New Insights into the Ligninolytic Capability of a Wood Decay Ascomycete

Wood-grown cultures of Daldinia concentrica oxidized a permethylatedß-¹⁴C-labeled synthetic lignin to ¹⁴CO₂ and also cleaveda permethylated ${alpha}$ -¹³C-labeled synthetic lignin to give C-C_ßcleavage products that were detected by ¹³C nuclear magneticresonance spectrometry. Therefore, this ascomycete resembleswhite-rot basidiomycetes in attacking the recalcitrant nonphenolicstructures that predominate in lignin.

INTRODUCTION

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INTRODUCTION

The degradation of lignocellulose by ascomycetes, a processgenerally called soft rot, is an important route for carboncycling in plant litter and soils (2). Little is known aboutthe decay mechanisms of these fungi, in contrast to those ofthe better-studied lignocellulolytic basidiomycetes that degradewood. Nevertheless, it is clear that some soft-rot fungi candegrade lignin, because they erode the secondary cell wall anddecrease the content of acid-insoluble material (Klason lignin)in angiosperm wood (13, 21). Some ascomycetes have also beenshown to mineralize radiolabeled synthetic lignins (known asdehydrogenative polymerizates [DHPs]), but the low extents of¹⁴CO₂ production reported—invariably less than 10% ofthe lignin—indicate that the ligninolytic capabilitiesof ascomycetes are more limited than those of white-rot basidiomycetes(11, 17).

One possibility is that ascomycetes attack only the phenolicunits in lignin, which comprise roughly 10% of the polymer andare chemically more labile than the ether-linked, nonphenolicunits that make up the remainder (Fig. 1). Phenolic lignin structuresare oxidized and directly cleaved by a variety of mild biologicaloxidants such as manganic chelates and phenol oxidases (7, 20).By contrast, the cleavage of nonphenolic lignin structures requiresthe action of stronger oxidants, including oxyradicals producedby the action of peroxidases or laccases on various redox mediators.The production of these more vigorous ligninolytic systems istypical of white-rot basidiomycetes (5, 12) and has not yetbeen convincingly shown in soft-rot ascomycetes. However, theproduction of phenol oxidases that could act directly on phenoliclignin is widespread among ascomycetes (11, 14, 19).

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FIG. 1. Principal structures of natural lignins and DHPs. (A) Arylglycerol-ß-aryl ether structures. (B) Phenylcoumaran structures. (C) Resinol structures. R₁ is H for guaiacyl structures and OCH₃ for syringyl structures. R₂ is H for phenolic structures, CH₃ for permethylated phenolic structures, and lignin for nonphenolic structures.

Mineralization of DHPs.

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Mineralization of DHPs.

To address this question, we compared the extents to which Daldiniaconcentrica mineralized phenolic and nonphenolic DHPs. D. concentricais a xylariaceous ascomycete that produces phenol oxidase activity(14) and degrades angiosperm wood extensively (13). Becausechemical analyses of wood degraded by this fungus have shownthat it degrades syringyl lignin units faster than guaiacylones (Fig. 1) (13), we performed these experiments with a syringyl/guaiacylDHP (i.e., a polymeric model of angiosperm lignin) that was¹⁴C labeled at C_ß on the side chains of its syringylunits. The radiolabeled DHP, prepared as described earlier (4,9), had a specific activity of 0.01 mCi/mmol of syringyl subunitsand a syringyl/guaiacyl ratio of approximately 4:1. A portionof this phenolic syringyl/guaiacyl DHP was then permethylatedwith diazomethane as described earlier (8) to obtain a nonphenolicsyringyl/guaiacyl DHP, i.e., a polymer in which all of the phenolichydroxyl groups had been blocked as methyl ethers. In addition,a phenolic guaiacyl DHP (i.e., a polymeric model of gymnospermlignin), prepared previously with ¹⁴C at C_ß of itsside chains (0.01 mCi/mmol of guaiacyl subunits) (4), was includedin the experiment for comparison.

Each DHP was fractionated by gel permeation chromatography (GPC)on Sephadex LH-20 in N,N-dimethylformamide, and the excludedfractions were taken to eliminate low-molecular-mass material( $≤$ $~$ 1 kDa) that might have been susceptible to uptake and intracellularmetabolism. This step is essential in microbiological experimentswith DHPs because ligninolysis is strictly an extracellularprocess (9). The excluded fractions were then subjected againto GPC on a 1.8- by 33-cm column of Sephacryl S-100 in N,N-dimethylformamide(15), and fractions eluting within the sieving range of thecolumn were pooled so that the phenolic and nonphenolic DHPshad similar molecular mass distributions. We included this extrapurification step in case the size of a DHP might influenceits bioavailability. Figure 2 shows the molecular mass distributionsof the three purified DHPs as determined by analytical GPC onSephacryl S-100 in N,N-dimethylformamide-0.1 M LiCl (15).

