Vol. 46, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1983, p. 191-1970099-2240/83/070191-07$02.00/0

Degradation of Phenolic Compounds and Ring Cleavage ofCatechol by Phanerochaete chrysosporiumGARY F. LEATHAM,' R. L. CRAWFORD,2 AND T. KENT KIRK'*

Forest Products Laboratory, Forest Service, U.S. Department of Agriculture, Madison, Wisconsin 53705,1and Gray Freshwater Biological Institute and Department of Microbiology, University of Minnesota,

Navarre, Minnesota 553922

Received 22 December 1982/Accepted 20 April 1983

POL-88, a mutant of the white-rot fungus Phanerochaete chrysosporium, was

selected for diminished phenol-oxidizing enzyme activity. A wide variety ofphenolic compounds were degraded by ligninolytic cultures of this mutant. Withseveral o-diphenolic substrates, degradation intermediates were produced thathad UV spectra consistent with muconic acids. Extensive spectrophotometric andpolarographic assays failed to detect classical ring-cleaving dioxygenases in cellhomogenates or in extracts from ligninolytic cultures. Even so, a sensitive carrier-trapping assay showed that intact cultures degraded [U-14C]catechol to [14C]mu-conic acid, establishing the presence of a system capable of 1,2-intradiol fission.Significant accumulation of [14C]muconic acid into carrier occurred only whenevolution of 14CO2 from [14C]catechol was inhibited by treating cultures withexcess nutrient nitrogen (e.g., L-glutamic acid) or with cycloheximide. L-Glutamicacid is known from past work to repress the ligninolytic system in P. chrysospor-ium and to mimic the effect of cycloheximide. The results here indicate, therefore,that the enzyme system responsible for degrading ring-cleavage products to CO2turns over faster than does the system responsible for ring cleavage.

White-rot wood-destroying basidiomycetesare among the most important degraders oflignin. An important step during lignin degrada-tion is aromatic ring cleavage, which probablyoccurs in the polymer (8, 11, 18), as well as inlow-molecular-weight phenolics (4). To facilitatestudies of individual chemical reactions involvedin ligninolytic activity, low-molecular-weightphenolic compounds structurally related to lig-nin are often used as substrates. However, phe-nol-oxidizing enzymes commonly produced bywhite-rot fungi hamper detailed studies by cata-lyzing oxidative coupling and polymerization.Phanerochaete chrysosporium, an active lignin-degrading fungus, has been chosen for degrada-tion studies in part because it has low phenol-oxidizing enzyme (PO) activity (17).Nevertheless, with wild-type strains of P. chry-sosporium, even the low titer of PO activityhampers detailed studies.The purpose of this investigation was to char-

acterize the ability of a mutant of P. chrysospor-ium with ligninolytic activity and diminished POactivity to degrade phenolic compounds and tocleave aromatic rings. Ligninolytic activity in P.chrysosporium was induced by nitrogen starva-tion (17).

MATERIALS AND METHODS

Chemicals. 3,4-Dimethylcatechol and 3,5-dimethyl-catechol were prepared by the method of Loudon andScott (24). 2-Chloroisovanillic acid had been preparedearlier (9). cis,cis-Muconic acid was prepared by themethod of Pandell (27), and cis,trans-muconic acidwas prepared by the method of Elvidge et al. (12);these were verified by proton magnetic resonancespectroscopy. The dimethyl esters of muconic acidswere prepared by methylation for 15 min with an ethersolution of diazomethane at room temperature. Excessdiazomethane and diethylether were removed by gen-tle heating. [U-14C]catechol (30 mCi/mmol) was ob-tained from Research Products International Corp.(Elk Grove Village, Ill.). [U-ring-14C]acetovanillonewas prepared from [U-ring-14C]guaiacol and aceticacid (23). Other chemicals were reagent grade fromcommercial sources.Organism and production and selection of mutants.P.

chrysosporium Burds. ME-446 (wild type; ATCC34540) was obtained from the Center for Forest Mycol-ogy Research, Forest Products Laboratory, Madison,Wis. Strain POL-88 and 10 other mutants were pro-duced from ME-446 by UV light-induced mutagenesisof conidia. Conidia were suspended in sterile distilledwater, filtered through glass wool, and diluted to anabsorbance at 650 nm of 0.5 (-2 x 106 conidia perml); 10 ml of the conidial suspension in a 9-cm Petridish was exposed to UV light (Mineralight UVS-11,

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192 LEATHAM, CRAWFORD, AND KIRK

