oxoglutarate (K(i), 127.0 microM). Although adenine nucleotides and other compounds, including some of the metabolic intermediates of glyoxylate and tricarboxylic acid cycles, had no or only slight inhibition, a mixture of oxaloacetate and glyoxylate potently inhibited the enzyme activity and the inhibition pattern was a mixed type.

Bioscience, Biotechnology, and Biochemistry


A metabolic role of the gloxylate and TCA cycles for development of the copper-tolerant brown-rot fungus Fomitopsis palustris

Fruit bodies of the copper-tolerant brown-rot fungus Fomitopsis palustris were produced in liquid medium for the first time. To induce fruit body formation of this fungus, it was important to inoculate the liquid medium with mycelia grown on potato dextrose agar plates and also to adjust the initial pH of the medium to 5.0. The metabolic role of the glyoxylate and tricarboxylic acid cycles during fungal development in the liquid culture was investigated in relation to oxalate biosynthesis. The enzymes for the glyoxylate cycle and oxalate biosynthesis in mycelium showed greater activities at the vegetative growth stage than at the fruiting stage. The ratios of isocitrate dehydrogenase activity to isocitrate lyase activity in mycelium were 0.3 and 4.0 at the vegetative and fruiting stage, respectively. Thus, isocitrate lyase of the glyoxylate cycle played a more important role in oxalate synthesis at the earlier stage of the cultivation, whereas isocitrate dehydrogenase played a major role in glutamate synthesis during fruit body formation.


A possible role of the key enzymes of the glyoxylate and gluconeogenesis pathways for fruit-body formation of the wood-rotting basidiomycete Flammulina velutipes


Purification and Characterization of Malate Synthase from the Glucose-grown Wood-rotting Basidiomycete Fomitopsis palustris(Biochemistry & Molecular Biology)

Malate synthase (EC 4. 1. 3. 2), the key enzyme of the glyoxylate cycle, was purified to a homogeneous protein from the wood-rotting basidiomycete Fomitopsis palustris grown on glucose. The purified enzyme, with a molecular mass of 520 kDa, was found to consist of eight 65-kDa subunits, and to have K_m of 45 and 2.2 μM for glyoxylate and acetyl-CoA, respectively. The enzyme activity was competitively inhibited by oxalate (K_i, 8.5 μM) and glycolate (K_i, 17 μM), and uncompetitively by coenzyme A (K_i, 100 μM). The potent inhibition of the activity by p-chloromercuribenzoate suggests that the enzyme has a sulfhydryl group at the active center. However, the enzyme was inhibited moderately by adenine nucleotides and weakly by some of the metabolic intermediates of glycolysis and tricarboxylic acid cycle. The enzyme was completely inactive in the absence of metal ions and was maximally activated by Mg^<2+> (K_m, 0.4 μM), which also served to significantly prevent enzyme inactivation during storage.


Purification and characterization of isocitrate lyase from the wood-destroying basidiomycete Fomitopsis palustris grown on glucose.

Isocitrate lyase (EC 4.1.3.1), a key enzyme in the glyoxylate cycle, was purified 76-fold with 23% yield as an electrophoretically homogeneous protein from the wood-destroying basidiomycete Fomitopsis palustris grown on glucose. The native enzyme has a molecular mass of 186 kDa, consisting of three identical subunits of 60 kDa. The K(m) for DL-isocitrate was found to be 1.6 mM at the optimum pH (7.0). The enzyme required Mg(2+) (K(m) 92 microM) and sulfhydryl compounds for optimal activity. The enzyme activity was strongly inhibited by oxalate and itaconate with a K(i) of 37 and 68 microM, respectively. The inhibition by the glycolysis and tricarboxylic acid cycle intermediates and related compounds suggested that the isocitrate lyase was a regulatory enzyme playing a crucial role in the fungal growth.


