Phanerochaete chrysosporium and Other Lignin-Degrading Fungi

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 199, p /9/ $2./ Copyright 199, American Society for Microbiology Vol. 56, No. 11 Chloromethane, Methyl Donor in Veratryl Alcohol Biosynthesis in Phanerochaete chrysosporium and Other Lignin-Degrading Fungi DAVID B. HARPER,l12* JOHN A. BUSWELL,3t JAMES T. KENNEDY,2 AND JOHN T. G. HAMILTON2 Department of Food and Agricultural Chemistry, The Queen's University of Belfast,l* and Food and Agricultural Chemistry Research Division, Department of Agriculture for Northern Ireland,2 Newforge Lane, Belfast B79 5PX, Northern Ireland, and Department of Biology, Paisley College of Technology, Paisley PA] 2BE, Scotland,3 United Kingdom Received 13 June 199/Accepted 27 August 199 Chloromethane, a gaseous natural product implicated in methylation processes in PheUinus pomaceus, has been shown to act as methyl donor in veratryl alcohol biosynthesis in the lignin-degrading fungi Phanerochaete chrysosporium, Phlebia radiata, and Coriolus versicolor, none of which released detectable amounts of CH3Cl during growth. When P. chrysosporium was grown in a medium containing C2H3Cl, levels of C2H3 incorporation into the 3- and 4--methyl groups of veratryl alcohol were very high and initially similar to those observed when the medium was supplemented with L-[methyl-2H3]methionine. When C2H3Cl was added to cultures actively synthesizing veratryl alcohol, incorporation of C2H3 was very rapid, with 81% of veratryl alcohol labeled after 12 h. By contrast, incorporation of C2H3 from L-[methyl-2H3]methionine was comparatively slow, attaining 1% after 12 h. It is proposed that these lignin-degrading fungi possess a tightly channeled multienzyme system in which CH3C1 biosynthesis is closely coupled to CH3Cl utilization for methylation of veratryl alcohol precursors. Many species of the family Hymenochaetaceae, a widely distributed group of wood-rotting bracket fungi, release large quantities of gaseous CH3Cl during secondary metabolism (5, 14, 17, 18). Although CH3C1 has been shown to be derived from methionine (15, 35), no clear physiological role had been defined for it until Harper et al. (16) demonstrated recently that it can act as the methyl donor for biosynthesis of methyl benzoate and methyl furoate during primary metabolism of Phellinus pomaceus. The broad-specificity methylating system can esterify a wide range of aromatic and aliphatic acids. Initially CH3Cl biosynthesis is closely coupled to utilization, but the system becomes less tightly channeled in late trophophase, at which stage the release of gaseous CH3Cl by the fungus begins. Hence, although CH3Cl ostensibly appears to be a characteristic secondary metabolite, its liberation in the idiophase would seem merely to reflect the breakdown of the strict coordination of biosynthesis and utilization of the compound which exists in the primary metabolic phase. A CH3Cl-utilizing system capable of methylating many phenols and thiophenol was also found in Phellinus pomaceus (16), although the natural substrate for the system was not identified. The observation that C2H3Cl was incorporated into methyl benzoate by a non-ch3cl-releasing fungus, Fomitopsis pinicola (a member of the family Polyporaceae), led Harper et al. (16) to speculate that CH3Cl biosynthesis and utilization as the methyl donor might not be confined to members of the Hymenochaetaceae but could exist more generally in nature. Several white rot fungi (2, 22, 26) are known to produce the secondary metabolite veratryl alcohol (3,4-dimethoxybenzyl alcohol), which is believed to be an important component of the lignin-degrading system. Veratryl alcohol * Corresponding author. t Present address: Department of Biology, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. 345 induces elements of the ligninolytic system (8, 9, 25), prevents inactivation of lignin peroxidase (13, 32, 33), and is reported to enhance the oxidation of lignin by lignin peroxidase in vitro (12). It may also function as a natural oneelectron redox mediator in the depolymerization process, possibly promoting oxidation by creation of activated oxygen species (19, 28). The key roles assigned to veratryl alcohol in lignin biodegradation render the biosynthetic route of the compound and its regulation of critical importance. Biosynthesis of veratryl alcohol in Phanerochaete chrysosporium proceeds via phenylalanine, 3,4-dimethoxycinnamyl alcohol, and veratryl glycerol; trapping experiments indicate that the -methyl groups are derived from methionine (29). 4--Methylation of lignin degradation products such as vanillic acid by ligninolytic cultures of this fungus with methionine as the methyl donor has also been reported (4). In the present study the labeling of veratryl alcohol produced during culture of P. chrysosporium and two other lignin-degrading fungi was investigated in the presence of C2H3Cl and, in some instances, L-[methyl-2H3]methionine to determine whether CH3Cl served as an intermediate in 3- and 4--methylation during the synthesis of veratryl alcohol. MATERIALS AND METHODS Organisms and maintenance media. P. chrysosporium Burds INA-12 (CNCM 1-398) was a strain used in investigations by Buswell et al. (3). Coriolus versicolor Quel. Ps 4a was acquired from S. Kawai, Wood Research Institute, Kyoto University, Uji, Kyoto, Japan. Phlebia radiata Fr. 79 (ATCC 64658) was also used in some experiments. Fungi were maintained on 4% (wt/vol) malt extract agar (Oxoid Ltd., London, United Kingdom). Chemicals. C2H3Cl (99.9 atom% 2H), C2H31 (99.5 atom% 2H), and L-[methyl-2H3]methionine (99.4 atom% 2H) were purchased from MSD Isotopes, Montreal, Quebec, Canada. C2H32H (99.5 atom% 2H), (C2H3)2SO4 (99 atom% 2H), veratryl alcohol, 3,4,5-trimethoxybenzyl alcohol, and other Downloaded from on September 18, 218 by guest