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FIG. 2. Gel permeation chromatography of the radiolabeled DHPs used in mineralization experiments. The distributions shown, with their weight average molecular masses (M_w) and number average molecular masses (M_n), are as follows: phenolic syringyl/guaiacyl DHP (solid line), M_w = 33 kDa, M_n = 16 kDa; nonphenolic syringyl/guaiacyl DHP (long-dash line), M_w = 25 kDa M_n = 14 kDa; phenolic guaiacyl DHP (short-dash line), M_w = 21 kDa, M_n = 5 kDa. Elution positions and masses of standards are shown for 35-kDa, 15-kDa, and 0.5-kDa polystyrenes; a 1.1-kDa lignin model tetramer (15); and 3,4-dimethoxybenzaldehyde (0.17 kDa). V_o indicates the excluded volume of the column.

The sized DHPs were dissolved in acetone-water (4:1), and 80,000dpm of each was added to the end grain of replicate sterileaspen wafers (approximately 300 mg each, 3 by 10 by 30 mm, withthe large face perpendicular to the grain). Additional sterilewater was added to each block to bring the final volume of addedwater to 300 µl, and the acetone was allowed to evaporate.The wafers were then placed on spacers over potato dextroseagar in 125-ml Erlenmeyer flasks that already contained a lawnof D. concentrica (FP-140074-sp; Center for Forest Mycology,USDA Forest Products Laboratory). This medium was chosen becauselignocellulose decay by ascomycetes is generally stimulatedwhen auxiliary sources of carbon and nitrogen are present (21).An agar plug of the fungus was placed atop each wafer, and theflasks were stoppered with gassing manifolds. The cultures wereincubated at 26°C and flushed with humidified, sterile airat 2- or 3-day intervals. The headspace gas from each flaskwas sparged through an alkaline scintillation cocktail to trap¹⁴CO₂ evolved from the DHPs, and the amount of radiocarbon ineach sample was then determined by scintillation counting (10).

We grew these cultures on wood wafers because weight loss inthe substrate provides an independent test of whether the fungushas expressed its entire lignocellulolytic system. The extensiveloss that occurred over the 12-week experiment—55% onaverage—leaves no doubt that this was the case. The mineralizationdata (Table 1) show that the phenolic syringyl/guaiacyl DHPwas degraded more extensively than the phenolic guaiacyl DHP,in agreement with previous analyses of the lignin from wooddegraded by D. concentrica (13). Nevertheless, the results establishthat this fungus can degrade guaiacyl lignin structures extracellularly,because the proportion of ¹⁴C with a molecular mass less than1 kDa in our guaiacyl DHP was only about 2% (Fig. 2). Most interestingly,the data also show that blockage of the phenolic groups in thesyringyl/guaiacyl DHP had no significant effect on its biodegradation.Therefore, contrary to our initial hypothesis, attack on nonphenoliclignin structures is a significant route for ligninolysis byD. concentrica.

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TABLE 1. Mineralization of ß-¹⁴C-labeled synthetic lignins by D. concentrica on wood

Identification of cleavage products in a nonphenolic DHP.

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Identification of cleavage...

To address the question of how D. concentrica cleaves nonphenoliclignin structures, we supplemented aspen wafers with a nonphenolicDHP enriched with ¹³C at C of its propyl side chain, extractedthis DHP from the wood after decay, and then identified chemicalchanges at C by ¹³C nuclear magnetic resonance (NMR) spectrometry.The [ ${alpha}$ -¹³C]DHP was a guaiacyl polymer prepared earlier (4) witha ¹³C content greater than 99% at C. It was first permethylatedto block its phenolic structures and then fractionated on SephadexLH-20 as described above to provide a high-molecular-mass fraction.The ¹³C NMR spectrum of this fraction was obtained, after whichtwo sterile aspen wafers (dry weights: 334 mg and 350 mg) wereeach infused with 9 mg of it in acetone-water and then inoculatedwith D. concentrica as described above. At the same time, fivereplicate cultures were set up on aspen wafers (average dryweight: 385 ± 22 mg) without [ ${alpha}$ -¹³C]DHP so that the contributionof endogenous lignin to the NMR spectra could be assessed. Thecultures were covered with aluminum foil and incubated at 26°Cand 70% relative humidity for 7 weeks.

