Production of New Unsaturated Lipids during Wood Decay by Ligninolytic Basidiomycetes

Lipids were analyzed by gas chromatography-mass spectrometryfor a 7-week in vitro decay of eucalypt wood by four ligninolyticbasidiomycetes. The sound wood contained up to 75 mg of lipophiliccompounds per 100 g of wood. Hydrolysis of sterol esters, whichrepresented 38% of total wood lipids, occurred during the fungaldecay. The initial increase of linoleic and other free unsaturatedfatty acids paralleled the decrease of sterol esters. Moreover,new lipid compounds were found at advanced stages of wood decaythat were identified from their mass spectra as unsaturateddicarboxylic acids consisting of a long aliphatic chain attachedto the C-3 position of itaconic acid. These dicarboxylic acidswere especially abundant in the wood treated with Ceriporiopsissubvermispora (up to 24 mg per 100 g of wood) but also wereproduced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkanderaadusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic,cis-7-hexadecenylitaconic, and hexadecylitaconic acids) aresynthesized by fungi in condensation reactions involving palmitic,oleic, and stearic acids. We suggest that both wood unsaturatedfatty acids (present in free form or released from esters duringnatural decay) and unsaturated metabolites synthesized by fungicould serve as a source for peroxidizable lipids in lignin degradationby white rot basidiomycetes.

INTRODUCTION

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Introduction

Fungi produce a variety of lipids including fatty acids in freeor esterified form, e.g., glycerides, phospholipids, glycolipids,sterol esters, sphingolipids, or simple esters, as well as otherlipids, e.g., free sterols, sterol glycosides, and hydrocarbons(46, 54, 56). Vegetative mycelium (from pure cultures) and fruitbodies of basidiomycetes differ in both lipid content and fattyacid profiles. Linoleic (18:2^9c,12c), oleic (18:1^9c), and palmitic(16:0) acids are the main fatty acids in most cases. Linoleicacid is very abundant in fruit bodies and may account for asmuch as 70 to 80% of the lipids present (1, 42, 48, 49). Othersaturated and unsaturated fatty acids are present in basidiomycetesat lower levels. As reported for other filamentous fungi, unsaturatedfatty acids could help basidiomycetes adapt to low growth temperatures(11, 12). These fatty acids also may have a role in lignin degradation.

Lipid peroxidation reactions could be a part of the lignin degradationprocess by white rot basidiomycetes. When unsaturated fattyacids are oxidized in the presence of manganese peroxidase (MnP),a ligninolytic peroxidase that oxidizes Mn²⁺ to Mn³⁺, lipidhydroperoxides and free radicals are formed. This reaction isconsidered to be sufficient for lignin degradation by some ligninolyticfungi, e.g., Ceriporiopsis (syn. Poria) subvermispora, thatlack lignin peroxidase (LiP) (4, 31-33). LiP, which has a highredox potential that enables direct oxidation of nonphenoliclignin units, was described in the white rot fungus Phanerochaetechrysosporium together with MnP (37). However, LiP has not beendetected in some efficient lignin-degrading fungi (27, 45),and unlike MnP, LiP is rarely found during fungal decay of lignocellulosicsubstrates.

Recent studies of MnP-mediated peroxidation of lipids have shownthat a free carboxyl group is required for linoleic acid peroxidationin a reaction that is initiated by the Mn³⁺ generated by MnP(55). The acyl radical formed reacts with oxygen to generatea lipid peroxyl radical (LOO·) that can react with otherfatty acid molecules, thereby propagating the reaction. Theresulting lipid hydroperoxide (LOOH) acts as an MnP-oxidizingsubstrate in the place of H₂O₂ and can also react with metals.Among the products of metal reactions are lipid alkoxyl radicals(LO·) that are stronger oxidants than the peroxyl radicals.The role of these lipid-derived radicals in lignin degradationis not well understood; however, one hypothesis is that MnP-mediatedlipid peroxidation is a common mechanism used for lignin degradationby many basidiomycetes (32, 33).