2 VOL. 56, 199 CHLOROMETHANE IS A METHYL DONOR IN P. CHRYSOSPORIUM 3451 substituted benzyl alcohols and benzaldehydes were obtained from Aldrich Chemical Co., Gillingham, Dorset, United Kingdom. Caffeic, ferulic, isoferulic, and 3,4-dimethoxycinnamic acids were also acquired from Aldrich. N- Methyl-N-trimethylsilyltrifluoroacetamide reagent (MSTFA) was obtained from Pierce Chemical Co., Rockford, Ill. [4-methoxy-2H3]veratryl alcohol (bp 166 C at 11 mm Hg [1,467 Pa]) and [3-methoxy-2H3]veratryl alcohol (bp 164 to 165 C at 14 mm Hg [1,867 Pa] were prepared by methylation of vanillin and isovanillin in aqueous NaOH with (C2H3)2SO4 by standard procedures followed by reduction of the resulting aldehydes in methanol with hydrogen in the presence of Raney nickel at room temperature and pressure. [3,4- dimethoxy-2h6]veratryl alcohol (bp 165 C at 11 mm Hg [1,467 Pa]) was prepared by methylation of 3,4-dihydroxybenzaldehyde in dimethyl sulfoxide with (C2H3)2SO4 (11) and reduction of the aldehyde with hydrogen in the presence of Raney nickel. Culture media and culture conditions. P. chrysosporium was grown without agitation at 37 C in 1-ml conical flasks in 1 ml of medium which contained the following (in grams per liter): KH2PO4,.2; MgSO4.7H2,.5; NaCl,.59; CaCl2.2H2,.13; thiamine,.25; yeast extract (Difco),.1; glycerol, 1; L-asparagine monohydrate, 1.; NH4NO3,.5; and 2,2-dimethylsuccinic acid, It also contained 1 ml of trace elements solution per liter (1). The medium was adjusted to ph 5. with 2 M KOH, filter sterilized, and inoculated with approximately 5 x 15 conidia per liter prepared as described by Eriksson and Johnsrud (7). Flasks were flushed with 1% 2 for 2 min before being stoppered with a polytetrafluoroethylene (PTFE)-coated rubber stopper. In some experiments the growth medium was supplemented with 1 mm L-[methyl-2H3]methionine (i.e., 1,umol flask-1). When the growth medium required supplementation with C2H3Cl, the following technique was used. From the PTFE-coated rubber stopper was suspended an aluminum clamp in which a Durham tube was fitted. Immediately before sealing the flasks, 3 mm aqueous C2H3Cl (1.67 ml) was pipetted into the tube to give a concentration of.77 mm C2H3Cl in the culture medium after equilibration of gaseous and aqueous phases (i.e., 5 ilmol of C2H3Cl flask-') (17). When the incorporation of precursors added to cultures after 72 h of growth was investigated, a different procedure was adopted. The PTFE-coated rubber stopper of each flask was fitted with two stoppered glass injection ports, one of which consisted of a capillary tube extending to within 2 mm of the base of the flask. After 72 h of growth the medium was supplemented with either 1 mm L-[methyl-2H3]methionine or.77 mm C2H3Cl by injection through the capillary injection port of 1 mm methionine (1.1 ml) or 3 mm C2H3Cl (1.67 ml), respectively. This technique was also used for supplementation of the culture medium with additional methionine (.5 mm) after 96 h of incubation. Phlebia radiata was grown under similar conditions to P. chrysosporium, except that a temperature of 29 C was used and the medium contained the following (in grams per liter): KH2PO4,.2; MgSO4 7H2O,.5; CaCl2 2H2,.1; NH4NO3,.39; L-asparagine,.69; glucose, 1; and 2,2- dimethylsuccinic acid, It also contained 1 ml of trace elements solution per liter (21) and 1 ml of vitamin solution per liter (23). The medium was adjusted to ph 4.5 with 2 M KOH, filter sterilized, and inoculated with a mycelial suspension (1 ml) prepared by manually shaking.25 g of mycelium with 1 ml of sterile water and 5 g of glass beads (diameter, 6 mm) in a 4-ml vial. Flasks were flushed with 1% 2 for 2 min before being stoppered with a PTFEcoated rubber stopper suspended from which, as described above, was a Durham tube containing a volume of aqueous C2H3CI such as to give a concentration of.77 mm C2H3Cl in the medium. After 7 days, cultures were harvested, veratryl alcohol in the supernatant was assayed, and the percent C2H3 incorporation into the compound was determined. C. versicolor was grown at 29 C under 2 as outlined for P. chrysosporium, except that the culture medium contained the following (in grams per liter): KH2PO4,.2; MgSO4 7H2,.5; CaCI2,.1; NH4NO3,.52; L-asparagine,.86; glucose, 1; and polyacrylic acid (.1 M in carboxyl),.72. It also contained 1 ml of mineral solution per liter (23) and.5 ml of vitamin solution per liter (23). The medium was adjusted to ph 4.5, filter sterilized, and inoculated with a mycelial suspension (1 ml) prepared as described for Phlebia radiata. Flasks were flushed with 1% 2 and stoppered with a PTFE-coated rubber stopper equipped with a C2H3Clcontaining Durham tube giving a concentration of.77 mm C2H3C1 in the medium. After 7 days, cultures were harvested, veratryl alcohol in the supernatant was assayed, and the percent C2H3 incorporation into the compound was determined. Lignin peroxidase assay. The activity of lignin peroxidase in culture