Alkylitaconic Acids Research Articles

Caprylic acid enhances hydroxyhexylitaconic acid production in Aspergillus niger S17-5.

Aspergillus niger S17-5 produces two alkylitaconic acids, 9-hydroxyhexylitaconic acid (9-HHIA) and 10-hydroxyhexylitaconic acid (10-HHIA), which have cytotoxic and polymer building block properties. In this study, we characterized the production of 9-HHIA and 10-HHIA by addition of their expected precursor, caprylic acid, to a culture of A. niger S17-5, and demonstrated batch fermentation of 9-HHIA and 10-HHIA in a jar fermenter with DO-stat. Production titres of 9-HHIA and 10-HHIA from 3% glucose in a flask after 25days cultivation were 0·35 and 1·01gl-1 respectively. Addition of 0·22gl-1 of caprylic acid to a suspension of resting cells of A. niger S17-5 led to 32% enhancement of total 9-HHIA and 10-HHIA production compared to no addition. No enhancement of the production of 9-HHIA or 10-HHIA by the addition of oxaloacetic acid was observed. Addition of caprylic acid to the culture at mid-growth phase was more suitable for 9-HHIA and 10-HHIA production due to less cell growth inhibition by caprylic acid. DO-stat batch fermentation with 3% glucose and 14·4gl-1 of caprylic acid in a 1·5l jar fermenter resulted in the production titres of 9-HHIA and 10-HHIA being 0·48 and 1·54gl-1 respectively after 10days of cultivation. Addition of caprylic acid to the culture of A. niger S17-5 enhances 9-HHIA and 10-HHIA production. These results suggest that 9-HHIA and 10-HHIA are synthesized with octanoyl-CoA derived from caprylic acid, and that the supply of octanoyl-CoA is a rate-limiting step in 9-HHIA and 10-HHIA production. To the best of our knowledge, this is the first report regarding the fermentation of naturally occurring itaconic acid derivatives in a jar fermenter.

Itaconic acid derivatives: structure, function, biosynthesis, and perspectives

Itaconic acid possessing a vinylidene group, which is mainly produced by fungi, is used as a biobased platform chemical and shows distinctive bioactivities. On the other hand, some fungi and lichens produce itaconic acid derivatives possessing itaconic acid skeleton, and the number of the derivatives is currently more than seventy. Based on the molecular structures, they can be categorized into two groups, alkylitaconic acids and α-methylene-γ-butyrolactones. Interestingly, some itaconic acid derivatives show versatile functions such as antimicrobial, anti-inflammatory, antitumor, and plant growth-regulating activities. The vinylidene group of itaconic acid derivatives likely participates in these functions. It is suggested that α-methylene-γ-butyrolactones are biosynthesized from alkylitaconic acids which are first biosynthesized from acyl-CoA and oxaloacetic acid. Some modifying enzymes such as hydroxylase and dehydratase are likely involved in the further modification after biosynthesis of their precursors. This contributes to the diversity of itaconic acid derivatives. In this review, we summarize their structures, functions, and biosynthetic pathways together with a discussion of a strategy for the industrial use. KEY POINTS: • Itaconic acid derivatives can be categorized into alkylitaconic acids and α-methylene-γ-butyrolactones. • The vinylidene group of itaconic acid derivatives likely participates in their versatile function. • It is suggested that α-methylene-γ-butyrolactones are biosynthesized from alkylitaconic acids which are first synthesized from acyl-CoA and oxaloacetic acid.

New Alkylitaconic Acid Derivatives from Nodulisporium sp. A21 and Their Auxin Herbicidal Activities on Weed Seeds.

Five alkylitaconic acid (AA) derivatives, including two novel compounds, epideoxysporothric acid (2) and sporochartine F (5), and three known compounds, deoxysporothric acid (1), deoxyisosporothric acid (3), and 1-undecen-2,3-dicarboxylic acid (4), were obtained from the fermentation culture of the endophytic fungus Nodulisporium sp. A21. The auxin herbicidal activities of compounds 1-4 against weed seeds were investigated under laboratory conditions. In general, the tested compounds displayed radicle growth promoting activity at low doses and inhibitory activity at higher doses. Compounds 1 and 2 could significantly inhibit the radicle growth of dicotyledon weeds, Eclipta prostrata and Veronica persica, at a concentration range from 50 to 200 μg mL-1, while 3 notably stimulated radicle growth at the same concentration range. The results suggested that these AA derivatives have the potential to be used as the lead scaffold for novel auxin herbicide development. In addition, the biosynthetic pathways of 1-4 were deduced based on 13C labeling experiment.