Fungal metabolism is one source of unsaturated lipids for peroxidationreactions (7, 34), but peroxidizable plant lipids also couldbe used. In previous studies, we have analyzed eucalypt woodlipids involved in pitch deposit formation during the manufacturingof chlorine-free eucalypt pulp (14, 15, 17, 23). Simultaneously,a large number of fungi were screened for the biotechnologicalcontrol of pitch (43), and four basidiomycetes were selectedbecause of their ability to efficiently remove these compoundsfrom eucalypt wood, resulting in reduced levels in pulp (24,26). When the removal of wood lipophilic compounds by thesefungi was analyzed, several new lipids were detected and wereespecially abundant in the C. subvermispora cultures. Thesecompounds were identified by mass spectrometry (MS) as a novelseries of alkylitaconic acids synthesized by the fungi. Theseand other unsaturated lipids could have a role in wood lignindegradation by C. subvermispora and other white rot fungi vialipid radicals generated by peroxidation reactions.

MATERIALS AND METHODS

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Materials and Methods

Fungi.
We used four strains from the Centraalbureau voor Schimmelcultures(CBS) (Baarn, The Netherlands): Bjerkandera adusta CBS 230.93,Phlebia radiata CBS 184.83, Pleurotus pulmonarius CBS 507.85,and C. subvermispora CBS 347.63. C. subvermispora IJFM A716(FP90031) and IJFM A718 (CZ-3) were received from K.-E. Eriksson.

Wood decay.
We evaluated in vitro decay of Eucalyptus globulus wood (obtainedfrom the ENCE mill in Pontevedra, Spain) under solid-state fermentationconditions. Two grams (dry weight) of small chips (1 to 2 by10 to 20 mm) and 4 ml of water were placed in 100-ml Erlenmeyerflasks, sterilized for 20 min at 120°C, and then inoculatedwith pellets from a fungal culture; the culture was grown inglucose-peptone medium (36) and had been washed and resuspendedin 1.5 ml of sterile water (the dry weight of inoculum was 0.5mg per g of wood). The cotton-plugged flasks were covered withaluminum foil to maintain the humidity and incubated (static)at 28°C for 7 weeks. The time course of lipophilic compoundsin the decaying wood samples was followed by analyzing the contentsof three complete flasks per fungus after 0, 1, 2, 4, and 7weeks of incubation. The basidiomycete biomass in the decayedwood was calculated from the total content of ergosterol. Anaverage ergosterol content of 0.4 mg g of mycelium^-1 was used,as reported for the related white rot fungus P. chrysosporium(13).

Lipid analysis.
The wood samples were dried in a forced-air oven at 60°C,milled using a knife mill (Janke and Kunkel, Staufen, Germany),and extracted with acetone in a Soxhlet extraction apparatusfor 8 h (51). The extracts were evaporated to dryness and resuspendedin chloroform for chromatographic analysis of the lipophilicfraction. The gas chromatography (GC) analyses were performedon a model GC 8000 Top gas chromatograph (Thermo Finnigan, SanJose, Calif.) coupled to a quadruple mass spectrometer detector(model Voyager; ThermoQuest Finnigan) equipped with a fusedsilica capillary column (model DB-5HT; 15 m by 0.25 mm [innerdiameter]; film thickness, 0.1 µm; J&W). The ovenwas heated from 120°C (1 min) to 380°C (5 min) at 10°Cmin^-1. The injector and transfer line temperatures were setat 300 and 350°C, respectively. Helium was used as the carriergas, and the injection was performed in splitless mode. Peakswere quantified by area, and a mixture of standards was usedto elaborate calibration curves (the fatty acid calibrationcurve was used for alkylitaconic acids). Trimethylsilyldiazomethanemethylation and bis(trimethylsilyl)trifluoroacetamide (BSTFA)silylation, alone or subsequently applied, were used to elucidatethe molecular structure of new fungal metabolites. Double-bondposition in the alkyl chain of alkylitaconic acids was determinedby hydroxylation with osmium tetroxide followed by silylationof the diol derivative using BSTFA (16). Compounds were identifiedby comparing their mass spectra with mass spectra in the Wileyand Nist libraries, by mass "fragmentography" and, when possible,by comparison with standards.

