Absolute configuration of ceriporic acids, the iron redox-silencing metabolites produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora

Ceriporic acids are a class of alk(en)ylitaconic acids produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora. The unique function of alkylitaconic acid is the redox silencing of the Fenton reaction system by inhibiting reduction of Fe³⁺. Ceriporic acids have an asymmetric centre at carbon-3, but absolute configuration has not been determined. We have isolated a series of ceriporic acids from the cultures of C. subvermispora, and measured their NMR spectra using a chiral shift reagent. In comparison with NMR spectra of (R)-(−)- and (S)-(+)-methylsuccinic acid and those of natural and chemically synthesized racemic mixtures of ceriporic acids, we have determined the absolute configuration of ceriporic acids as (R)-3-tetradecylitaconic acid (ceriporic acid A), (R)-3-hexadecylitaconic acid (ceriporic acid B) and (R,Z)-2-(hexadec-7-enyl)-3-itaconic acid (ceriporic acid C). We herein discuss their stereoselective biosynthetic pathway and the structural diversity of fungal secondary metabolites.     

Chemical synthesis,iron redox interactions and charge transfer complex formation of alkylitaconic acids from Ceriporiopsis subvermispora

In 1999, we first reported that a white rot fungus, Ceriporiopsis subvermispora produced a series of novel alkylitaconic acids (ceriporic acids). In the present paper we synthesized the metabolite, 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B) by Grignard reaction to analyze chemical properties of the alkylitaconates. Mass spectrometer (MS) and nuclear magnetic resonance (NMR) spectra of the synthetic compound was identical to those of the fungal metabolite isolated. The dicarboxylic acid inhibited autoxidation of Fe²⁺ to Fe³⁺ as well as reduction of Fe³⁺ to Fe²⁺ by the strong natural reductants, cysteine, glutathione, and ascorbic acid. The formation of charge transfer complexes (CTCs) between 1-heptadecene-2,3-dicarboxylic acid and oxidized intermediates from phenolic substrates were also observed. Thus, we herein report that the new class of lipid-related metabolites produced by C. subvermispora are potential metabolites participating in the control of iron redox reactions and CTCs formation from oxidized lignin fragments.     

Diverse rare lipid-related metabolites including ω-7 and ω-9 alkenylitaconic acids (ceriporic acids) secreted by a selective white rot fungus, Ceriporiopsis subvermispora

Ceriporiopsis subvermispora is a selective white-rot fungus that secretes alk(en)ylitaconic acids named ceriporic acids, known as ion redox silencers. In this study, we analysed a series of extracellular lipid-related metabolites produced by the fungus and found that a wide variety of ceriporic acids and fatty acids, including those with odd-numbered and very long-chains, were produced in wood meal cultures. Two new ceriporic acids, (R)-3-[(Z)-tetradec-7-enyl]-itaconic acid (ceriporic acid E) and (R)-3-[(Z)-tetradec-5-enyl]-itaconic acid (ceriporic acid F), were for the first time identified by dimethyl disulfide derivatisation, followed by GC/EI-MS, (1)H and homonuclear J-resolved 2D NMR and feeding experiments with [(13)C-U] glucose coupled with multiple-stage mass spectrometry. In separation by GC and LC, a reversed correlation of elution sequences between a nonpolar GC column and an ODS-LC column for cis and trans isomers of ω7 and ω9 lipids was found, and the elution of new metabolites was in accordance with the prevailing theory. The biosynthetic precursors of ceriporic acid F can be proposed as oxaloacetate and 16:1Δ7-CoA. Because fatty acids biosynthesised from 16:1Δ7-CoA have been reported for only a limited number of organisms, the highly individual structure of ceriporic acid F is highlighted.     

Ceriporic acid C, a hexadecenylitaconate produced by a lignin-degrading fungus, Ceriporiopsis subvermispora

A lignin-degrading basidiomycete, Ceriporiopsis subvermispora produces a series of alkyl- and alkenylitaconates (ceriporic acids). Previously, two alkylitaconic acids with tetradecyl and hexadecyl side chains were isolated and identified as 1-heptadecene-2,3-dicarboxylic acid (ceriporic acid A) and 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B). In the present study, one hexadecenylitaconate (ceriporic acid C) was isolated and its chemical structure was analyzed by glycolation and subsequent (1) trimethylsilation, or (2) acetalation with acetone and acetone-d6. Analyses of the isolated metabolite demonstrated that the hexadecenylitaconic acid was (Z)-1,10-nonadecadiene-2,3-dicarboxylic acid. The structure of the side chain in ceriporic acid C was the same as that of hexadecenylcitraconate, chaetomellic acid B. Thus, it was found that ceriporic acids share close structural similarity with alk(en)yl citraconate derivatives, chaetomellic acids and other lichen lactones, protolichesterinic, lichesterinic, and murolic acids.     