supernatants was determined by measuring the increase in A31 due to the oxidation of veratryl alcohol to veratraldehyde in the presence of H22 (31). In the assay, supernatant (1 ml) was incubated with 4 mm veratryl alcohol and.27 mm H22 in 6 mm sodium tartrate buffer ph 3. at 37 C. Activities are expressed as nanomoles of veratraldehyde formed per minute per milliliter of culture supernatant. Assay of veratryl alcohol and determination of percent C21H3 incorporation. Culture supernatant (1 ml), after addition of 3,4,5-trimethoxbenzyl alcohol (.2 ml of a 5-,ug/ml solution in acetone) as an internal standard, was extracted three times with chloroform (15 ml per extraction), and the bulked extract was washed with saturated NaCl solution (1 ml). The extract was dried over anhydrous MgSO4 and evaporated to.5 ml under reduced pressure. Finally, the solution was made up to 5 ml with diethyl ether. Veratryl alcohol in the extract was determined by gas chromatography-mass spectrometry (GC-MS) on a Hewlett- Packard 589 gas chromatograph linked to an HP597 massselective detector controlled by an HP3 series computer. The gas chromatograph was equipped with an Ultra 1 fused-silica wall-coated open tubular capillary column (25 m by.2 mm) with 1% dimethylpolysiloxane as the bonded phase. Helium was used as the carrier gas at a flow rate of 1.5 ml min-1, and a split ratio of 2:1 was used. After injection the oven temperature was held at 1 C for 1 min and then programmed at 1 C min-1 up to 16 C followed by 3 C min-1 up to 3 C. To quantify veratryl alcohol, ion currents at mle 168, 171, and 174 were monitored and their sum at the retention time of veratryl alcohol was compared with that given by an authentic sample of the compound. The incorporation of C2H3 into the 3- and 4--methyl groups of veratryl alcohol was measured as the relative proportions of the three ions after correction for a small proportion of (M - 3) ion (approximate intensity, 5%) present in spectra of both labeled and unlabeled compounds. As it was not possible on the basis of the MS fragmentation pattern to distinguish between veratryl alcohol monosubstituted with C2H3 in the 3-position from that monosubstituted in the 4-position, veratryl alcohol with a single C2H3 substitutent is referred to simply as monolabeled. Thus, the compound giving an ion of Downloaded from on September 18, 218 by guest

3 3452 HARPER ET AL. mle 168 (substitution OCH3, OCH3) is referred to henceforth as unlabeled compound, that giving an ion of mle 171 (substitution OCH3, OC2H3) is referred to as monolabeled, and that giving an ion of mle 174 (substitution OC2H3, OC2H3) is referred to as dilabeled. In experiments on utilization of C2H3Cl and L-[methyl- 2H3]methionine during growth of P. chrysosporium, values given are the mean of two replicates. In experiments involving the addition of labeled precursor to actively growing cultures of P. chrysosporium, the percent incorporations are expressed as the mean + standard deviation of three to five replicates. Results given for Phlebia radiata are the mean of five replicates. Veratryl alcohol in C. versicolor was determined in the bulked supernatants of five replicate cultures. Determination of nonenzymatic methylation. The extent of possible nonenzymatic methylation of phenolic precursors of veratryl alcohol was investigated by the following procedure. Phenolic substrate (.5 mm) was incubated in sterile culture medium under conditions identical to those of fungal culture at 37 C in the presence of either 1 mm C2H3Cl or.25 mm C2H31. After a 7-day incubation a solution of the possible methylated product in unlabeled form was added to each flask to give a final concentration of 5 pum. The culture medium was then acidified to ph 3. and extracted with chloroform, and the extract was reduced to a small volume as described above. For aldehydes and alcohols the solution was made up to 1 ml in ether and examined by GC-MS under the conditions described above for the veratryl alcohol assay. Ion currents at mle values corresponding to the molecular ions (M) of possible methylated products, the M + 3 ions, and in some cases the M + 6 ions were monitored. The ratios of the last two ions to the molecular ion at the retention time of the methylated product were a measure of the nonbiological incorporation of C2H3 from C2H3Cl into the monomethylated and dimethylated products, respectively. For the substituted cinnamic acids the solution was taken to dryness and the residue was silylated with 1 ml of MSTFA. The solution was examined by using GC-MS as above, except that the gas chromatograph was equipped with an Ultra 2 fused-silica wall-coated open tubular column (12 m by.2 mm) with 95% dimethylpolysiloxane-5% diphenyl as the bonded phase. Helium was used as the carrier gas at a flow rate of 1 ml min-', and a split ratio of 2:1 was employed. After injection the oven temperature was held at 1 C for 1 min and then programmed at 2 C min-' up to 3 C and held at this temperature for 5 min. Ion currents at mle values corresponding to the molecular ion (M) and the M + 3 and M + 6 ions were monitored on a Hewlett-Packard 597 mass-selective detector, and the ratios of the last two ions relative to the molecular ion were again used as a measure of nonenzymatic methylation. The following conversions were investigated by this technique: caffeic acid to ferulic, isoferulic, and 3,4-dimethoxycinnamic acids; ferulic acid and isoferulic acid to 3,4- dimethoxycinnamic acid; 3,4-dihydroxybenzaldehyde to vanillin, isovanillin, and veratraldehyde; vanillin and isovanillin to veratraldehyde; and 3-hydroxy-4-methoxybenzyl alcohol and 3-methoxy-4-hydroxybenzyl alcohol to veratryl alcohol. NMR. 