At the conclusion of the experiment, the two supplemented wafershad a combined dry weight of 464 mg and showed weight lossesof 31% and 33%, compared with a mean value of 46% ± 3%for the five control wafers without [ ${alpha}$ -¹³C]DHP. Apparently, thepresence of DHP inhibited wood decay somewhat, but it is evidentthat a complete biodegradative system was nevertheless expressedin supplemented wafers. Two wafers, weighing a total of 461mg, were chosen from the decayed set lacking [ ${alpha}$ -¹³C]DHP, andeach pair was then ball milled. The milled wood samples weretreated with a crude cellulase mixture (Cellulysin; Calbiochem,San Diego, CA) for 48 h thrice as described elsewhere (16),after which the lignin-enriched fraction was extracted for 24h thrice with dioxane-water (9:1) to solubilize as much ligninas possible. The two wafers originally supplemented with [ ${alpha}$ -¹³C]DHPyielded 97 mg of solubles, whereas the two unsupplemented wafersyielded 82 mg.

Qualitative ¹³C NMR analyses of these samples were obtained,and the normalized spectra (Fig. 3) were inspected for signalsindicative of ligninolysis. No significant changes were discerniblein the alcohol and ether range (70 to 90 ppm), correspondingto C in major structures (Fig. 1) of the undegraded DHP, butnew structures were apparent in the carbonyl (190 to 205 ppmand 165 to 175 ppm) regions. Figure 3a shows the spectrum inthese regions for the [ ${alpha}$ -¹³C]DHP that was extracted from degradedwood, and Fig. 3b shows the spectrum in the same regions forthe extract from degraded wood that had received no [ ${alpha}$ -¹³C]DHP.Figure 3c shows the difference between these two spectra andis thus a spectrum of degraded [ ${alpha}$ -¹³C]DHP that has been correctedfor the small contribution made by material originating fromthe wood. To see the changes that D. concentrica caused in thenonphenolic [ ${alpha}$ -¹³C]DHP, the difference spectrum in Fig. 3c shouldbe compared with Fig. 3d, which shows the spectrum of the original,undegraded, permethylated polymer.

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FIG. 3. ¹³C NMR results showing (a) a spectrum of the extract from wood decayed by D. concentrica in the presence of permethylated guaiacyl [ ${alpha}$ -¹³C]DHP, (b) a spectrum of the extract from decayed wood that received no DHP, (c) the difference between spectra a and b, (d) a spectrum of undegraded, permethylated, guaiacyl [ ${alpha}$ -¹³C]DHP, and (e) a DEPT spectrum of the extract from wood decayed in the presence of permethylated guaiacyl [ ${alpha}$ -¹³C]DHP. Spectra a and b were normalized with respect to the amount of wood lignin present by equalizing the signal from C-2/C-6 in syringyl aromatic carbons (guaiacyl DHP does not contribute in this region). Spectra a and d were normalized with respect to the amount of DHP present by equalizing the signal from C in resinol structures (this carbon was present at a very low abundance in the wood lignin). The samples were dissolved in 400 µl of acetone-d₆-D₂O (4:1), and the spectra were obtained with a Bruker DPX 250-MHz spectrometer (62.9 MHz for ¹³C). The qualitative experiments shown in spectra a to d were acquired with a standard power-gated sequence, a 0.5-s delay, and 150,000 scans. Quantitative experiments (not shown) were performed with an inverse-gated sequence, a 15-s delay, a 90° pulse, and 25,000 scans. For spectrum e, the standard Bruker DEPT135 sequence was run for 30,000 scans. In all cases, the acetone signal at 29.8 ppm was used as the internal reference. Structures corresponding to signal assignments 1 to 3 are shown below the spectra. The assignments were confirmed by measuring carbonyl chemical shifts ( ${delta}$ ) for 3,4-dimethoxybenzaldehyde, 3,4-dimethoxybenzoic acid, and 3,4-dimethoxybenzoate methyl ester. The new signals at 172 to 174 ppm in spectrum c may represent aliphatic carboxyl groups resulting from C-aryl cleavage of the DHP (1).