Linoleic acid peroxidation initiated by Fe-reducing compounds recovered from Eucalyptus grandis biotreated with Ceriporiopsis subvermispora

This work evaluates linoleic acid peroxidation reactions initiated by Fe³⁺-reducing compounds recovered from Eucalyptus grandis, biotreated with the biopulping fungus Ceriporiopsis subvermispora. The aqueous extracts from biotreated wood had the ability to reduce Fe³⁺ ions from freshly prepared solutions. The compounds responsible for the Fe³⁺-reducing activity corresponded to UV-absorbing substances with apparent molar masses from 3 kDa to 5 kDa. Linoleic acid peroxidation reactions conducted in the presence of Fe³⁺ ions and the Fe³⁺-reducing compounds showed that the rate of O₂ consumption during peroxidation was proportional to the Fe³⁺-reducing activity present in each extract obtained from biotreated wood. This peroxidation reaction was coupled with in-vitro treatment of ball-milled E. grandis wood. Ultraviolet data showed that the reaction system released lignin fragments from the milled wood. Size exclusion chromatography data indicated that the solubilized material contained a minor fraction representing high-molar-mass molecules excluded by the column and a main low-molar-mass peak. Overall evaluation of the data suggested that the Fe³⁺-reducing compounds formed during wood biodegradation by C. subvermispora can mediate lignin degradation through linoleic acid peroxidation.     

De novo synthesis of (Z)- and (E)-7-hexadecenylitaconic acids by a selective lignin-degrading fungus, Ceriporiopsis subvermispora

Ceriporic acids are a class of alk(en)ylitaconic acids produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora. Their structural units have similarity with biologically important lichen acids, such as chaetomellic and protolichesterinic acids. The unique function of alkylitaconic acid is the redox silencing of the Fenton reaction system by inhibiting reduction of Fe(3+). As estimated by the catalytic function of Delta9-desaturases, 7-hexadecenyl derivatives bearing a trans configuration have not been reported in the family of alk(en)ylitaconic acids, i.e. the structurally similar lichen acids-alk(en)ylcitraconic and paraconic acids. In this paper, we discuss the isolation of an itaconic acid derivative with an (E)-7-hexadecenyl chain from cultures of C. subvermispora. To identify the natural metabolite, (E)- and (Z)-7-hexadecenylitaconic acids were chemically synthesised. The isolated metabolite was identical to the synthetic (E)-hexadecenylitaconic acid and was designated as ceriporic acid D. Administration of (13)C-[U]-glucose demonstrated that ceriporic acid C and trans-7-hexadecenylitaconic acid (ceriporic acid D) were biosynthesised de novo by C. subvermispora.     

Lipid Peroxidation Induced by Phenolics in Conjunction with Aluminum Ions

Using the whole plant and model systems, we demonstrate that the aluminum ions (Al³⁺) stimulate phenolic-dependent lipid peroxidation. Lipid peroxidation in barley (Hordeum vulgare L. cv. Donor) roots was 30 % higher under AlCl₃ treatment than without Al. Major decomposition product of lipid peroxidation was 4-hydroxynonenal (4-HNE) but not thiobarbituric acid reactive substances (TBARS), a widely used markers for lipid peroxidation. Similarly, AlCl₃ stimulated lipid peroxidation of soybean liposomes in the presence of chlorogenic acid (CGA) and H₂O₂/horseradish peroxidase system which can oxidize phenolics. Al³⁺ was found to enhance lipid peroxidation induced by oxidized CGA. Intermediates of lignin biosynthesis in plants, including p-coumaric acid, ferulic acid, sinapic acid and coniferyl alcohol, also showed similar effects. These results suggest that Al³⁺ has a potential to induce oxidative stress in plants by stimulating the prooxidant nature of endogenous phenolic compounds.     