Ultra-Violet Products, Inc., San Gabriel, Calif.) at adistance of 20 cm for 5 min, with stirring once perminute; this gave 99% mortality (1). Surviving conidiawere plated on a medium that restricted colony growth(15). It contained (per liter of distilled water): 40 g ofsorbose, 2.5 g of citric acid, 0.1 g of desoxy-cholicacid, 5 g of KH2PO4, 2 g of NH4Cl, 5 mg of thiamine-hydrochloride, 1 ml of a trace metals solution (29), and15 g of agar (Difco Laboratories, Detroit, Mich.); thefinal pH was adjusted to 4.0 with KOH. Approximate-ly 25 colonies were introduced per plate. After incuba-tion at 37°C for at least 10 days, plates were floodedwith 1 mM o-anisidine in sterile water so that a liquidfilm surrounded the colonies. After an additional 1 to 2days of incubation, phenol-oxidizing colonies becamepigmented due to oxidative polymerization of the o-anisidine. Colorless colonies were selected as poten-tial PO-negative mutants.

Culture conditions. All strains were maintained atroom temperature on glucose-malt extract-peptone-yeast extract agar slants, which promoted rapid, heavyconidiation. This agar contained (per liter of distilledwater): 10 g of glucose, 10 g of malt extract, 2 g ofpeptone, 2 g of yeast extract, 1 g of L-asparagine-hydrochloride, 2 g of KH2PO4, 1 g of MgSO4 * 7H20, 1mg of thiamine-hydrochloride, and 20 g of agar. Thelow-nitrogen liquid medium used was that of Kirk etal. (22), except that the only vitamin was thiamine-hydrochloride (0.1 mg/liter), and polyacrylic acid (0.01M in COOH groups, adjusted to pH 4.2 with KOH)was used as buffer (25). All components were preparedas 10-fold-concentrated solutions and were sterilizedby filtration, except for the buffer, which was auto-claved. Medium (10 ml) plus -106 conidia were addedto 125-ml Erlenmeyer flasks. Cultures were incubatedat 39 ± 1°C without agitation and were flushed everythird day with 02- Cultures became nitrogen starvedand developed ligninolytic activity (i.e., the capabilityof degrading [14C]lignin to '4CO2 after 3 to 4 days [17]).

Biodegradation assays. Radiorespirometric assayprocedures for monitoring the degradation of 14C-labeled compounds to 14CO2 were described previous-ly (22). Assays for the degradation of unlabeled aro-matic compounds were carried out by the addition of 1mg of compound in 10 ,ul of N,N-dimethylformamideto a 7-day-old ligninolytic culture. After 3 days ofincubation at 39°C under 02, 1 ml of culture filtratewas removed and diluted with 5 ml of 95% ethanol.After 4 h, the UV-visible absorption spectrum wasrecorded. The percent degradation for each compoundwas measured by the reduction in absorbance at thewavelength of maximal absorbance (260 to 300 nm)relative to the absorbance of the compound incubatedwith an identical heat-killed culture. Heat killing wasaccomplished by autoclaving cultures at 116°C for 5min. Assay variation was always 10% or less (of thevalues shown) between duplicates and 20% or less onrepetition of experiments.

Assay of catechol ring-cleaving activity. Unless other-wise stated, 1 ,ug (10' dpm) of [U-14C]catechol in 100,ul of distilled water and 1,000 ,ug of unlabeled cis,cis-muconic acid in 10 ,ul ofN,N-dimethylformamide wereadded to a 7-day-old ligninolytic culture (final concen-trations, 0.9 puM and 0.7 mM, respectively). After 6 hat 39°C under 02, reactions were terminated by acidi-fying cultures to pH 1.8 (with 100 pul of concentratedHCl per culture) and adding 10 ml of 1:1 (vol/vol)