RESULTS

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Results

Lipid analysis during eucalypt wood decay by white rot fungi.
The major lipids (Fig. 1A) in the sound eucalypt wood were (i)sitosterol and other minor sterols, e.g., stigmastanol, citrostadienol,fucosterol, cycloartenol, and 24-methylenecycloartanol, eitherin free form or as esters with unsaturated and saturated fattyacids, e.g., linoleic, oleic, palmitic, and palmitoleic acids(16:1^9c), or glucosides; (ii) free fatty acids, the most abundantbeing palmitic, oleic, linoleic, and stearic (18:0) acids (minorpeaks of other free fatty acids are indicated on the chromatogram);(iii) various steroid hydrocarbons, e.g., stigmasta-3,5-diene,and ketones, e.g., stigmast-4-en-3-one and stigmasta-3,5-dien-7-one;and (iv) squalene. The amount of these compounds in the woodchanged dramatically during fungal growth. As decay progressed(the highest wood degradation rate occurred between 2 and 4weeks of incubation), wood lipophilic compounds were degradedand new lipid compounds were identified. These compounds wereespecially common in the wood degraded by C. subvermispora (Fig.1B to E), where three new lipids (peaks I to III) were the majorcompounds at advanced stages of decay.

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FIG. 1. Total-ion GC-MS chromatograms of the lipophilic fraction of acetone extract from eucalypt wood decayed by C. subvermispora. (A) Control wood; (B) 1-week decay (1% weight loss); (C) 2-week decay (2% weight loss); (D) 4-week decay (10% weight loss); (E) 7-week decay (14% weight loss). Sterol glucosides are not shown because they appear only in silylated samples. Linoleic and oleic acids coeluted in the same peak (their respective abundances were obtained from single-ion chromatographic profiles). The free ergosterol and ergosterol ester peaks in the decayed wood are indicated by one and two asterisks, respectively.

The most extensive degradation of wood lipids was achieved withP. radiata and C. subvermispora (Table 1), which also causedthe highest loss of wood weight. The lowest lipid and wood degradationoccurred with P. pulmonarius. Among the different lipid classes,free and esterified sterols were the most extensively degraded,while significant amounts of triglycerides, steroid ketones,and hydrocarbons remained after the fungal decay of wood. Thenew lipids (peaks I to III) were not found in sound eucalyptwood but were found in the samples treated with each of thefour fungi.

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TABLE 1. Composition of lipophilic extracts including three alkylitaconic-type metabolites in sound eucalypt wood and after 7-week decay with four basidiomycetes

Chemical identification of new lipids in the decayed wood.
The chemical structure of the new lipids was proposed from themass spectra of underivatized, methylated, and silylated extractsfrom the wood decayed by C. subvermispora for 4 and 7 weeks.These lipids are unsaturated dicarboxylic acids consisting ofa long aliphatic chain attached to the C-3 position of itaconicacid (methylenebutanedioic acid). The three major peaks, I,II, and III, were identified as 2-methylene-3-carboxyheptadecanoic(tetradecylitaconic) acid, cis-2-methylene-3-carboxy-10-nonadecenoic(cis-7-hexadecenylitaconic) acid, and 2-methylene-3-carboxynonadecanoic(hexadecylitaconic) acid, respectively (Fig. 2).

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FIG. 2. Mass spectra and formulae of the three lipid metabolites present in eucalypt wood decayed by different basidiomycetes as the corresponding methyl esters: I, methyl 2-methylene-3-carboxyheptadecanoate (methyl tetradecylitaconate); II, methyl cis-2-methylene-3-carboxy-10-nonadecenoate (methyl cis-7-hexadecenylitaconate); III, methyl 2-methylene-3-carboxynonadecanoate (methyl hexadecylitaconate).