'H nuclear magnetic resonance (NMR) spectra of C2H3-labeled veratryl alcohols were recorded in C2HC13 at 2 C with tetramethylsilane as the internal standard on a GE Omega 5-MHz NMR spectrometer, with 16,384 datum points and a sweep width of 5 Hz. Assessment of growth. Mycelial growth was measured as APPL. ENVIRON. MICROBIOL. the weight of mycelia harvested by filtration at various stages of incubation and dried at 1 C. CH3C1 assay. CH3Cl was determined by GC, using the headspace technique described by Harper and Kennedy (17). A water/air partition coefficient (wt/vol per wt/vol) of 2.21 for CH3C1 at 37 C was used in calculations. For confirmation of the presence of CH3Cl in the headspace of fungal cultures at concentrations down to.2 ng ml-', the GC-MS technique described by Harper et al. (18) was used. Methionine analysis. Culture medium was filtered through a.22-,um membrane filter (Millipore Corp.), and filtrate (8 ml) was evaporated to dryness on a Hetovac VRI vacuum concentrator fitted with a CT 6 cooling trap. The residue was dissolved in 5 ml of lithium citrate buffer (ph 2.2) containing 2 mm norleucine as internal standard. The sample (15 [li) was then chromatographed on an LKB model 44 amino acid analyzer by using a lithium citrate buffer elution program (2). The concentration of methionine was calculated from the peak areas by using an internal standard procedure, a calibration standard being chromatographed after every five samples. RESULTS AND DISCUSSION C2H3 incorporation into veratryl alcohol from L-[methyl- 2H3]methionine in growth medium. Figure 1A shows the incorporation of C2H3 into veratryl alcohol at various stages during growth of P. chrysosporium on a medium containing 1 mm L-[methyl-2H3]methionine; Fig. 2 records the veratryl alcohol concentration in the culture medium and mycelial dry weight during this period. In the early stages of growth the -methyl groups in the 3 and 4 positions of veratryl alcohol were almost wholly labeled with C2H3, 83% of the compound present being dilabeled and 17% monolabeled. This finding confirms the conclusion of Shimada et al. (29) that the -methyl groups of veratryl alcohol originate from methionine. Between 96 and 12 h the level of labeling fell sharply, presumably reflecting exhaustion of labeled methionine in the culture medium and the utilization of endogenous unlabeled methionine for methylation. The rapidity of this decline implies a high rate of veratryl alcohol turnover in the culture medium as previously observed (24). This is consistent with the key metabolic role proposed for the compound. That the decrease in labeling is attributable to the depletion of labeled methionine in the culture medium is clearly demonstrated by an experiment in which the culture medium of the fungus grown as in Fig. IA was supplemented with additional L-[methyl-2H3]methionine (.5 mm) after 96 h. Under these conditions the level of labeling in veratryl alcohol quickly recovered to its original high value, which was maintained until the experiment was terminated at 29 h (Fig. 1B). C2H3 incorporation into veratryl alcohol from C2H3Cl in growth medium. Figure 3A illustrates the labeling pattern found in veratryl alcohol present at various stages during growth of the fungus in medium containing.77 mm C2H3Cl, whereas Fig. 3B shows the veratryl alcohol concentration in the culture medium and mycelial dry weight during the experiment. High levels of incorporation of C2H3 into veratryl alcohol were observed throughout the whole incubation period. CH3Cl is clearly as effective a precursor as methionine for 3- and 4--methylation. Maintenance of high levels of labeling throughout incubation can be attributed to the equilibrium existing between C2H3Cl in the flask headspace (initially 41,umol) and that in the aqueous phase (initially 9,umol), which ensures that C2H3C1 in the medium utilized in Downloaded from on September 18, 218 by guest

4 VOL. 56, 199 CHLOROMETHANE IS A METHYL DONOR IN P. CHRYSOSPORIUM B 4,., 6 - w.. I Incubation time (hours ) FIG. 1. Incorporation of C2H3 from L-[methyl-2H3]methionine into the 3- and 4-methoxyl groups of veratryl alcohol during growth of P. chrysosporium. (A) Culture medium contained an initial concentration of 1 mm L-[methyl-2H3]methionine; (B) conditions were similar to panel A, except that a further supplement of L-[methyl-2H3]methionine (.5 mm) was added at 96 h. Symbols:, percent dilabeled (di-c2h3) veratryl alcohol; A, percent monolabeled (CH3, C2H3) veratryl alcohol;, percent unlabeled (di-ch3) veratryl alcohol. Cultures of P. chrysosporium were grown at 37 C in 1-ml conical flasks containing glycerol-high-nitrogen medium (1 ml) under 1% 2 in the presence of 1 mm L-[methyl-2H3]methionine as described in Materials and Methods. In panel B, flasks were supplemented with additional L-[methyl-2H3]methionine at 96 h (arrowed). At intervals during growth, the contents of individual flasks were harvested for measurement of mycelial dry weight, veratryl alcohol concentration, and percent C2H3 incorporation into the compound. metabolism is largely replaced from the gaseous