chloroform:acetone per flask. Assay flasks were ex-tracted 1 to 4 days after termination. Under theseconditions, the cis,cis-muconic acid isomerized to themore stable cis,trans-isomer (cf. reference 12). Dupli-cate cultures were pooled and extracted three timeswith 30 ml of 1:1 (vol/vol) chloroform:acetone (total,90 ml) and then once with 30 ml of chloroform, andevaporated to dryness. The residues recovered byevaporation were redissolved ih 25 ml of anhydrousmethanol, the solvent again was removed, and theresidues were redissolved in methanol. Efficiencies of14C extraction (-80%) were determined by removingaliquots of the methanol solutions and counting in adioxane-naphthalene scintillation fluid (19). The re-maining methanol solution was concentrated by evap-oration to 0.5 ml, transferred to a small vial, andmethylated with diazomethane. After methylation,samples were evaporated to 0.1 to 0.2 ml. A 5-pIsample of the concentrate was applied as a band onto aprecoated silica gel-60 thin-layer chromatographyplate (2 by 10 cm; 0.2 mm thick; aluminum support)containing a fluorescent indicator (E. Merck, no. 5539;Darmstadt, West Germany). The plate was developedto 9 cm in 16:1 (vol/vol) methylene chloride:ethylace-tate. Rf values for cis,trans-muconic acid dimethyles-ter, catechol, guaiacol, and veratrol (1,2-dimethoxy-benzene) were 0.43, 0.16, 0.50, and 0.53, respectively.Muconic acids did not migrate under these conditions.Guaiacol and veratrol were included as standardsbecause they are formed by methylation of residualcatechol. Formation of muconic acid was quantitatedas the percentage of radioactivity that comigrated onthin-layer chromatography plates with the dimethyles-ter of cis,trans-muconic acid. Its identity was estab-lished by co-chromatographing the labeled productwith cis,trans-muconic acid in several solvent systems(both as the free acid and the dimethylester) and byobserving consistent co-crystallization of the 14C me-tabolite with cis,trans-muconic acid; four successiverecrystallizations did not change the ratio of radioac-tivity to absorbance at 260 nm.

RESULTS AND DISCUSSIONCharacteristics of strain POL-88. Eleven mu-

tant strains of P. chtysosporium were selectedfor their inability to polymerize o-anisidine oxi-datively on agar plates. The frequency of thisphenotype was about 1 in 103 among survivors,as also reported by Ander and Erikssori (1).Spectrophotometriz assays, using catechol,guaiacol, L-dopa, p-phenylenediamine, o-toli-dine, and syringaldezine as substrates, revealedno PO activity in strain POL-88. However, 7-day-old cultures did cause limited dimerizationof 4-t-butylguaiacol during a 24-h incubation at39°C under 02. This highly sensitive but qualita-tive test indicated that some PO activity waspresent (17). Nevertheless, the activity wasweak; phenolic compounds listed in Table 1were not polymerized (colored) by 7-day-oldcultures, even with 2- to 3-day incubations under02. For instance, when 500 ,ug of [U-ring-14C]ace-tovanillone was added to cultures of ME-446(wild-type strain), 30% had polymerized within 5

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PHENOL COMPOUND DEGRADATION BY P. CHRYSOSPORIUM 193

h, soating the cell walls with a dioxane-extract-able radioactive pigment. In contrast, only 20%of that amount of pigment was formed by strainPOL-88 after 48 h of incubation.The degradative abilities of POL-88 were su-

perior to those of many other mutants andsimilar to those of wild-type strain ME-446, asdetermined by evolution of 14CO2 from [U-ring-"4C]acetovanillone, [U-14C]catechol, and [U-ring-14C]synthetic lignin. With this strain, thelower titer of PO activity did not result in lowerligninolytic activity, as seen with the mutantscharacterized by Ander and Ericksson (1) andby Gold et al. (16). It is unclear whether POactivity has an essential role in ligninolytic activ-ity.

Degradation of aromatic compounds by strainPOL-88. Thirty-six aromatic compounds weretested for degradability by ligninolytic cultures(Table 1). P. chrysosporium appears to beamong the most versatile and nonspecific de-graders of aromatic molecules yet examined.Based on disappearance of UV absorbance,using 1 mg of compound per culture, 16 com-pounds were degraded by more than 50% and 28compounds were degraded by at least 20% in 3days. The degradation assay used here wasbased on the difference in soluble phenolic com-pound concentration between live and heat-killed controls. Similar results were obtainedwhen control cultures were killed with 1 mMsodium azide instead of heat. This effectivelyrules out artifacts caused by phenolic com-pounds binding to the mycelium. This degrada-tion assay probably underestimated the extent ofdegradation, because UV-absorbing degradationproducts are not distinguishable from startingmaterials, and especially because some of theprobable intermediates-e.g., muconic acids-have higher extinction coefficients than do thestarting materials. Furthermore, at the high con-centrations tested, toxicity of an aromatic com-pound might cause an underestimation of de-gradability as compared with degradation ratesat lower concentrations-e.g., subsequent studyshowed that 4-methylcatechol is toxic.The most extensively degraded substrates

were the p-hydroxybenzoic acids (e.g., vanillicacid) and compounds that were probably con-verted to them, such as vanillin (4-hydroxy-3-methoxybenzaldehyde). Vanillic acid and relat-ed compounds are oxidatively decarboxylatedby P. chrysosporium (2, 3, 5, 6, 30) and subse-quently may be demethylated to form hydroxy-quinol (1, 2, 4-trihydroxybenzene). The aromat-ic ring of the latter is rapidly cleaved andmetabolized further by P. chrysosporium (4).Ander et al. (2) and Buswell et al. (5), however,presented evidence that ring clevage occurswithout demethylation, the ring-cleavage sub-