The formation of the methyl ester derivatives of these lipidsintroduced two methyl groups, indicating that these compoundsare dicarboxylic acids. Subsequent silylation of the methylesters did not introduce any trimethylsilyl group, confirmingthe absence of further free hydroxyl groups after methylation.The mass spectra of the methyl derivatives of compounds I andIII are very similar, with similar fragmentation patterns anda difference of two methylene groups in the molecular ion. Thesemass spectra have as a base peak intense fragments at m/z 157from the itaconate group that was formed by cleavage of theallylic C-C. The fragment at m/z 171 was formed in a similarmanner from the citraconic isomer. A McLafferty rearrangementof the methyl ester produced the fragment at m/z 158 (35). Thefragment at m/z 126 may arise from a loss of methoxyl from thefragment at m/z 157. The molecular ions, at m/z 354 and 382for compounds I and III, respectively, are clearly present inthe mass spectra. Loss of a methoxyl group (M-OCH₃) or a carboxylategroup (M-COOCH₃) gives rise to the fragments at m/z 323 and295 for peak I and m/z 351 and 323 for peak III, which are prominentin the mass spectra. On the other hand, the mass spectrum ofthe methyl derivative of peak II is identical to that reportedpreviously (19) for the methyl derivative of a compound isolatedfrom a culture of C. subvermispora grown on beech wood and identifiedas (Z)-1,7-nonadecadien-2,3-dicarboxylic acid (which also maybe termed cis-4-hexadecenylitaconic acid). The molecular ionat m/z 380 is very weak, but the fragments arising from lossesof methoxy and carboxylate groups at m/z 349 and m/z 321, respectively,are clearly present. The itaconate fragment at m/z 157 alsois clearly visible in the mass spectrum of the methyl derivativeof peak II. The chemical structures deduced from mass spectraof methylated derivatives of the three fungal lipids were confirmedby the mass spectra of the underivatized and silylated compounds.

Hydroxylation of the double bond of hexadecenylitaconic acidfollowed by silylation and GC-MS analysis yielded peaks withan intense m/z 215 fragment corresponding to the cleavage ofa diol located at C-7 of the alkyl chain and indicated thatcompound II is cis-7-hexadecenylitaconic acid.

In addition to these three major compounds (with retention timesof 9.1, 10.5 and 10.8 min after GC-MS of underivatized extracts),other alkylitaconic acids, present as minor peaks or traces,were tentatively identified from their mass spectra as 2-methylene-3-carboxypentadecanoic(dodecylitaconic) acid at 7.2 min, 2-methylene-3-carboxyheptadecenoic(tetradecenylitaconic) acid at 8.8 min, 2-methylene-3-carboxyeneicosenoic(octadecenylitaconic) acid at 12.2 min, and 2-methylene-3-carboxyeneicosanoic(octadecylitaconic) acid at 12.3 min.

Time course of lipids in the wood cultures of C. subvermispora.
Free unsaturated linoleic and oleic acids (Fig. 3A), which aremajor constituents of wood sterol esters, increased during thefirst week of decay, paralleling the decrease in sterol esters(Fig. 3B). Saturated palmitic and stearic acids progressivelydecreased and triglycerides increased as decay progressed (Fig.3A), while the amount of free sterols decreased (Fig. 3B). Lesspronounced decreases occurred in the amount of steroid hydrocarbons,even though the level of steroid ketones increased during thefirst week, and comparatively high levels of these compoundsremained at the end of the 7-week period (Fig. 3B).

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FIG. 3. Lipids and fungal biomass during 7-week decay of eucalypt wood by C. subvermispora. (A) Palmitic ( ${lozenge}$ ), stearic ( ${circ}$ ), oleic ( ${blacktriangleup}$ ), and linoleic ( ${blacksquare}$ ) acids and total triglycerides ( ${square}$ ). (B) Free ( ${blacktriangleup}$ ) and esterified ( ${blacklozenge}$ ) sterols, steroid ketones ( ${circ}$ ), and hydrocarbons ( ${square}$ ). (C) Alkylitaconic acids I ( ${lozenge}$ ), II ( ${blacksquare}$ ), and III ( ${triangleup}$ ). (D) Free ( ${lozenge}$ ) and esterified ( ${square}$ ) ergosterol and fungal biomass ( ${blacktriangleup}$ ). The 95% confidence limits for all values are below 20% of the means (three replicates).