phase. Therefore, at the termination of the experiment at 216 h, the C2H3Cl concentration in the medium was still.16 mm. Unequivocal confirmation that label was incorporated into the methoxyl groups of veratryl alcohol was obtained by chemical synthesis of the various possible methoxyl-labeled veratryl alcohols and comparison of the mass spectrum of an appropriate mixture of these compounds with that of the veratryl alcohol synthesized by the fungus in the presence of C2H3Cl (Fig. 4). The mass spectra of unlabeled and dilabeled compounds (Fig. 4A and B, respectively) are quite different, and both are distinct from the two monolabeled compounds (Fig. 4C and D). However, the latter compounds give practically identical mass spectra and therefore cannot be distinguished by the technique. Figure 4E is the mass spectrum of veratryl alcohol extracted from culture medium after growth of P. chrysosporium for 168 h in the presence of.77 mm C2H3Cl. On the basis of the relative intensity of the molecular ions at mle 168, 171, and 174 (after correction for la Incubation time (hours ) FIG. 2. Veratryl alcohol concentration during growth of P. chrysosporium in the presence of 1 mm L-[methyl-2H3]methionine. Symbols: O, growth measured as mycelial dry weight; *, total veratryl alcohol concentration. Cultures of P. chrysosporium were grown as described for Fig. 1A E b the M - 3 ion as described in Materials and Methods), it was concluded that the ratio of dilabeled to monolabeled to unlabeled compound was 69:27:4. Figure 4F is the mass spectrum of a mixture of compounds from Fig. 4B, C, and A in the above proportion and is virtually indistinguishable from the fungal product, demonstrating that C2H3 is incorporated wholly into the methoxyl groups of veratryl alcohol and validating the ion-monitoring technique used to measure the proportions of the various labeled species. That methylation was not due to a nonenzymatic reaction of C2H3CI with a veratryl alcohol precursor produced by the fungus was demonstrated by the isotopic dilution technique described in Materials and Methods. None of the possible products of methylation were found at concentrations above the detection limit of.25,um when.5 mm caffeic and ferulic acids (the precursors postulated by Shimada et al. [29]) and isoferulic acid were incubated for 7 days in sterile culture medium at 37 C with 1 mm C2H3Cl. Additionally, when.5 mm 3,4-dihydroxybenzaldehyde, vanillin, isovanillin, 3-methoxy-4-hydroxybenzyl alcohol, and 3-hydroxy-4- methoxybenzyl alcohol were incubated under similar conditions, no evidence of formation of methylated product was found at concentrations above the detection limit of.1,um. Even when the more potent chemical methylating agent iodomethane was incubated at a concentration of.25 mm under similar conditions with.5 mm solutions of the above substrates, no methylated products were observed at concentrations above the detection limit of.25,um. These findings are not unexpected in view of the very weakly nucleophilic nature of unionized phenols. As veratryl alcohol was typically present at.2 mm in the culture medium of the fungus, it is clear that the substantial labeling obtained in the veratryl alcohol biosynthesized in the presence of C2H3Cl is not explicable in terms of nonenzymatic methylation. The possibility that methylation proceeds by hydrolytic dehalogenation of CH3Cl to methanol followed by utilization of methanol as the methyl donor, a route which was eliminated in investigations with Phellinus pomaceus (16), is also rendered highly improbable in P. chrysosporium since addition of.5 mm C2H3OH to the growth medium failed to Downloaded from on September 18, 218 by guest

5 3454 HARPER ET AL. APPL. ENVIRON. MICROBIOL. -. X - -c _." M c e > E :6._e 8 A " Incubation I 4 E. 2 time ( hours ) FIG. 3. (A) Incorporation of C2H3 from.77 mm C2H3C1 into the 3- and 4-methoxyl groups of veratryl alcohol at stages during growth of P. chrysosporium. (B) Veratryl alcohol concentration and mycelial dry weight during growth of P. chrysosporium in presence of.77 mm C2H3Cl. Symbols: O, growth measured as mycelial dry weight; *, total veratryl alcohol concentration;, percent dilabeled (di-c2h3) compound; A, percent monolabeled (CH3, C2H3) compound; *, percent unlabeled (di-ch3) compound. Cultures of P. chrysosporium were grown at 37 C in 1-ml conical flasks containing glycerol-high-nitrogen medium (1 ml) under 1% 2 in the presence of.77 mm C2H3Cl as described in Materials and Methods. At intervals during growth the contents of individual flasks were harvested for measurement of mycelial dry weight, veratryl alcohol concentration, and the percent C2H3 incorporation into the compound. result in detectable incorporation of C2H3 into veratryl alcohol. C2H3 substitution pattern in monolabeled compound. In view of the failure of GC-MS to distinguish between OC2H3 label in the 3- and 4-positions of veratryl alcohol, 'H NMR was used to establish the comparative proportions of 3- and 4-labeled compound in the monolabeled veratryl alcohol produced by the fungus in the presence of C2H3Cl. By recording spectra of synthetic 3- and 4-OC2H3-labeled veratryl alcohols, it was possible to assign the singlet signals observed at 3.88 and 3.9 ppm (tetramethylsilane as the internal standard) to the 4- and 3-methoxyl groups, respectively. The relative proportions of 3- and 4-OC2H3 label in the monolabeled compound present