TABLE 1. Degradation of aromatic compounds byligninolytic cultures of P. chrysosporium POL-88

Compound %Degradation'

Monophenols, m- and p-diphenolsVanillic acid........................Ferulic acid ........................2,4-Dihydroxybenzoic acid...........4-Hydroxy-3-methoxybenzaldehyde ...Isovanillic acid .....................Syringic acid .......................Curcumin ..........................4-Hydroxy-3-methoxyphenylacetic acid7-Hydroxy-4-methylcoumarin .........2,6-Dihydroxybenzoic acid...........2'-Hydroxy-3'-methoxyacetophenone4'-Hydroxy-3 '-methoxyacetophenone6,7-Dimethoxycoumarin .............7-Hydroxycoumarin.Gentisic acid .......................Guaiacol ...........................4-Hydroxy-3-methoxymandelic acid...2-Chloroisovanillic acid..............

o-Di- and triphenols (catechols)Protocatechuic acid .................3',4'-Dihydroxyacetophenone.Gallic acid .........................2',3'-Dihydroxy-4'-methoxy-acetophenone .....................

6,7-Dihydroxy-4-methylcoumarin .....3,5-Dimethylcatechol ................2',3',4'-Trihydroxyacetophenone .2,3-Dihydroxybenzoic acid...........3,4-Dihydroxyphenylacetic acid.......4-Isopropylcatechol .................7,8-Dihydroxy-4-methylcoumarin .....2,3,4-Trihydroxybenzoic acid.........Pyrogallol ..........................Catechol ...........................3-Methylcatechol .3,4-Dimethylcatechol ................3,5-Disulfonylcatechol ...............4-Methylcatechol ....................

1001009896837974615541393825201911104

947474

67655451413736353029221911118

a Average percent degradation (loss of UV absor-bance) of 1 mg of aromatic compound per cultureduring a 3-day incubation at 39°C under 02 relative toduplicate heat-killed controls. Duplicate cultures var-ied less than ±10% of the values shown.

strate probably being a hydroxylated methoxy-hydroquinone. Because vanillic and related ac-ids are prominent intermediates of lignin degra-dation (7), enzymes involved in vanillic aciddegradation are clearly of importance in lignindegradation.

Strain POL-88 did not significantly degrade 2-chloroisovanillic acid, which is in accord withearlier studies with the white-rot fungus Poly-porus dichrous (21).The UV spectra of many of the phenolic

compounds examined were not altered duringdegradation; such changes are commonly ob-

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194 LEATHAM, CRAWFORD, AND KIRK

served with Fusarium solani (26). Instead, theabsorbance simply diminished. Some of the cat-echols, however, underwent substantial spectralchanges. Transient peaks at 258 to 268 nm wereobserved with catechol, 3,4-dimethylcatechol,3,5-dimethylcatechol, and 2',3'-dihydroxy-4'-methoxy-acetophenone. Appearance of thesepeaks was consistent with formation of muconicacid intermediates. When tested by the UVspectrum disappearance method, cis,cis-mu-conic acid and cis,trans-muconic acid were de-graded by ligninolytic cultures to 54 and 86%,respectively, in 3 days.

Assay of aromatic ring-cleavage activity. Po-larographic and spectrophotometric assay tech-niques (9,10), using catechol, protocatechuicacid, gentisic acid, homogentisic acid, methoxy-hydroquinone and other o- and p-dihydroxyben-zenoid compounds as substrates, failed to revealany ring-cleavage dioxygenases in many differ-ent enzyme preparations from ligninolytic cul-tures. Because these cultures could respire ringcarbons, failure to detect activity indicated ei-ther that it was lost during preparation of crudeextracts or that the assays were not sufficientlysensitive. Therefore, a sensitive isotope-trap-ping method was developed involving intactcultures and [U-14C]catechol. With this assay,ring-cleavage activity was detected.We initially determined the response of cul-