Metabolism of auxin in pine tissues: Indole-3-acetic acid conjugation

Incubation of sections of various tissues of Pinus pinea L. with a relatively low concentration (3.6 μM) of indole-3-acetic acid-2-¹⁴C (IAA) resulted in the formation of two major metabolites. The first, which has not been identified, seemed to be a polar acidic compound and the second was identified as indole-3-acetylaspartic acid (IAAsp). The polar acidic metabolite has been found to be the major metabolite in needles, shoot wood and roots, while IAAsp has been found to be the major metabolite in shoot bark. Increasing the concentration of IAA in the incubation medium resulted in an increase in the formation of a third metabolite which proved to be l-O-(indole-3-acetyl)-β-d -glucose (IAGlu) and a concomitant decrease in the amount of the polar acidic metabolite. This phenomenon was prominent particularly in needles. IAGlu was isolated from needles and IAAsp was isolated from shoot bark by means of polyvinylpolypyrrolidone column chromatography and preparative thin-layer chromatography. IAGlu was identified by comparison with authentic material by co-chromatography in three different solvent systems and by ¹H-nuclear magnetic resonance analysis. IAAsp was identified by comparison with authentic material by gas-liquid chromatography and ¹H-nuclear magnetic resonance analysis. Several aspects of formation, separation and isolation of IAA metabolites are discussed.     

Oxidation of biphenyl by the Cyanobacterium,Oscillatoria sp., strain JCM

The oxidation of biphenyl by Cyanobacterium, Oscillatoria sp., strain JCM was studied. The organism grown photoautotrophically in the presence of biphenyl oxidized biphenyl to form 4-hydroxybiphenyl. The structure of the metabolite was elucidated by ultraviolet and mass spectra and shown to be identical to authentic 4-hydroxybiphenyl. In addition this metabolite had properties indentical to 4-hydroxybiphenyl when analyzed by thin-layer and high-pressure liquid chromatography. Experiments with [¹⁴C]-biphenyl showed that over a 24 h period the organism oxidized 2.9% of the added biphenyl to ethyl acetate-soluble products.Abbreviations tlc thin-layer chromatography - hplc high pressure liquid chromatography     

Ceriporic acid B, an extracellular metabolite of Ceriporiopsis subvermispora, suppresses the depolymerization of cellulose by the Fenton reaction

The white rot fungus, Ceriporiopsis subvermispora, is able to degrade lignin in wood without intensive damage to cellulose. Since lignin biodegradation by white rot fungi proceeds by radical reactions, accompanied by the production of a large amount of Fe3+-reductant phenols and reductive radical species in the presence of iron ions, molecular oxygen, and H2O2, C. subvermispora has been proposed to possess a biological system which suppresses the production of a cellulolytic active oxygen species, *OH, by the Fenton reaction. In the present paper, we demonstrate that 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B), an extracellular metabolite of C. subvermispora, strongly inhibited *OH production and the depolymerization of cellulose by the Fenton reaction in the presence of iron ions, cellulose, H2O2, and a reductant for Fe3+, hydroquinone (HQ), at the physiological pH of the fungus.     

Relationship between hydroxycinnamic acid content,lignin composition and digestibility of maize silages in sheep

Cell wall-bound hydroxycinnamic acids and the composition of lignin were studied in relation to the digestibility of a collection of 91 maize silages in wethers. Total lignin and guaiacyl content showed the highest correlation coefficients with digestibility. Using the above-mentioned chemical parameters, eight equations were also developed to predict digestibility. The prediction of organic matter digestibility produced a high adjusted R ² value (0.487) using total lignin, guaiacyl, esterified ferulic acid and esterified p-coumaric acid content as predictors. The prediction of in vivo dry matter digestibility produced a higher adjusted R ² value (0.516) using the same variables as predictors. Cell wall digestibility depends on a multiplicity of factors and it is not possible to attribute a causal effect on in vivo digestibility to any single factor. However, total lignin, guaiacyl and p-coumaric acid content emerge as good predictors of digestibility.     