In [ ${alpha}$ -¹³C]DHP recovered from degraded wood, a signal in the regioncharacteristic of benzaldehyde carbonyls was apparent (Fig.3a and 3c, signal 1) and was confirmed to be from a protonated(i.e., aldehyde as opposed to ketone) carbonyl in a distortionlessenhancement with polarization transfer (DEPT) experiment (18)(Fig. 3e). This benzaldehyde signal was absent from the ligninextracted from degraded wood that had received no [ ${alpha}$ -¹³C]DHP(Fig. 3b) and therefore must have originated from the DHP. Benzaldehyderesidues were also present in the original, undegraded [ ${alpha}$ -¹³C]DHP(Fig. 3d), as also found in earlier research on another DHP(3), but they occurred at a much higher level in the degradedsample.

Similarly, the [ ${alpha}$ -¹³C]DHP from degraded wood exhibited benzoicacid carboxyl signals (Fig. 3a and c, signal 2) that were detectableneither in the lignin from wood without [ ${alpha}$ -¹³C]DHP (Fig. 3b)nor in the original undegraded [ ${alpha}$ -¹³C]DHP (Fig. 3d). The undegraded[ ${alpha}$ -¹³C]DHP did originally contain benzoic acid residues, in agreementwith earlier work (3), but they all appear in our spectra asmethyl benzoates (Fig. 3d, signal 3) because the [ ${alpha}$ -¹³C]DHP hadbeen permethylated before the experiment. Since these preexistingmethyl benzoates were unaffected by fungal decay (Fig. 3a and c)they cannot have been the source of the new benzoic acid residues.

There are two possible sources for new benzaldehydes and benzoicacids in a biodegraded lignin. First, if the polymer originallycontained primary benzyl alcohols, these might simply becomeoxidized at C. Such reactions would not be ligninolytic, butwe can rule them out as an explanation for our data becausea two-dimensional ¹H-¹³C heteronuclear spin quantum correlation(HSQC) NMR spectrum of the original permethylated [ ${alpha}$ -¹³C]DHPshowed that it lacked the ¹H-¹³C cross-peak at a ${delta}$ of 4.6, 65ppm, that is expected for primary benzyl alcohols (data notshown; for typical NMR chemical shifts of lignin-related structuressee the U.S. Dairy Forage Research Center NMR Database of Ligninand Cell Wall Model Compounds at http://ars.usda.gov/Services/docs.htm?docid=10491).This result leaves the second explanation, which is that thenew benzaldehydes and benzoic acids arose via biodegradativeC-C_ß cleavage of propyl side chains in the [ ${alpha}$ -¹³C]DHP.A quantitative ¹³C NMR spectrum (data not shown) showed thatnew benzoic acid residues were roughly 10-fold more abundantthan new benzaldehyde residues in the degraded [ ${alpha}$ -¹³C]DHP andthat approximately 1% of the residual polymer had been cleavedbetween C and C_ß.

Conclusion.

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Conclusion.

Our results show that D. concentrica degrades the recalcitrantnonphenolic structures in lignin and that C-C_ß cleavageof the lignin propyl side chain is one of the processes responsible.The ligninolytic agents that D. concentrica produces remainunidentified because no method has yet been found to elicittheir production on any medium except wood. However, it is reasonableto predict that they resemble some of the extracellular one-electronoxidants produced by white-rot basidiomycetes, because the latterfungi and their isolated ligninolytic systems also cleave nonphenoliclignin structures between C and C_ß (1, 4). The relativelylow ability of ascomycetes to depolymerize guaiacyl lignin structureshas led researchers to suggest that they produce weaker ligninolyticoxidants (13), but this inference is contradicted by the observationthat guaiacyl lignin structures are actually slightly easierto oxidize than syringyl structures (6). We consider it morelikely that the ascomycetes produce a relatively small quantityof ligninolytic oxidants and consequently fail to overcome thetendency that guaiacyl lignin fragments have to repolymerizeduring one-electron oxidation (4).

ACKNOWLEDGMENTS

We thank Carl Houtman for calculating the DHP molecular massdistributions, Hoon Kim for milling the wood samples, and JohnRalph for advice on sample preparation for NMR analysis.

This study was supported by U.S. Department of Energy grantsDE-FG02-94ER20140 and DE-AI02-07ER64480 to K.E.H.

FOOTNOTES

* Corresponding author. Mailing address: Institute for Microbial and Biochemical Sciences, USDA Forest Products Laboratory, One Gifford Pinchot Dr., Madison, WI 53726. Phone: (608) 231-9528. Fax: (608) 231-9262. E-mail: kehammel{at}wisc.edu

Published ahead of print on 31 August 2007.

REFERENCES

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