A physiological role for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris

A metabolic mechanism for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris has been proposed on the basis of biochemical analyses of glucose metabolism. There was a strong correlation between glucose consumption and oxalate production. Oxalic acid was found to accumulate in the culture fluid in about 80% of the theoretical yield or about 5-fold, on the basis of the fungal biomass harvested. The results clearly indicate that glucose was not completely oxidized to CO(2) by the tricarboxylic acid (TCA) cycle but converted mainly to oxalate. The determination of the 12 enzymes concerned has revealed the occurrence of the unprecedented metabolic coupling of the TCA and glyoxylate cycles that support oxalate biosynthesis. In this metabolic system, isocitrate lyase (EC ), together with oxaloacetase (EC ), was found to play a pivotal role in yielding oxalate from oxaloacetate via the acetate-recycling routes. Moreover, malate dehydrogenase (EC ), with an extraordinarily high activity among the enzymes tested, was shown to play an important role in generating NADH by oxidation of malate to oxaloacetate. Thus, it is proposed that the wood-rotting basidiomycete acquires biochemical energy by oxidizing glucose to oxalate.

Proceedings of the National Academy of Sciences of the United States of America


New role for glyoxylate cycle enzymes in wood-rotting basidiomycetes in relation to biosynthesis of oxalic acid


Induction of L-phenylalanine ammonia-lyase and suppression of veratryl alcohol biosynthesis by exogenously added L-phenylalanine in a white-rot fungus phanerochaete chrysosporium

The effects of exogenously added L-phenylalanine (L-Phe) on the activities of L-phenylalanine ammonia-lyase (PAL) and 3, 4-dimethoxybenzyl alcohol (veratryl alcohol, VA) biosynthesis in ligninolytic cultures of Phanerochaete chrysosporium were investigated. Increasing PAL activity was detected in low nitrogen (LN) culture but not in high nitrogen (HN) culture. The addition of L-Phe into the LN culture caused a 25-fold increase in enzyme activity, which clearly shows that L-Phe, a substrate of the enzyme, served as an inducer of PAL. The increase in activity of PAL triggered by nitrogen starvation was correlated with biosynthesis of VA. However, PAL induced by the added L-Phe did not promote VA biosynthesis but suppressed the biosynthesis probably due to NH(4)(+) released from L-Phe.


Purification and characteristics of a novel cytochrome c dependent glyoxylate dehydrogenase from a wood-destroying fungus Tyromyces palustris


Cloning of Phanerochaete chrysosporium leu2 by Complementation of Bacterial Auxotrophs and Transformation of Fungal Auxotrophs

A Phanerochaete chrysosporium cDNA library was constructed in an expression vector that allows expression in both Escherichia coli and Saccharomyces cerevisiae. This expression vector, lambda YES, contains the lacZ promoter for expression in E. coli and the GAL1 promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the beta-isopropylmalate dehydrogenase from P. chrysosporium was characterized further. The genomic clone (gleu2) was subsequently isolated and was used successfully as a selectable marker to transform P. chrysosporium auxotrophs for LEU2. Protoplasts for transformation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous P. chrysosporium transformation procedures. The method described here allows other genes to be isolated from P. chrysosporium for use as selectable markers.


Degradation of non-phenolic β-O-4 type Lignin carbohydrate complex model compounds by lignin peroxidase from the white-rot fungus Phanerochaete Chrysosporium


A New Biomimetic Degradation of Nonphenolic α-Carbonyl β-O-4 Lignin Model Compounds with Mn(3)in the Presence of Oxalate and Dimethylsulfoxide


Formate and oxalate esters in lignin obtained from bagasse degraded by Lentinus edodes


Characterization of Antiviral Water-Soluble Lignin from Bagasse Degraded by Lentinus edodes


Kinetic Analysis of the Noncompetitive Inhibition of the Lignin-Peroxidase-catalyzed Reaction by Oxalic Acid

Oxalic acid was found to inhibit noncompetitively the Cα-Cβ bond cleavage of veratrylglycerol catalyzed by a lignin peroxidase (LiP) isozyme of the white-rot fungus P.chrysosporium. With greater amounts of oxalic acid in the LiP system, the substrate was not converted to veratraldehyde but was almost all recovered. Oxalic acid was shown to be decomposed to CO_2 during the enzymatic reaction. The results clearly indicate that oxalic acid reduced the cation radical intermediate formed in the reaction back to the substrate to block the production of veratraldehyde. A novel equation has been derived to explain the mechanism for this unique non-competitive inhibition that is different from the classical noncompetitive one. The inhibition constant K_i obtained here, which is different from the classical inhibition constant K_i, is defined as the ratio of the rate constant (K_p) for product formation to the rate constant (K_i) for the reduction of the cation radical to the substrate.