tures to different concentrations of catechol.Experiments in which increasing concentrationswere added to ligninolytic cultures showed com-

plex kinetics for the evolution of 14CO2 from [U-14C]catechol. Two distinct respiration responseswere observed, both of which followed satura-tion kinetics. The first response showed half-saturation (Kn) at 13 ,uM catechol and had aVmax of 9.4 nM/min catechol evolved as CO2 perculture. The second response was half-saturatedat 218 ,uM catechol and had a Vmax of 22.2 nM/min evolved as CO2 per culture. Because of thepresence of the second, low-affinity response,evolution of CO2 was higher than if the firstresponse (higher-affinity system) alone had beensaturated. In subsequent studies, a low concen-

tration of substrate was used (0.9 ,uM) to ensurethat only the high-affinity system was signifi-cantly involved.

Incubation of [U-14C]catechol in ligninolyticcultures in the presence of excess unlabeledcis,cis-muconic acid resulted in the trapping ofonly a small amount of labeled muconic acid-i.e., there was no strong pooling effect (Table 2).Heat-killed cultures and the medium alone gave

similar yields. Most of the [U-14C]catechol de-graded by live cultures was trapped as 14CO2.The remaining 14C was predominantly in startingmaterial (determined by thin-layer chromoto-graphy). Further experiments revealed that

TABLE 2. Ring cleavage and respiration of[U-'4C]catechol by ligninolytic cultures of

P. chrysosporium POL-88 during a 6-h assay inthe presence of cis,cis-muconic acid carrier

Total radioactivityas:

Culturecis,trans-Mu- CO2conic acid (%) (%)

Medium only 2 0Heat-killed control 2 0Culturea 2 16Culture + L-glutamic acidb (12 h 17 7

earlier)Culture + cycloheximide (25 27 1mM, 12 h earlier)

Culturea under N2 instead of 02 0 0Culture + sodium azide (1 mM, 0 0

5 min earlier)a Control culture in the defined medium, under 02-b Three times more nitrogen than originally present.

"4Co2 formation from [U-14C]catechol could notbe suppressed by the addition of common meta-bolically important organic acids (e.g., succinic,malic, oxalic, or glycolic acids) with whichcleavage fragments might pool. However, selec-tive suppression of 14CO2 formation was ob-tained by pretreating the cultures with cyclohex-imide, a potent protein synthesis inhibitor.Although unlabeled cis,cis-muconic acid did notin itself suppress 14Co2 evolution from [U-14C]catechol, significant accumulation of la-beled product occurred only when unlabeledcis,cis-muconic acid was present in cyclohexi-mide-treated cultures. Under these conditions,labeled muconic acid was trapped in good yieldas the major product of degradation. L-Glutamicacid had an effect similar to that of cyclohexi-mide (Table 2).

Failure to trap labeled muconic acid in thepresence of a large excess of unlabeled carrier,in the absence of cycloheximide or glutamicacid, suggests that the ring-cleavage activity wascompartmentalized and that sufficient unlabeledmuconic acid (or the other acids tested) couldnot enter the compartment for a significant pool-ing effect. In the presence of cycloheximide andthe absence of the unlabeled carrier, labeledmuconic acid also did not accumulate. Thisresult shows that the product, when releasedfrom the cleavage site, could pool with thecarrier (if present), and that muconic acid couldbe further altered even by cycloheximide-treat-ed cells. The blocked release of CO2 from cis,cis-muconic acid in the presence of cyclohexi-mide indicates that an enzyme(s) involved incatechol degradation beyond the point of ringcleavage turns over more rapidly than the sys-tem responsible for cleavage. Protein turnover is

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PHENOL COMPOUND DEGRADATION BY P. CHRYSOSPORIUM 195

known to be rapid in nitrogen-starved ligninoly-tic cultures (5 to 7%/h) (14).An alternative interpretation for the cyclohex-

imide effect is that the antibiotic eliminated thecompartmentalization, permitting pooling of la-beled and unlabeled muconic acids. The fact thatglutamic acid had an effect similar to that ofcycloheximide argues against this interpretation,however, because it is unlikely that the glutamicacid would affect compartmentalization. It isprobable that glutamic acid, like cycloheximide,stopped synthesis of certain proteins. This andcertain other amino acids are powerful repres-

sors of the ligninolytic system in nitrogen-starved cultures of P. chrysosporium (13). Byshifting cultures to primary metabolism, theyapparently act as general repressors of second-ary metabolism, including lignin degradation(14, 28). The inhibitory effect of excess glutamicacid on ligninolytic activity is physiologicallyindistinguishable from that of cycloheximide(13).