Chemical changes and increased degradability of wheat straw and oak wood chips treated with the white rot fungi Ceriporiopsis subvermispora and Lentinula edodes

Wheat straw and oak wood chips were incubated with Ceriporiopsis subvermispora and Lentinula edodes for 8 weeks. Samples from the fungal treated substrates were collected every week for chemical characterization. L. edodes continuously grew during the 8 weeks on both wheat straw and oak wood chips, as determined by the ergosterol mass fraction of the dry biomass. C. subvermispora colonized both substrates during the first week, stopped growing on oak wood chips, and resumed growth after 6 weeks on wheat straw. Detergent fiber analysis and pyrolysis coupled to gas chromatography/mass spectrometry showed a selective lignin degradation in wheat straw, although some carbohydrates were also degraded. L. edodes continuously degraded lignin and hemicelluloses in wheat straw while C. subvermispora degraded lignin and hemicelluloses only during the first 5 weeks of treatment after which cellulose degradation started. Both fungi selectively degraded lignin in wood chips. After 4 weeks of treatment, no significant changes in chemical composition were detected. In contrast to L. edodes, C. subvermispora produced alkylitaconic acids during fungal treatment, which paralleled the degradation and modification of lignin indicating the importance of these compounds in delignification. Light microscopy visualized a dense structure of wood chips which was difficult to penetrate by the fungi, explaining the relative lower lignin degradation compared to wheat straw measured by chemical analysis. All these changes resulted in an increased in in vitro rumen degradability of wheat straw and oak wood chips. In addition, more glucose and xylose were released after enzymatic saccharification of fungal treated wheat straw compared to untreated material.

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, 1H and homonuclear J-resolved 2D NMR and feeding experiments with [ 13C-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.

Epoxy ceriporic acid produced by selective lignin-degrading fungus Ceriporiopsis subvermispora

Ceriporiopsis subvermispora is a selective white rot basidiomycete which degrades lignin in wood at a distance far from enzymes. Low molecular mass metabolites play a central role in the oxidative degradation of lignin. To understand the unique wood-decaying mechanism, we surveyed the oxidized derivatives of ceriporic acids (alk(en)ylitaconic acids) produced by C. subvermispora using high-resolution liquid chromatography multiple-stage mass spectrometry (HR-LC/MSn). The analysis of the precursor and product ions from the extract suggested that an epoxidized derivative of ceriporic acid is produced by the fungus. To identify the new metabolite, an authentic compound of ceriporic acid epoxide was synthesized in vitro by reacting (R)-3-[(Z)-hexadec-7-enyl]-itaconic acid (ceriporic acid C) with m-chloroperbenzoic acid. The precursor and product ions from the natural metabolite and authentic epoxide were identical and distinguishable from those of hydroxy and hydroperoxy derivatives after reduction with NaBD4. Feeding experiments with [U-13C]-glucose, 99% and the subsequent analyses of the first and second generation product ions demonstrated that the oxidized ceriporic acid was (R)-3-(7,8-epoxy-hexadecyl)-itaconic acid. To our knowledge, this study is the first to report that natural alkylitaconic acid bears an epoxy group on its side chain.

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 Fe 3+. 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.

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 Δ9-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 13C-[U]-glucose demonstrated that ceriporic acid C and trans-7-hexadecenylitaconic acid (ceriporic acid D) were biosynthesised de novo by C. subvermispora.

Relevance of extractives and wood transformation products on the biodegradation of Pinus taeda by Ceriporiopsis subvermispora

Ceriporiopsis subvermispora is a promising white-rot fungus for biopulping. However, the underlying biochemistry involved in lignin removal and insignificant cellulose degradation by this species is not completely understood. This paper addresses this topic focusing on the involvement of ethanol-soluble extractives and wood transformation products in the biodegradation process. Cultures containing ethanol-extracted or in natura wood chips presented similar levels of extracellular enzymes and degradation of wood components. Fe 3+-reducing compounds present in undecayed Pinus taeda were rapidly diminished by fungal degradation. Lignin-degradation products released during biodegradation restored part of the Fe 3+-reducing activity. However, Fe 3+ reduction was ineffective in presence of 0.5 mM oxalate at pH 4.5. Fungal consumption of Fe 3+-reducing compounds and secretion of oxalic acid minimized the significance of Fenton's reaction in the initial stages of wood biotreatment. This would explain limited polysaccharide degradation by the fungus that also lacks a complete set of hydrolytic enzymes. Scientific relevance of the paper Ceriporiopsis subvermispora is a white-rot fungus suitable for biopulping processes because it degrades lignin selectively and causes significant structural changes on the wood components during the earlier decay stages. However, the intricate mechanism to explain lignin transformation and insignificant cellulose degradation by this species remains poorly understood. Some recent evidences pointed out for lipid peroxidation reactions as an initiating process explaining lignin degradation. On the other hand, alkylitaconic acids produced by the fungus via transformations of fatty acids occurring in wood showed to prevent polysaccharide degradation in Fenton reactions. In this context, one may conclude that the involvement of native wood substances or their transformation products in the overall wood biodegradation process induced by C. subvermispora is still a matter of discussion. While free and esterified fatty acids present in wood extractives may be involved in the biosynthesis of alkylitaconic acids and in lipid peroxidation reactions, some extractives and lignin degradation products can reduce Fe 3+, providing Fe 2+ species needed to form OH radical via Fenton's reaction. The present study focuses on this topic by evaluating the relevance of ethanol-soluble extractives and wood transformation products on the biodegradation of P. taeda by C. subvermispora. For this, solid-state cultures containing ethanol-extracted and in natura wood chips were evaluated in details for up to 4 weeks.