In contrast to most wood lipids, which strongly decreased duringfungal decay, the main alkylitaconic acids (Fig. 3C) increasedafter the second week, attaining total amounts similar to thoseof the major lipid classes in the sound eucalypt wood, i.e.,free and esterified sterols. The three alkylitaconic acids werealso found in C. subvermispora CZ-3 (IJFM A718) and FP90031(IJFM A716) cultures grown under the same conditions (data notshown). Free and esterified ergosterol were not detectable inthe sound eucalypt wood and were relatively minor peaks in thedecayed wood (asterisks in Fig. 1) but were used to estimatebasidiomycete biomass. Free ergosterol was detected only inthe 1 or 2 week samples, while the ester peaked after 3 weeksand remained stable for the remainder of the 7-week period (Fig.3D). Since the free form was a low percentage of the total ergosterolin the decayed wood, the time course of fungal biomass paralleledthat of esterified ergosterol.

DISCUSSION

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Discussion

Biodegradation of wood lipids.
Extractives, i.e., the compounds extractable from wood withorganic solvents, consist of both polar and lipophilic molecules,with the amount and composition of the two fractions varyingby plant species (21, 28). The accumulation of lipophilic extractivesin pulp and paper mills (forming the so-called pitch deposits)results in low-quality pulp and blockages that can halt operationsand lead to economic losses (29). In the present study, as before(23, 50), sterol esters (consisting mainly of sitosterol esterifiedwith unsaturated fatty acids) and free sitosterol were the mainextractable lipids from E. globulus wood. Higher amounts oftriglycerides characterize the lipid fractions from other hardwoods,while softwoods commonly contain triglycerides and resin acids(21).

Wood triglycerides can be hydrolyzed by various microorganisms,including both ascomycete- and basidiomycete-type fungi (43).The released fatty acids can be metabolized or used to synthesizemicrobial lipids. Steroids, however, are more recalcitrant towardsmicrobial degradation. Esterases that hydrolyze sterol estersare much rarer than are microbial lipases (25). In the presentstudy, we found that the four basidiomycetes efficiently degradedcommon eucalypt wood lipids, including free and esterified sitosterol,the highest degradation rates being caused by P. radiata andC. subvermispora. C. subvermispora produced more biomass thandid the other three basidiomycetes. P. radiata efficiently removedextractives despite its low biomass production on the eucalyptwood.

The time course of changes in lipid classes during wood decayshowed an initial increase in unsaturated free fatty acids.This was mainly related to sterol ester hydrolysis, since triglycerideswere minor lipid components in the eucalypt wood. The amountof triglycerides increased during the studied decay period,suggesting fungal esterification of fatty acids. The steroidketones also increased during wood decay, probably because theyare among the first products of oxidative degradation of sitosteroland other eucalypt sterols (18, 47). The constant value forbiomass of C. subvermispora (0.25 to 0.45%) throughout wooddecay suggests continued fungal activity even after a colonizationphase, as reported for other wood-colonizing fungi (22).

Fungal production of alkylitaconic acids.
The new lipid compounds we found in the decayed wood were firstevaluated as possible degradation products of the main woodlipids. However, we concluded that they result from fungal biosyntheticactivity. We identified three main alkylitaconic acids in thewood decayed by all four basidiomycetes studied, but the amountof these compounds was 1 to 2 orders of magnitude higher inthe C. subvermispora wood cultures. These compounds were notdetected in sound eucalypt wood.

Hexadecenylitaconic acid (peak II) has also been isolated fromC. subvermispora grown on beech wood and identified using MS,and ¹H, ¹³C, and bidimensional nuclear magnetic resonance (NMR)(19). The amount of hexadecenylitaconic acid we obtained fromeucalypt wood (approximately 7 mg 100 g of wood^-1) was similarto that reported on beech wood preextracted with benzene-ethanol(2:1) and supplemented with glucose and ammonium (19). An itaconicacid-type microbial metabolite with a long alkyl chain, 15-hydroxyhexadecylitaconicacid (a hydroxylated form of compound III), also has been isolatedfrom the lichen Usnea aliphatica (35).