in veratryl alcohol extracted from culture medium of the fungus after 96 h of growth in the presence of.77 mm C2H3Cl were calculated from the relative areas of the two signals after correction for the percentage of unlabeled compound present in the sample as measured by GC-MS. The percentages of 3- and 4-OC2H3 label found were 57.3 and 42.7%, respectively, indicating a slight predominance of label in the 3-position in the monolabeled veratryl alcohol present. This finding may imply that methylation by CH3Cl is more tightly channeled in the 4-position than in the 3-position, possibly suggesting that two independent enzymes are involved or that the 3- and 4-monomethylated compounds have different effects on the kinetic parameters of a single enzyme system. Veratryl alcohol concentration and lignin peroxidase activity during growth in the presence of L-[methyl-2H3]methionine and C2H3Cl. The maximum veratryl alcohol concentration attained on 1 mm methionine-supplemented medium (Fig. 2) was considerably lower than on the.77 mm C2H3Clsupplemented medium (Fig. 3B), and veratryl alcohol biosynthesis appeared to be initiated earlier in the growth cycle in the latter medium. Inhibition of the production of a secondary metabolite by a readily utilized carbon and nitrogen source such as methionine is not unusual (6). When in a separate experiment (not shown) veratryl alcohol production on unsupplemented medium was compared with that on.5 mm CH3Cl-supplemented medium, maximum levels attained were not significantly different. However, veratryl alcohol biosynthesis was again initiated earlier in the CH3Clsupplemented medium, and the period over which concentrations remained maximal was longer. Veratryl alcohol formation by several lignin-degrading fungi is known to be associated with the development of lignin peroxidase activity (8, 9, 23, 25). When culture supernatants from the above experiment were assayed as described by Buswell et al. (3), cultures on unsupplemented medium showed a maximum lignin peroxidase activity of 146 nmol of veratraldehyde formed min-' ml-' whereas growth of the fungus in the presence of.5 mm CH3Cl gave a maximum activity of 228 nmol of veratraldehyde min-' ml-'. However, the extent of the increase in lignin peroxidase activity observed in this experiment in the presence of CH3Cl was variable and appeared critically dependent on CH3Cl concentration. Further investigations are clearly required to fully delineate the nature of the relationship between lignin peroxidase activity, veratryl alcohol, and CH3Cl concentrations. C2H3 incorporation into veratryl alcohol from precursors added to 72-h cultures. Incorporation of C2H3 into veratryl alcohol at intervals after addition of C2H3Cl or L-[methyl- 2H3]methionine to 72-h cultures actively synthesizing veratryl alcohol is presented in Table 1. Labeling was very swift after addition of C2H3Cl, with 33% of veratryl alcohol labeled after 2 h and 81% after 12 h. In contrast, incorporation of label from methionine was comparatively slow, with labeled compound reaching only 1% after 12 h. The reason for this difference is not immediately clear, as veratryl alcohol biosynthesis appeared to continue after each addition, with the concentration rising from 56 to 63 and 66 nmol culture-' after 12 h in the presence of C2H3Cl and L-[methyl-2H3]methionine, respectively. Actual veratryl alcohol biosynthesis was undoubtedly very much higher than these concentrations might at first suggest, as the rapid incorporation in the presence of C2H3Cl confirms the rapid turnover of the compound. The failure of methionine to act as a more effective precursor seems unlikely to be due to the inability of the fungus to take up methionine at this stage of growth, as an assay of the culture medium after addition of the amino acid under the conditions described in Table 1 w- I E 'a. a U -5 Downloaded from on September 18, 218 by guest

6 VOL. 56, 199 CHLOROMETHANE IS A METHYL DONOR IN P. CHRYSOSPORIUM 3455 a) c._ 1 in particular, protein biosynthesis. These results, when considered in association with those displayed in Fig. 3, provide compelling evidence that CH3Cl must participate as an intermediate in the methylation of veratryl alcohol precursors r% v,'.. 2 4_ n3 CH2H JOC2H3 O CH3 Ill..11 6t, i6 1,1,, F C.1..-I,.,1,1,.1,,, ,,., L CH2H OCH3 I, 6 CH2H 6 CH2H O C H3 OC'H3.,.1 1 'II, Ill.,. x, I Ill,,I IllIll,...14JII --Lllli * JOC2H3 ^f^e t ie l*i Xaek kelal llisefi _4la ^svl- A E I I ISO Ill.1111 lffil sl r ,.,ll, 1ll.,1l.,..-III 1 'Ii'' 6! ,1,lll---I l.,,1 A1ll,,1 1i, 6 1o 14 Mass/Charge FIG. 4. MS comparison of veratryl alcohol extracted from culture medium of P. chrysosporium grown in the presence of C2H3Cl with authentic C2H3-labeled and unlabeled standards. (A to D) Mass spectra of various labeled and unlabeled standards of veratryl alcohol. (E) Mass spectrum of veratryl alcohol extracted from fungal culture medium after growth of the fungus for 168 h in presence of.77 mm C2H3Cl. (F) Mass spectrum of a mixture of B, C, and A in the proportion 69:27: i B D.11 -Ai Downloaded from on September 18, 218 by guest showed a 29% fall in concentration within 12 h. Therefore, the most feasible explanation of the comparatively low incorporation of methionine observed in Table 1 is that exogenous amino acid taken up at this stage of growth is routed predominantly into competing biochemical pathways,