Redox silencing of the Fenton reaction system by an alkylitaconic acid, ceriporic acid B produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora

The selective lignin-degrading fungus, Ceriporiopsis subvermispora secretes alkylitaconic acids (ceriporic acids) during wood decay. We reported that ceriporic acid B (hexadecylitaconic acid) was protective against the depolymerization of cellulose by the Fenton reaction. To understand the redox silencing effects, we analyzed the physicochemical and redox properties of itaconic, octylitaconic and hexadecylitaconic acids. The initial rate of HO production by the Fenton system with Fe(3+), H(2)O(2) and L-cysteine was suppressed by hexadecylitaconic and octylitaconic acids by 0.04 and 0.16 of the reaction rate without chelators. ESR, O(2) uptake and the assay of Fe(2+) with BPS demonstrated that Fe(3+) reduction by L-cysteine was suppressed by hexadecylitaconic and octylitaconic acids while the reaction of Fe(2+) with H(2)O(2) was not suppressed by the two alkylitaconic acids. Ligand exchange experiments with NTA demonstrated that Fe(3+) chelation by two carboxyl groups of alkylitaconic acids is a critical step in iron redox modulation. In stark contrast, the production of HO* and reduction of Fe(3+) were not suppressed by itaconic acid due to HO*--initiated degradation of the chelator. The strong redox silencing effects by a series of alkylitaconic acids have attracted interest in controlling microbial plant cell wall degradation and chemoprotection against cellular oxidative injury.

Tensyuic Acids, New Antibiotics Produced by Aspergillus niger FKI-2342

Six new alkylitaconic acids, designated tensyuic acids A to F, were isolated from the culture broth of Aspergillus niger FKI-2342 by solvent extraction, silica gel column chromatography and HPLC. Their structures were elucidated by spectroscopic analysis including UV, NMR, and MS. They are all alkylitaconic acid derivatives. Only tesyuic acid C showed moderate antimicrobial activity against Bacillus subtilis.

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-d 6. 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.

A selective lignin-degrading fungus, Ceriporiopsis subvermispora, produces alkylitaconates that inhibit the production of a cellulolytic active oxygen species, hydroxyl radical in the presence of iron and H 2O 2

A cellulolytic active oxygen species, hydroxyl radicals ( OH ), play a leading role in the erosion of wood cell walls by brown-rot and non-selective white-rot fungi. In contrast, selective white-rot fungi have been considered to possess unknown systems for the suppression of OH production due to their wood decay pattern with a minimum loss of cellulose. In the present paper, we first report that 1-nonadecene-2,3-dicarboxylic acid, an alkylitaconic acid (ceriporic acid B) produced by the selective white-rot fungus Ceriporiopsis subvermispora intensively inhibited OH production by the Fenton reaction by direct interaction with Fe ions, while non-substituted itaconic acid promoted the Fenton reaction. Suppression of the Fenton reaction by the alkylitaconic acid was observed even in the presence of the Fe 3+ reductants, cysteine and hydroquinone. The inhibition of OH production by the diffusible fungal metabolite accounts for the extracellular system of the fungus that attenuates the formation of OH in the presence of iron, molecular oxygen, and free radicals produced during lignin biodegradation.

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 Fe 2+ to Fe 3+ as well as reduction of Fe 3+ to Fe 2+ 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.

Production of new unsaturated lipids during wood decay by ligninolytic basidiomycetes.

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.

Identification of a novel series of alkylitaconic acids in wood cultures of Ceriporiopsis subvermispora by gas chromatography/mass spectrometry.

A novel series of long-chain unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid has been identified by gas chromatography/mass spectrometry during in vitro decay of eucalypt wood by the white-rot basidiomycete Ceriporiopsis subvermispora. The major compounds were identified as tetradecyl-, 7-hexadecenyl- and hexadecylitaconic acids by their mass fragmentation patterns. Other members of the same compound series, identified as dodecanyl-, tridecanyl-, tetradecenyl-, pentadecanyl-, octadecenyl- and octadecanylitaconic acids, were present in very minor amounts or traces. Whereas hexadecenylitaconic acid has already been reported in cultures of C. subvermispora, to our knowledge this is the first report of the presence of the other alkylitaconic acids in fungal cultures. These new alkylitaconic-type metabolites may constitute a source for peroxidizable lipids involved in lignin degradation during wood decay by C. subvermispora and other white-rot basidiomycetes.