Characteristics of the ring-cleaving system.Use of the standard 6-h assay with whole lignin-olytic cultures of strain POL-88, incubated withcycloheximide and carrier cis,cis-muconic acid,allowed some of the characteristics of the cate-chol-cleaving system to be examined. The ring-cleavage activity was fully constituted in 1-day-old cultures. This was 1 day before the timecultures evolved CO2 from catechol, and 2 to 3days before the appearance of ligninolytic activi-ty ([U-ring-14C]synthetic lignin -k 14CO2). Cul-

tures that had been heat-killed, incubated under

N2, or treated with the electron transport inhibi-tors sodium azide (1 mM; a strong ligand of CU2+and Fe3+) or carbonyl cyanide-m-chlorophenolhydrazone (CCCP, 0.48 ,uM) did not exhibitring-cleaving activity and did not evolve 14CO2from either [U-14C]catechol or [6-14C]glucose.Ring-cleavage activity was stimulated by 02-

Maximal activity occurred in cultures assayedunder 100% 02; cultures under 20 and 60% 02

gave 27 and 52%, respectively, of the activityunder 100% 02. No activity was detected in cellpastes or in cell-free extracts.The effects of 02, N2, azide, and CCCP are

consistent with the presence of an oxygen- andtransition metal (e.g., Cu2+ or Fe3+)-dependentsystem capable of intradiol cleavage of catecholwith formation of cis,cis-muconic acid. The re-

sults also suggest that intact cultures and elec-tron transport are essential for ring clevage. Arequirement for active energy metabolism is alsosuggested because compounds that inhibitedglucose respiration also inhibited catechol cleav-age to a similar or greater extent (Table 3).White-rot fungi require a growth substrate suchas cellulose or glucose to degrade lignin (20),apparently because lignin degradation is a netconsumer of energy. Early expression of activitydoes not rule out participation of this activity inthe production of CO2 from either catechol or

lignin. The expression of another enzyme appar-ently limits the rate of CO2 production fromcatechol and perhaps lignin.Because P. chrysosporium can degrade a wide

variety of aromatic compounds, it was of inter-

TABLE 3. Effects of phenolic compounds on production of CO2 from glucose and catechol and on ringcleavage of catechol by ligninolytic cultures of P. chrysosporium POL-88

"4C02 production' from: Ring cleavage ofAdditiona [614C]glucosec [UEl4Ccatechold [U-l4CIcatechold

(relative rate) (relative rate) (relative rate)

No addition (control) 100 100 100Catechol 34 2 54-Methylcatechol 26 5 133,5-Dimethylcatechol 69 5 43',4'-Dihydroxyacetophenone 55 13 3Protocatechuic acid 86 34 63Gallic acid 61 22 71Vanillic acid 66 24 11Syringic acid 93 34 43Gentisic acid 85 31 30

a Phenolic compounds were at a final concentration of 1 mg per culture.b Rates are expressed as "4CO2 production relative to the no-addition control during a 6-h incubation under 02

at 390C.c [6-14C]glucose (1 mg per culture; approximately 200,000 dpm). Control cultures respired approximately 1 mg

of glucose during the 6-h assay.d [U-14C]catechol (1 mg per culture; 0.9 mM; approximately 100,000 dpm). Ring cleavage assays were in the

presence of 25 mM cycloheximide added 12 h before, and in the presence of 1 mg (0.7 mM) of cis,cis-muconicacid per culture. Rates are expressed as production of ['4C]muconic acid relative to the control during a 6-hincubation under 02 at 39°C.

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196 LEATHAM, CRAWFORD, AND KIRK