Microbial Delignification with White Rot Fungi Improves Forage Digestibility

Three wild-type white rot fungi and two cellulase-less mutants developed from Phanerochaete chrysosporium K-3 (formerly Sporotrichum pulverulentum) were tested for their ability to delignify grass cell walls and improve biodegradation by rumen microorganisms. Fungal-treated and control stems of Bermuda grass were analyzed for their content of ester- and ether-linked aromatics by using alkali extraction and gas chromatography, for in vitro dry weight digestion and production of volatile fatty acids in in vitro fermentations with mixed ruminal microorganisms, for loss of lignin and other aromatics from specific cell wall types by using microspectrophotometry, and for structural changes before and after in vitro degradation by rumen microorganisms by using transmission electron microscopy. P. chrysosporium K-3 and Ceriporiopsis subvermispora FP 90031-sp produced the greatest losses in lignin and improved the biodegradation of Bermuda grass over that of untreated control substrate. However, C. subvermispora removed the most lignin and significantly improved biodegradation over all other treatments. Phellinus pini RAB-83-19 and cellulase-less mutants 3113 and 85118 developed from P. chrysosporium K-3 did not improve the biodegradation of Bermuda grass lignocellulose. Results indicated that C. subvermispora extensively removed ester-linked p-coumaric and ferulic acids and also removed the greatest amount of non-ester-linked aromatics from plant cell walls. Microscopic observations further indicated that C. subvermispora removed esters from parenchyma cell walls as well as esters and lignin from the more recalcitrant cell walls (i.e., sclerenchyma and vascular tissues). C. subvermispora improved in vitro digestion and volatile fatty acid production by ruminal microorganisms by about 80%, while dry matter loss due to fungi was about 20% greater than loss in untreated control stems. The chemical and structural studies used identified sites of specific fungal attack and suggested mechanisms whereby improvement occurred.     

A novel enantioselective epoxide hydrolase for (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol

Bacillus sp. Z018, a novel strain producing epoxide hydrolase, was isolated from soil. The epoxide hydrolase catalyzed the stereospecific hydrolysis of (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol. Epoxide hydrolase from Bacillus sp. Z018 was inducible, and (R)-phenyl glycidyl ether was able to act as an inducer. The fermentation conditions for epoxide hydrolase were 35°C, pH 7.5 with glucose and NH₄Cl as the best carbon and nitrogen source, respectively. Under optimized conditions, the biotransformation yield of 45.8% and the enantiomeric excess of 96.3% were obtained for the product (R)-3-phenoxy-1,2-propanediol.     

Metabolism of Chiral Ionylideneacetic Acids on the Abscisic Acid Biosynthetic Pathway in Cercospora

A Chiralcel OJ column was used to determine the absolute configuration of naturally occurring α-ionylideneacetic acid from Cercospora rosicola and γ-ionylideneacetic acid from C. cruenta as (R) enantiomers in accordance with their biosynthetic product, (S)-ABA. Both enantiomers of [1, 2-¹³C₂]-α and γ-ionylideneacetic acids were prepared and fed to C. rosicola and C. cruenta. Six combinations of feeding experiments comparatively and unequivocally demonstrated stereoselectivity in the biosynthetic conversions, including stepwise hydroxylation at C-1′ and 4′. Enzymatic isomerization from the γ to α-intermediate was suggested not to be involved in ABA biosynthesis in C. rosicola.     

Chemical synthesis of the (25R)- and (25S)-epimers of 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid as well as their corresponding glycine and taurine conjugates

The (25R)- and (25S)-epimers of C₂₇ 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid as well as their corresponding N-acylamidate conjugates with glycine or taurine were prepared starting from cholic acid in 14 steps. The principal reactions involved were (1) reduction of a key intermediary C₂₄allo-cholic acid performate with NaBH₄/triethylamine/ethyl chloroformate, (2) iodination of the resulting 3,7,12-triformyloxy-5α-cholan-24-ol with I₂/triphenylphosphine; (3) nucleophilic substitution of the iodo derivative with diethylmethyl malonate/NaH; and (4) hydrolysis of the resulting 3,7,12-triformyloxy-25-methyl-26,27-diethyl ester with KOH, followed by decarboxylation of the geminal dicarboxylic acid with LiCl. N-Acylamidation of the resulting (25R)/(25S)-3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid mixture with glycine or taurine afforded the corresponding epimeric mixtures of the glycine and taurine conjugates. The (25R)- and (25S)-epimers of the three variants of unconjugated and conjugated 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid were efficiently separated by HPLC on a reversed-phase C₁₈ column and their structural characteristics, particularly the chiral center at C-25, delineated using ¹H and ¹³C NMR. These synthetic compounds should be useful as authentic reference standards for establishing their presence in bile as well as being useful in studies on the biosynthesis of allo-bile acids from cholesterol.     