Bioscience, Biotechnology, and Biochemistry


Sugar components in malt whisky I.Analysis by gas chromatography


Degraddation of a (B-O-4)-(5-5')lignin substructure model trimer by lignin peroxidase preparation


Degradation of lignin substrucuture model compounds in the presence of veratoryl alcohol or veratryl B-D-xyloside by lignin peroxidase


Degradation of phenolic and nonphenolic syringyl and biphenyl lignin model compounds by lignin peroxidase


Producution of secondary metabolites including a new metabolite p-methoxyphenylpropanol by the brown-rot fungus lentinus lepideus


Aromatic ring cleavage of vanillyl alcohol by lifnin peroxidace of Phanerochaete chrysisporium


A new biomimetic lignin degradation system developed with Mn/Co catalysts and its application to the chlorine free bleaching of kraft pulp


New Mechanism for Oxygenative Ring Cleavage of 3,4-Dimethoxybenzyl Alcohol Catalyzed by the Ligninase Model(Organic Chemistry)


Regiospecific oxygenations during ring cleavage of a secondary metabolite, 3,4-dimethoxybenzyl alcohol catalyzed by lignin peroxidase.

Enzymatic oxidation of veratryl alcohol yielded a new ring cleavage product (delta-lactone) in addition to the two known gamma-lactone products. The experiment with 18O-enriched water and dioxygen clearly showed that one oxygen atom each from water and dioxygen is specifically incorporated into the cleavage product at the original C3 or C4 position of 3,4-dimethoxybenzyl alcohol. A new type of reaction mechanism proposed for the ring cleavage of this compound is rationally explained in good accord with the one-electron transfer mechanism.


Simple measurement of starch degradation in Tricholoma matsutake

マツタケのデンプン分解能簡易測定法


Cloning and xpression analysis of a cDNA encoding an oxaloacetate acetylhydrolase from the brown-rot fungus Fomitopsis palustris


Biodiversity in the tropical plantation forest


きのこの代謝のひみつとその環境浄化への応用

きのこの代謝のひみつとその環境浄化への応用


Protocols for lignin analysis for Forest Biomass Analytical System of RISH, Kyoto University

森林バイオマス評価分析システムにおけるリグニン分析プロトコール


Conservation of biodiversity in the tropical plantation forest

熱帯大規模一斉植林における生物多様性の確保


Basic Studies towards Elucidation of Heartwood Formation Mechanisms

この論文は国立情報学研究所の学術雑誌公開支援事業により電子化されました．

この論文は国立情報学研究所の学術雑誌公開支援事業により電子化されました．


A Role of Formate Dehydrogenase in the Oxalate Metabolism in the Wood-destroying Basidiomycete Ceriporiopsis subvermispora


cDNA Cloning of Isocitrate Lyase from the Copper-Tolerant Wood-Rotting Basidiomycete Fomitopsis palustris


An Enzymatic Study on Isocitrate Metabolism in the Ectomycorrhizal Fungus Laccaria amethystea


A Possible Role of Oxalate Produced in the Symbiotic Culture System with a Host Plant Pinus densiflora and a Mycorrhizal Fungus Lactarius hatsudake


An Enzymatic Study of an Oxalate Producing System in Relation to the Glyoxylate Cycle in White-rot Fungus Phanerochaete chrysosporium


Dechlorination of Trichloroacetic Acid by Mn(III)/oxalate System and the Enzymatic System of Manganese Peroxidase/Mn(II)/oxalate


A Possible Growth Promoting Effect of the Organic Acids Produced in an Axenic Symbiotic Culture of Pinus densiflora and Lactarius hatsudake on Both of Symbionts