est to determine the specificity of the ring-cleavage system described here. Buswell andEriksson (4) reported a ring-cleavage system inP. chrysosporium capable of cleaving hydroxy-quinol. However, no activity was detected forcleavage of catechol, protocatechuic acid, orgentisic acid. In the present study, substratespecificity could not be assessed directly be-cause the various labeled catechols and corre-sponding muconic acids were unavailable. Anindirect approach was used. Phenolic com-pounds were examined for ability to inhibit (orcompete with) formation of labeled muconicacid or 14Co2 or both from [U-14C]catechol. Allphenols tested inhibited evolution of CO2 fromcatechol (Table 3). The compounds also inhibit-ed respiration of glucose, but the inhibition wasless than one-half that observed for the evolu-tion of CO2 from catechol. Thus, the inhibitionof evolution of CO2 from catechol by phenoliccompounds was selective-i.e., not due simplyto an overall toxic effect. Cleavage of the [U-14C]catechol to muconic acid was inhibited mostby catechol itself, and also by 3,5-dimethylcate-chol, 3',4'-dihydroxyacetophenone, 4-methylca-techol, and vanillic acid. Surprisingly, otherbenzoic acids such as protocatechuic and gallicacids, which are also catechols, were much lesssuppressive. All compounds studied except pro-tocatechuic and gallic acids inhibited both ringcleavage of catechol and evolution of 14CO2from [U-14C]catechol to a similar extent. This isconsistent with ring cleavage being a rate-limit-ing step in degradation. Pooling of carbon frag-ments after a different mechanism of cleavagemight explain the inhibition pattern observed forprotocatechuic and gallic acids.Although not conclusive, the selective inhibi-

tory effect of several dihydroxyaromatic com-pounds on catechol ring cleavage is consistentwith the presence of a relatively nonspecificintradiol ring-cleaving system in ligninolytic cul-tures of P. chrysosporium. It is not yet knownwhether this system is a classical 1,2-catecholdioxygenase, which could not be detected. Fur-ther studies into the localization, specificity, andthe mechanism of action of this ring-cleavagesystem, and its possible dependence on electrontranport, are of interest as they relate to themechanism(s) of aromatic compound degrada-tion and lignin degradation by P. chrysosporium.

ACKNOWLEDGMENTS

This collaborative project was supported by the U.S. ForestProducts Laboratory. G.F.L. was also supported by NationalScience Foundation grant AER 7701388 through the Bacteriol-ogy Department, College of Agriculture and Life Sciences,University of Wisconsin-Madison. The Forest Products Labo-ratory is maintained by the U.S. Department of Agriculture,Forest Service, in cooperation with the University of Wiscon-sin.

LITERATURE CITED

1. Ander, P., and K.-E. Eriksson. 1976. The importance ofphenol oxidase activity in lignin degradation by the white-rot fungus Sporotrichum pulverulentum. Arch. Microbiol.109:1-8.

2. Ander, P., A. Hatakka, and K.-E. Eriksson. 1980. Vanillicacid metabolism by the white-rot fungus Sporotrichumpulverulentum. Arch. Microbiol. 125:189-202.

3. Buswell, J. A., P. Ander, B. Pettersson, and K.-E. Eriks-son. 1979. Oxidative decarboxylation of vanillic acid bySporotrichum pulverulentum. FEBS Lett. 103:98-101.

4. Buswell, J. A., and K.-E. Eriksson. 1979. Aromatic ringcleavage by the white-rot fungus Sporotrichum pulveru-lentum. FEBS Lett. 104:258-260.

5. Buswell, J. A., K.-E. Eriksson, J. K. Gupta, S. G. Hamp,and I. Nordh. 1982. Vanillic acid metabolism by selectedsoft-rot, brown-rot, and white-rot fungi. Arch. Microbiol.131:366-374.

6. Buswell, J. A., K.-E. Eriksson, and B. Pettersson. 1981.Purification and partial characterization of vanillate hy-droxylase (decarboxylating) from Sporotrichum pulveru-lentum. J. Chromatogr. 215:99-108.

7. Chen, C.-L., H.-M. Chang, and T. K. Kirk. 1982. Aromat-ic acids produced during degradation of lignin in sprucewood by Phanerochaete chrysosporium. Holzforschung36:3-9.

8. Chua, M. G. S., C.-L. Chen, H.-M. Chang, and T. K.Kirk. 1982. 13C NMR spectroscopic study of lignin de-graded by Phanerochaete chrysosporium. 1. New struc-tures. Holzforschung 36:165-172.

9. Crawford, R. L., E. McCoy, J. M. Harkin, T. K. Kirk,and J. R. Obst. 1973. Degradation of methoxylated benzo-ic acids by a Nocardia from a lignin-rich environment:significance to lignin degradation and effect of chlorosubstituents. Appl. Microbiol. 26:176-184.

10. Dagley, S. 1977. Microbial degradation of organic com-pounds in the biosphere. Surv. Prog. Chem. 8:121-170.

11. Ellwardt, P.-C., K. Haider, and L. Ernst. 1981. Untersu-chung des microbiellen ligninabbaues durch 13C-NMR-spektroskopie an spezifisch '3C-angereichertem DHP-lignin aus coniferylalkohol. Holzforschung 35:103-109.