Linoleic acid peroxidation and lignin degradation by enzymes produced by Ceriporiopsis subvermispora grown on wood or in submerged liquid cultures

Ceriporiopsis subvermispora is a white-rot fungus used in biopulping processes and seems to use the fatty acid peroxidation reactions initiated by manganese-peroxidase (MnP) to start lignin degradation. The present work shows that C. subvermispora was able to peroxidize unsaturated fatty acids during wood biotreatment under biopulping conditions. In vitro assays showed that the extent of linoleic acid peroxidation was positively correlated with the level of MnP recovered from the biotreated wood chips. Milled wood was treated in vitro by partially purified MnP and linoleic acid. UV spectroscopy and size exclusion chromatography (SEC) showed that soluble compounds similar to lignin were released from the milled wood. SEC data showed a broad elution profile compatible with low molar mass lignin fractions. MnP-treated milled wood was analyzed by thioacidolysis. The yield of thioacidolysis monomers recovered from guaiacyl and syringyl units decreased by 33% and 20% in MnP-treated milled wood, respectively. This has suggested that lignin depolymerization reactions have occurred during the MnP/linoleic acid treatment.     

Influence of cultivation conditions on production of lignocellulolytic enzymes by Ceriporiopsis subvermispora

The aim of this work was to make a survey describing factors that influence the production of extracellular enzymes by white-rot fungus Ceriporiopsis subvermispora responsible for the degradation of lignocellulolytic materials. These factors were: carbon sources (glucose, cellulose, hemicellulose, lignin, maltose and starch), nitrogen sources (ammonium sulphate, potassium nitrate, urea, albumin and peptone), pH, temperature and addition of three different concentrations of Cu²⁺ and Mn²⁺. The cellulase and xylanase activities were similar in medium with different carbon sources and the highest cellulase and xylanase activities were measured in medium with urea and potassium nitrate as nitrogen sources, respectively. The highest laccase activity was observed in medium with lignin and peptone as carbon and nitrogen sources. In other experiments, time course of production of lignocellulolytic enzymes by white-rot fungus C. subvermispora in medium with lignin or glucose as carbon sources was observed.     

The biosynthesis and conjugation of indole-3-acetic acid in germinating seed and seedlings ofDalbergia dolichopetala

Germinating seed ofDalbergia dolichopetala converted both [²H₅]l-tryptophan and [²H₅]indole-3-ethanol to [²H₅]indole-3-acetic acid (IAA). Metabolism of [2-¹⁴C]IAA resulted in the production of indole-3-acetylaspartic acid (IAAsp), as well as several unidentified components, referred to as metabolites I, II, IV and V. Re-application of [¹⁴C]IAAsp to the germinating seed led to the accumulation of the polar, water-soluble compound, metabolite V, as the major metabolite, together with a small amount of IAA. Metabolites I, II and IV were not detected, nor were these compounds associated with the metabolism of [2-¹⁴C]IAA by shoots and excised cotyledons and roots from 26-d-oldD. dolichopetala seedlings. Both shoots and cotyledons converted IAA to IAAsp and metabolite V, while IAAsp was the only metabolite detected in extracts from excised roots. The available evidence indicates that inDalbergia, and other species, IAAsp may not act as a storage product that can be hydrolysed to provide the plant with a ready supply of IAA.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting - IAA indole-3-acetic acid - IAAsp indole-3-acetylaspartic acid - IAlnos 2-O-indole-3-acetyl-myo-inositol - IEt indole-3-ethanol     

Characterization of white zones produced on Pinus radiata wood chips by Ganoderma australe and Ceriporiopsis subvermispora

White zones produced on biodegraded Pinus radiata wood chips were characterized by micro-localized-FTIR (Fourier Transformed Infra Red) spectroscopy and scanning electron microscopy. Both techniques permitted assignment of the white zones to a selective lignin removal process. Although both fungi studied have degraded lignin selectively in these restricted superficial areas, chemical analysis of the wood chips indicated that Ganoderma australe removed 16% of the initial amount of glucan at the 20% weight loss level. Ceriporiopsis subvermispora did not remove glucan at weight loss values below 17%. Prolonged biodegradation resulted in reduction of white zones by G. australe, and increased white zones from C. subvermispora decayed samples. This revised version was published online in July 2006 with corrections to the Cover Date.