Annual Report of Interdisciplinary Research Institute of Environmental Sciences


A Possible Intramolecular Electron Transfer Pathway of Glyoxylate Dehydrogenase in a Brown-rot Fungus Tyromyces palustris


Mechanisms for Ectomycorrhizae Synthesis. (Part I). The Production of Organic Acids during the Symbiotic Cultivation of Pinus densiflora Associated with Lactarius hatsudake


The Role of Secondary Metabolite of Wood-Rotting Fungi and Mycorrhizal Fungi


A New Oxidative Degradation of A Bio-recalcitrant α-Carbonyl β-O-4 Lignin Model Compound with Mn(III)/Oxalate System


The Degradation of β-O-4 Type Lignin Carbohydrate Complex (LCC) Model Compounds by Lignin Peroxides


Occurrence of Enzyme Systems for Production and Decomposition of Oxalate in a White-Rot Fungus Coriolus versicolor and Some Characteristics of Glyoxylate Oxidase


Effect of Oxalic Acid on the Oxidative Breakdown of Cellulose by the Fenton Reaction


Biomimetic Oxidation of β-O-4 Lignin Model Compound with tert-Butylhydroperoxide and Fe(III)-Octacarboxyphthalocyanine


Lignin Peroxidase-Catalyzed Oxidation of Monomeric Lignin Model Substrates

この論文は国立情報学研究所の学術雑誌公開支援事業により電子化されました．The substrate specificity of lignin peroxidase (LiP) was investigated by determining the K_ms and V_<max>s of LiP for the four lignin model substrates of 3,4-dimethoxybenzyl alcohol (I), 3,4-dimethoxybenzyl glycerol (II), 3,4,5-trimethoxybenzyl alcohol (III), and 3, 4, 5-trimethoxybenzyl glycerol (IV). The K_ms (μM) and V_<max>s (turnover number, sec^<-1>) of LiP were determined. The K_m/V_<max> values calculated to be 4.7, 18.2, 29.9, and 76.7 for I, II, III, and IV, respectively. Among the substrates tested, I (veratryl alcohol), which is a secondary metabolite of white-rot fungi, was found to be the best substrate for LiP.


Aromatic Ring Opening of Lignin Monomeric Model Compounds by Lignin Peroxidase


Improvement of Lignin Peroxidase Production by Phanerochaete chrysosporium in Shaking Culture in the Presence of Polyurethane Foam Cubes

この論文は国立情報学研究所の学術雑誌公開支援事業により電子化されました．Effects of polyurethane foam cubes and MnSO_4 on lignin peroxidase (LiP) production by the white-rot fungus Phanerochaete chrysosporium was investigated by use of shaking culture. Addition of both polyurethane cubes and MnSO_4 greatly enhanced the production of LiP activity up to 950 U/l culture medium. The increase in the enzyme activity is about twice as compared with that obtained from the stationary culture cultivated in the absence of these additives.


The C_α-C_β Bond Cleavage of the Secondary Metabolite Veratryglycerol Catalized by A New Modified "Ligninase" Preparation from Phanerochaete chrysosporium

この論文は国立情報学研究所の学術雑誌公開支援事業により電子化されました．The various culture conditions for production of lignin peroxidase were examined by use of the white-rot fungus Phanerochaete chrysosporium grown for 4 days in the cultures with high carbon and low nitrogen (HC/LN)-, low carbon and high nitrogen (LC/HN)-, or low carbon and medial nitrogen (LC/MN)-media. Among them, LC/MN-culture with 0.1% Tween 80 produced the greatest amount of ligninase activity (356 units/l). A secondary metabolite veratrylglycerol occurring in the extra-cellular culture fluid underwent Cα-Cβ bond cleavage in the presence of ligninase and hydrogen peroxide, yielding veratraldehyde and glycolaldehyde. The oxidation rates of a threo- and a mixture of threo- and erythro-forms of veratrylglycerol synthesized were about 60% of that of veratryl alcohol. A possible relevance of this cleavage reaction to the Cα-Cβ bond cleavage of arylglycerol-β-aryl ether substructures of lignin polymer was discussed.