The mass spectrum of methylated peak II was nearly identicalto that previously reported as corresponding to (Z)-1,7-nonadecadiene-2,3-dicarboxylicacid (cis-4-hexadecenylitaconic acid) (19). However, the resultsobtained here suggest that the location of the double bond inthe unsaturated alkyl chain of this hexadecenylitaconic acidfrom C. subvermispora could be erroneously assigned by Enokiet al. (19). The m/z 215 fragment in the mass spectra of productsobtained after double-bond hydroxylation and silylation suggestsa double bond at C-7. An m/z 257 fragment should be obtainedfrom hydroxylation and silylation of cis-4-hexadecenylitaconicacid, which was not observed. As discussed by del Ríoet al. (16), this conclusion is also supported by the ¹³C NMRspectrum of this compound (19) that shows a shift differencebetween the two olefinic carbons of the alkyl chain (0.3 ppm)that is smaller than expected if the double bond was locatedat C-4, closer to the carboxylates. Similar differences havebeen reported between olefinic carbon signals in the ¹³C NMRspectra of oleic (18:1^9c) and petroselinic (18:1^6c) acids (38).

The two other alkylitaconic acids (peaks I and III) have notbeen previously reported. We deduced their chemical structures,and those of the four minor compounds (dodecylitaconic, tetradecenylitaconic,octadecenylitaconic, and octadecylitaconic acids), from themass spectra of underivatized, methylated, and silylated derivatives(16). The three dominant alkylitaconic acids also were foundin cultures of C. subvermispora CZ-3 and FP90031. The diunsaturatedone (peak II) had been described previously from FP90031 (19).

Proposed biosynthetic pathways.
This series of fungal alkylitaconic acids could be synthesizedby carbonyl-methylene condensation as reported for related long-chainaliphatic di- or tricarboxylic acids produced by lichens andfungi (52, 53). The seven alkylitaconic acids can be derivedformally by condensation of the carbonyl group of oxaloacetatewith the ${alpha}$ -methylene groups of myristic (14:0), palmitic, palmitoleic,stearic, oleic, arachidic (20:0), and eicosenoic (20:1) acids.Various 14- to 20-C fatty acids were found in the sound eucalyptwood but also can be synthesized by white rot basidiomycetes,although the 20-C fatty acids are rare (1, 54). When free fattyacids were analyzed in cultures of C. subvermispora grown onbeech wood, palmitic, linoleic, stearic, and two octadecenoicisomers were reported (20).

The proposed reaction is similar to oxaloacetate and acetyl-S-coenzymeA (-CoA) condensation for citric acid formation in the tricarboxylic-acidcycle. Therefore, fatty acids probably will be activated totheir CoA-SH thioesters (a requisite for most fatty acid reactions)before reacting with oxaloacetate. Citrate synthases have beencharacterized from a variety of organisms, but synthases catalyzingthe formation of alkylcitric acids are relatively rare, althoughtwo of them, producing decylcitric and decylhomocitric acids,have been purified from Penicillium lehmanii (syn. Penicilliumspiculisporum) (39, 40). Formation of alkylitaconic acids isprobably regulated by the substrate specificity of the alkylcitratesynthases. Although nothing is known about these enzymes inbasidiomycetes, the P. lehmanii decylcitrate synthase exhibitspreference for CoA thioesters of 12 C or shorter saturated fattyacids (39, 41).

The product of condensation of oxaloacetate with the ${alpha}$ -methylenegroup of the fatty acids is exemplified by agaricic (hexadecylcitric)acid, a long-chain aliphatic tricarboxylic metabolite firstdescribed in 1907 from Laricifomes officinalis (syn. Polyporusofficinalis), norcaperatic (tetradecylcitric) acid, or the twoP. lehmanii metabolites (10). Removal of the hydroxyl groupfrom alkylcitric acids would result in alkyl-cis-aconitic acidintermediates. Their decarboxylation, leaving a methylene groupas proposed for itaconic acid synthesis (8), would yield theseries of alkylitaconic acids described from the wood culturesof C. subvermispora.