12. Elvidge, J. A., R. P. Linstead, P. Sims, and B. Orkin.1950. The third isomeric (cis,trans)-muconic acid. J.Chem. Soc. (London) (1950):2235-2241.

13. Fenn, P., S. Choi, and T. K. Kirk. 1981. Ligninolyticactivity of Phanerochaete chrysosporium: physiology andsuppression by NH4' and L-glutamate. Arch. Microbiol.130:66-71.

14. Fenn, P., and T. K. Kirk. 1981. Relationship of nitrogento the onset and suppression of ligninolytic activity andsecondary metabolism in Phanerochaete chrysosporium.Arch. Microbiol. 130:59-65.

15. Gold, M. H., and T. M. Cheng. 1978. Induction of colo-nial growth and replica plating of the white rot basidiomy-cete Phanerochaete chrysosporium. Appl. Environ. Mi-crobiol. 35:1223-1225.

16. Gold, M. H., M. B. Mayfield, T. M. Cheng, K. Krisnang-kura, M. Shimada, A. Enoki, and J. K. Glenn. 1982. APhanerochaete chrysosporium mutant defective in lignindegradation as well as several other metabolic functions.Arch. Microbiol. 132:115-122.

17. Keyser, P., T. K. Kirk, and J. G. Zeikus. 1978. Ligninoly-tic enzyme system of Phanerochaete chrysosporium: syn-thesized in the absence of lignin in response to nitrogenstarvation. J. Bacteriol. 135:790-797.

18. Kirk, T. K., and H.-m. Chang. 1975. Decomposition oflignin by white-rot fungi. II. Characterization of heavily-degraded lignins from decayed spruce. Holzforschung29:56-64.

19. Kirk, T. K., W. J. Connors, R. D. Bleam, W. F. Hackett,and J. G. Zeikus. 1975. Preparation and microbial decom-position of synthetic ["C] lignins. Proc. NatI. Acad. Sci.U.S.A. 72:2515-2519.

20. Kirk, T. K., W. J. Connors, and J. G. Zeikus. 1976.Requirement for a growth substrate during lignin decom-

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position by two wood-rotting fungi. Appl. Environ. Mi-crobiol. 32:192-194.

21. Kirk, T. K., and L. F. Lorenz. 1973. Methoxyhydroquin-one, an intermediate of vanillate catabolism by Polyporusdichrous. Appl. Microbiol. 26:173-175.

22. Kirk, T. K., E. Schultz, W. J. Connors, L. F. Lorenz, andJ. G. Zeikus. 1978. Influence of culture parameters on

lignin metabolism by Phanerochaete chrysosporium.Arch. Microbiol. 117:277-285.

23. Landucci, L. L., S. A. Geddes, and T. K. Kirk. 1981.Synthesis of '4C-labeled 3-methoxy-4-hydroxy-ct-(2-meth-oxy-phenoxy)-3-hydroxypropiophenone, a lignin modelcompound. Holzforschung 35:66-69.

24. Loudon, J. D., and J. A. Scott. 1953. Ortho-hydroxylationof phenols. II. Derivatives of catechol. J. Chem. Soc.(London) (1953):265-285.

25. Nakatsubo, F., T. K. Kirk, M. Shimada, and T. Higuchi.1981. Metabolism of a phenyl-coumaran substructurelignin model compound in ligninolytic cultures of Phaner-

ochaete chrysosporium. Arch. Microbiol. 128:416-420.26. Ohta, M., T. Higuchi, and S. Iwahara. 1979. Microbial

degradation of dehydrodiconiferyl alcohol, a lignin sub-structure model. Arch. Microbiol. 121:23-28.

27. Pandell, A. J. 1976. Enzyme-like aromatic oxidations.Metal-catalyzed peracetic acid oxidation of phenol andcatechol to cis,cis-muconic acid. J. Org. Chem. 41:3992-3996.

28. Shimada, M., F. Nakatsubo, T. K. Kirk, and T. Higuchi.1981. Biosynthesis of the secondary metabolite veratrylalcohol in relation to lignin degradation in Phanerochaetechrysosporium. Arch. Microbiol. 129:321-324.

29. Wolin, E. A., M. J. Wolin, and R. S. Wolfe. 1963. Forma-tion of methane by bacterial extracts. J. Biol. Chem.238:2886-2888.

30. Yajima, Y., A. Enoki, M. B. Mayfield, and M. H. Gold.1979. Vanillate hydroxylase from the white-rot basidio-mycete Phanerochaete chrysosporium. Arch. Microbiol.123:319-321.

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