7 3456 HARPER ET AL. APPL. ENVIRON. MICROBIOL. Time (h) after TABLE 1. Incorporation of C2H3 into veratryl alcohol at intervals after addition of C2H3Cl or L-[methyl-2H3]methionine to 72-h cultures' of P. chrysosporium % C2H3 substitutionb in 3- and 4-positions of veratryl alcohol ± SD addition of C2H3CI (.77 mm) L-[methyl-2H3Jmethionine (1 mm) labeled precursor Di-CH3 CH3, C2H3 Di-C2H3 Di-CH3 CH3, C2H3 Di-C2H ± ± ± ± ± ± ± ± ± ± ± ± 3 a Cultures were grown as described in Materials and Methods for 72 h on unsupplemented glycerol-high-nitrogen medium. b Di-CH3, unlabeled compound; CH3, C2H3, monolabeled compound; Di-C2H3, dilabeled compound. C213 incorporation into veratryl alcohol from C2H3CI in other white rot fungi. Two other white rot fungi, Phlebia radiata and Coriolus versicolor, both widely used in investigations of lignin degradation and reported to produce veratryl alcohol (2, 22), were also examined for the presence of a CH3Cl-utilizing methylation system. Veratryl alcohol produced at a mean concentration of 5 nmol culture-1 after 7 days of growth of Phlebia radiata in the presence of.77 mm C2H3Cl as described in Materials and Methods gave a mean ratio of dilabeled to monolabeled to unlabeled compound of 82:17:1. Growth of C. versicolor for 7 days in the presence of.77 mm C2H3Cl as described in Materials and Methods resulted in a veratryl alcohol concentration of 4 nmol culture-1 with a ratio of dilabeled to monolabeled to unlabeled compound of 45:41:14. These findings demonstrate that a CH3Cl-utilizing methylation system is also operative in both these fungi, albeit rather more tightly channeled in C. versicolor than in P. chrysosporium or Phlebia radiata. CH3C1 release by fungal cultures. Despite this evidence for CH3Cl as a metabolic intermediate in all three species investigated, the release of CH3Cl as a natural product into the flask headspace could not be detected during the growth of any of the fungi at concentrations above.2 ng ml-', which represents the limit of detection of the GC-MS technique used. Similarly, screening of the three species for CH3Cl release on three different solid media, containing glucose, malt extract, and cellulose, respectively, as the main carbon source by the methods of Harper et al. (18) failed to demonstrate the presence of free CH3Cl in the culture headspace. This suggests that, as with the methylation of benzoic acid by CH3Cl in Fomitopsis pinicola (15), biosynthesis of CH3Cl in these species is closely coupled to its utilization by a high-affinity methylation system. Such channeling could involve direct transfer of CH3Cl from the biosynthetic enzyme to the methylation system or, alternatively, could entail restriction of CH3Cl to an unmixed layer within the cell (1). In either case, the compound is effectively confined to a multienzyme complex and never attains the status of a freely diffusible intermediate. Nevertheless, the fact that exogenous CH3Cl can act as the methyl donor indicates that access to the methylating enzyme is not restricted solely to endogenous CH3Cl. An alternative explanation for the failure to detect CH3Cl as a natural product of these fungi is that rather than being a genuine metabolic iptermediate, the compound is involved in exchange reactions with the true substrate of the methyltransferase enzyme, which could be either S-adenosylmethionine or an enzyme-bound activated methyl group. However, to achieve the high labeling levels exhibited in Fig. 3 and the speed of incorporation apparent in Table 1, exchange would have to be very rapid, and this seems exceedingly unlikely on thermodynamic grounds. Significance of CH3Cl in fungal metabolism. Participation of CH3Cl in the biosynthesis of a compound of such central importance in lignin degradation affords strong support for the suggestion by Harper et al. (16) that CH3Cl may have a widespread, if previously unsuspected, role in methylation processes in many fungi and even perhaps higher plants. The part played by the compound as methyl donor may have escaped recognition hitherto because CH3Cl is a relatively chemically unreactive gas that is readily overlooked by normal analytical procedures and is usually synthesized and utilized in tightly channeled multienzyme complexes from which the release of free CH3Cl is seldom or never observed. Although this report considers only the role of CH3C1 in methylation of veratryl alcohol, the presence of CH3Clutilizing systems in lignin-degrading fungi may have a much wider significance. Although ligninases are able to degrade lignin in vitro to a limited extent (3), Haemmerli et al. (12) have shown that repolymerization outweighs degradation, probably owing to the phenoloxidizing activity associated with the enzyme. Accordingly, they and others postulate that, inter alia, systems must exist in vivo to prevent polymerization (12, 34). It is conceivable that these include an extracellular methylation system that uses CH3C1, which could provide the rapid methylating capability necessary to halt repolymerization of phenolic cleavage products of ligninase action. A sufficiently high-affinity enzyme might even be able to use natural CH3Cl normally present at a concentration of about 1 ppb (vol/vol) in the atmosphere (27). ACKNOWLEDGMENTS We thank Don Annette for methionine analyses and Paul Stevenson, Chemistry Department, The Queen's University of Belfast, for NMR analyses. LITERATURE CITED 1. Ander, P., and K.-E. Eriksson The importance of phenol oxidase activity in lignin degradation by the white rot fungus Sporotrichum pulverulentum. Arch. Microbiol. 19: Andrews, R. P Analysis of human serum and plasma using the LKB 44 amino acid analyser. Technical Leaflet PCN12. LKB Instruments Ltd., Croydon, United Kingdom. 3. Buswell, J. A., B. Molletr and E. Odier Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sufficiency. FEMS Microbiol. Lett. 25: Chen, C. L., H. M. Chan, and T. K. Kirk Aromatic acids produced during degradation of lignin in spruce wood by Phanerochaete chrysosporium. Holzforschung 36: Cowan, M. I., T. A. Glen, S. A. Hutchinson, M. E. MacCartney, J. M. Mackintosh, and A. M. Moss Production of volatile Downloaded from on September 18, 218 by guest