For example (Fig. 4), tetradecylitaconic acid (peak I) couldbe synthesized from palmitic acid via three steps: (i) condensationof oxaloacetic acid and palmitoyl-S-CoA; (ii) hydroxyl removaland double-bond formation; and (iii) decarboxylation yieldingtetradecylitaconic acid. Similar biosynthetic pathways couldbe postulated for the other lipid metabolites described. Thisbiosynthetic scheme is also compatible with the structure thatwe propose for compound II, cis-7-hexadecenylitaconic acid insteadof cis-4-hexadecenylitaconic acid as previously proposed (19).The cis-7-hexadecenylitaconic acid would be formed from oleicacid, which is present in high amounts in the lipophilic extractsof both eucalypt wood and C. subvermispora biomass, whereasthe cis-4-hexadecenylitaconic acid probably would be formedfrom petroselinic acid, which is not common in nature and hasnot been reported in either eucalypt wood or solid state culturesof C. subvermispora. In lichens and some other fungi a relatedbiosynthetic pathway leads to a series of lactone-type metabolites(52, 53).

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FIG. 4. Proposed biosynthetic scheme for tetradecylitaconic acid including: condensation of the oxaloacetic acid (step 2) carbonyl group with the ${alpha}$ -methylene group of palmitoyl-S-CoA (step 1), hydroxyl removal from the resulting tetradecylcitric acid (step 3) and double bond formation, and decarboxylation of resulting tetradecyl-cis-aconitic acid (step 4), forming tetradecylitaconic acid (step 5).

In conclusion, we found that esters of the unsaturated sterolsitosterol with linoleic and oleic acids are the main lipidsin eucalypt wood and that their biodegradation results in atransient increase of free unsaturated fatty acids that areconsumed in biosynthetic or degradative reactions. The roleof sterol radicals in wood decay remains to be determined. Inparallel to lipid degradation, white rot fungi synthesize anovel series of unsaturated alkyldicarboxylic acids derivedfrom fatty acids. These acids are the major lipids at the advancedstages of wood decay by C. subvermispora. The presence of lipidhydroperoxides and thiobarbituric acid reactive substances,an indicator of lipid peroxidation, is characteristic of thesestages (20). The involvement of alkylitaconic acids in peroxidationreactions, together with unsaturated fatty acids, is suggestedby both the presence of one or two double bonds and their dicarboxylicnature, but this involvement remains to be confirmed (9, 30).It has been proposed that lipid radicals from MnP-mediated lipidperoxidation are involved in lignin (4, 31-33) and xenobioticdegradation (5-7, 44) by C. subvermispora and other fungi. Moreover,the ability of white rot fungi to degrade lignin has been relatedto higher production of unsaturated lipids compared with thebrown rot species (34). However, more studies are required toidentify the lipid peroxidation mechanisms operating in nature,and to establish a relationship between lipid metabolism andthe ability of C. subvermispora to selectively remove ligninfrom lignocellulosic materials (2, 3, 20).

ACKNOWLEDGMENTS

We thank K.-E. Eriksson (STFI, Stockholm, Sweden) for C. subvermisporastrains CZ-3 and FP90031, J. Romero (ENCE, Pontevedra, Spain)for providing E. globulus wood chips, and F. Guillén(CIB, CSIC, Madrid) for useful discussions on the role of freeradicals in lignin degradation.

This study was supported by the European Commission (contractQLK5-99-1357) and the Spanish CICYT (FEDER project 1FD97-0742).A.G. is thankful for a "Ramòn y Cajal" contract of theSpanish MCYT.

FOOTNOTES

* Corresponding author. Mailing address: Centro de Investigaciones Biológicas, CSIC, Velázquez 144, E-28006 Madrid, Spain. Phone: 34 915611800. Fax: 34 915627518. E-mail: ATMartinez{at}cib.csic.es.

Present address: Instituto Madrileno de Investigaciones Agrariasy Alimentarias, Alcalá de Henares, E-28800 Madrid, Spain.

REFERENCES

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