8 VOL. 56, 199 CHLOROMETHANE IS A METHYL DONOR IN P. CHRYSOSPORIUM 3457 metabolites by species of Fomes. Trans. Br. Mycol. Soc. 6: Drew, S. W., and A. L. Demain Effect of primary metabolites on secondary metabolism. Annu. Rev. Microbiol. 31: Eriksson, K.-E., and S. C. Johnsrud Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and P-D-glucosidase production. Enzyme Microb. Technol. 5: Faison, B. D., and T. K. Kirk Factors involved in the regulation of ligninase activity in Phanerochaete chrysosporium. Appl. Environ. Microbiol. 49: Faison, B. D., T. K. Kirk, and R. L. Farrell Role of veratryl alcohol in regulating ligninase activity in Phanerochaete chrysosporium. Appl. Environ. Microbiol. 52: Gaertner, F. H Unique catalytic properties of enzyme clusters. Trends Biochem. Sci. 3: Gillis, R. G Trideuteromethylation in dimethyl sulphoxide. Tetrahedron Lett. 1968: Haemmerli, S. D., M. S. A. Leisola, and A. Fiechter Polymerisation of lignins by ligninases. FEMS Microbiol. Lett. 35: Haemmerli, S. D., M. S. A. Leisola, D. Sanglard, and A. Feichter Oxidation of benzo (a) pyrene by extracellular ligninases of Phanerochaete chrysosporium. J. Biol. Chem. 261: Harper, D. B Halomethane from halide ion-a highly efficient fungal conversion of environmental significance. Nature (London) 315: Harper, D. B., and J. T. G. Hamilton Biosynthesis of chloromethane in Phellinus pomaceus. J. Gen. Microbiol. 134: Harper, D. B., J. T. G. Hamilton, J. T. Kennedy, and K. J. McNally Chloromethane, a novel methyl donor for biosynthesis of esters and anisoles in Phellinus pomaceus. Appl. Environ. Microbiol. 55: Harper, D. B., and J. T. Kennedy Effect of growth conditions on halomethane production by Phellinus species: biological and environmental implications. J. Gen. Microbiol. 132: Harper, D. B., J. T. Kennedy, and J. T. G. Hamilton Chloromethane biosynthesis in poroid fungi. Phytochemistry 27: Harvey, P. J., H. E. Schoemaker, and J. M. Palmer Veratryl alcohol as a mediator and the role of radical cations in lignin biodegradation by Phanerochaete chrysosporium. FEBS Lett. 195: Hatakka, A., A. Kantelinen, A. Tervila-Wilo, and L. Viikari Production of ligninases by Phlebia radiata in agitated cultures p In E. Odier (ed.), Lignin enzymic and microbial degradation. International Symposium, Paris (April 23-24), INRA Publications, Paris. 21. Hatakka, A. I., and A. K. Uusi Ravva Degradation of 14C-labelled poplar wood lignins by selected white-rot fungi. Eur. J. Appl. Microbiol. Biotechnol. 17: Kawai, S., T. Umezawa, and T. Higuchi De novo synthesis of veratryl alcohol by Coriolus versicolor. Wood Res. 73: Kirk, T. K., E. Schultz, W. J. Connors, L. F. Lorenz, and J. G. Zeikus Influence of cultural parameters on lignin metabolism by Phanerochaete chrysosporium. Arch. Microbiol. 117: Leisola, M. S. A., B. Schmidt, U. Thanei-Wyss, and A. Fiechter Aromatic ring cleavage of veratryl alcohol by Phanerochaete chrysosporium. FEBS Lett. 189: Leisola, M. S. A., D. C. Ulmer, R. Waldner, and A. Fiechter Role of veratryl alcohol in lignin degradation by Phanerochaete chrysosporium. J. Biotechnol. 1: Lundquist, K., and T. K. Kirk De novo synthesis and decomposition of veratryl alcohol by a lignin degrading basidiomycete. Phytochemistry 17: Rasmussen, R. A., L. E. Rasmussen, M. A. K. Khalil, and R. W. Dalluge Concentration distribution of methyl chloride in the atmosphere. J. Geophys. Res. 85: Schoemaker, H. E., P. J. Harvey, R. M. Bowen, and J. M. Palmer On the mechanism of enzymatic lignin breakdown. FEBS Lett. 183: Shimada, M., F. Nakatsubo, T. K. Kirk, and T. Higuchi Biosynthesis of the secondary metabolite veratryl alcohol in relation to lignin degradation in Phanerochaete chrysosporium. Arch. Microbiol. 129: Tien, M., and T. K. Kirk Lignin-degrading enzyme from the hymenomycete Phanerochaete chrysosporium Burds. Science 221: Tien, M., and T. K. Kirk Lignin degrading enzyme from Phanerochaete chrysosporium. Purification, characterization, and catalytic properties of a unique H22-requiring oxygenase. Proc. Natl. Acad. Sci. USA 81: Tonon, F., and E. Odier Influence of veratryl alcohol and hydrogen peroxide on ligninase activity and ligninase production by Phanerochaete chrysosporium. Appi. Environ. Microbiol. 54: Wariishi, H., and M. H. Gold Lignin peroxidase compound III formation, inactivation and conversion to the native enzyme. FEBS Lett. 243: Westermark, U., and K.-E. Eriksson Carbohydrate-dependent enzymic quinone reduction during lignin degradation. Acta Chem. Scand. B28: White, R. H Biosynthesis of methyl chloride in the fungus Phellinus pomaceus. Arch. Microbiol. 132:1-12. Downloaded from on September 18, 218 by guest