Chlorometabolite production by the ecologically important white rot fungus Bjerkandera adusta

Chemosphere 44 (2001) 1603±1616

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Chlorometabolite production by the ecologically important white rot fungus Bjerkandera adusta P.J. Silk a

a,* ,

C. Aubry b, G.C. Lonergan a, J.B. Macaulay

a

Chemical and Biotechnical Services Department, Research and Productivity Council, 921 College Hill Road, Fredericton, NB, Canada E3B 6Z9 b Chemistry Department, University of Ottawa, 10 Marie-Curie, Ottawa, Ont., Canada KIN 6N5 Received 24 May 2000; accepted 7 October 2000 Dedication to Professor Alex Fallis on the occasion of his 60th birthday

Abstract Two strains of the basidiomycete, Bjerkandera adusta (DAOM 215869 and BOS55) produce in static liquid culture, phenyl, veratryl, anisyl and chloroanisyl metabolites (CAM's) (alcohols, acids and aldehydes) as well as a series of compounds not previously known to be produced by Bjerkandera species: 1-phenyl, 1-anisyl, 1-(3-chloro-4-methoxy) and 1-(3,5-dichloro-4-methoxy) propan-1,2-diols, predominantly as erythro diastereomers with 1R, 2S absolute con®gurations. 1-Anisyl-propan-1,2-diol and 1-(3,5-dichloro-4-methoxy)-propan-1,2-diol are new metabolites for which the names Bjerkanderol A and B, respectively, are proposed. Experiments with static liquid cultures supplied with 13 C6 - and 13 C9 -L-phenylalanine showed that all identi®ed aromatic compounds (with the exception of phenol) can be derived from L-phenylalanine. For the aryl propane diols, the 13 C label appeared only in the phenyl ring and the benzylic carbon, suggesting a stereoselective re-synthesis from a C7 and a C2 -unit, likely aromatic aldehyde and decarboxylated pyruvate, respectively. Other compounds newly discovered to be derived from phenylalanine by this white rot fungus include phenylacetaldehyde and phenylpyruvic, phenylacetic, phenyllactic, mandelic and phenyl glyoxylic (benzoyl formic) acids. For both strains, cultures supplied with Na37 Cl showed incorporation of 37 Cl in all identi®ed chlorometabolites. Veratryl alcohol and the CAM alcohols, which occur in both strains and can be derived from L-phenylalanine (all 13 C-labelled), have reported important physiological functions in this white rot fungus. Possible mechanisms for their formation through the newly discovered compounds are discussed. Ó 2001 Elsevier Science Ltd. All rights reserved.

1. Introduction Environmental organohalogen compounds were considered to be only of anthropogenic origin, but now it is certain that a ubiquitous capacity exists for their production in nature. This has been documented in unpolluted marine and terrestrial environments (Neidleman and Geigert, 1986; Grimvall and de Leer, 1995;

*

Corresponding author. Tel.: +1-506-452-1366; fax: +1-506452-1395. E-mail address: [email protected] (P.J. Silk).

Gribble, 1988). Both natural and anthropogenic organohalogens likely meet similar environmental fates. Organochlorine production (particularly chlorinated aromatics) is common among basidiomycetous fungi dominated by species within the genera Hypholoma, Mycena and Bjerkandera (Field et al., 1995; Verhagen et al., 1996; deJong and Field, 1997). The organochlorine metabolites produced have important physiological roles as methyl donors, antibiotics and as substrates for H2 O2 -generating oxidases, for e.g., and are ``not biological accidents'' (deJong and Field, 1997). Halomethanes, halogenated aromatics and haloaliphatic compounds have all been documented to occur in fungal

0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 0 ) 0 0 5 3 7 - 3

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culture and in the environment (de Jong et al., 1994a,b,c). Of particular interest to us are the chlorinated aromatics of fungal origin such as chlorinated anisyl (CAM's), chlorinated hydroquinone (CHM's) and chlorinated orcinol (COM's) metabolites. These naturally occurring chlorinated aromatic methyl ethers can ®nd their way into environmental compartments where they can, on microbial demethylation, become incorporated into humus (deJong et al., 1994c) or by (oxidative) coupling, potentially form chlorinated dioxins and furans (Silk et al., 1997; Gribble, 1994, 1998). Among the ecologically signi®cant group of organisms that degrade lignocellulosic material, the smokey polypore, Bjerkandera adusta, is one of the most studied of the basidiomycetes. Over 20 chlorinated aromatics have been identi®ed to date from Bjerkandera sp. BOS55 and B. adusta (deJong and Field, 1997). Many of these chlorometabolites are 3- or 3,5-chlorinated anisyl alcohols, aldehydes and acids (CAM's). Veratryl, anisyl, 3chloroanisyl and 3,5-dichloroanisyl alcohols and the corresponding aldehydes are all produced de novo from glucose (deJong et al., 1992) simultaneously with the extracellular ligninolytic enzymes. Biosynthesis likely proceeds through the phenylpropanoid pathway since CAM production is increased 10-fold when phenylalanine is added to cultures of Bjerkandera sp. (Mester et al., 1997). Hydroxylation and methylation have been proposed in the production of veratryl alcohol via benzoic acid in which methylation of the phenolic hydroxyl is thought to occur through S-adenosyl methionine (SAM)-dependent methyl transferase and/or through a chloromethane-dependent transferase (Harper et al., 1996). How and when chlorination takes place through the phenylpropanoid pathway is unknown. Electrophilic aromatic substitution via a chloroperoxidase has been suggested since chlorination is ortho to the p-hydroxy/ methoxy group (Field et al., 1995) although these enzymes have not been detected in the extracellular ¯uids of basidiomycetes. It has, however, recently been suggested that NADH-dependent halogenases are more likely to be involved in halometabolite biosynthesis than haloperoxidases (Hohaus et al., 1997). The biosynthetic pathway of aryl metabolites in white-rot fungi is not completely understood. Veratryl alcohol, which displays a diverse array of functions in conjunction with lignin peroxidase (LiP) in ligninolytic activity, originates through the shikimate pathway via phenylalanine with cinnamate, benzoate and/or benzaldehyde as purported biosynthetic intermediates in the case of Phanerochaete chrysosporium (Jensen et al., 1994). Similarly, in B. adusta, anisyl and chloroanisyl metabolites are likely derived through the phenylpropanoid pathway from aromatic amino acids since exogenous tyrosine increases their production in culture (Spinnler et al., 1994) and deuterated 4-hydroxy and

3-chloro-4-hydroxy benzoates stimulate aryl metabolite production and lead to deuterated CAM metabolites (Mester et al., 1997). The objectives of this present study were to determine if aryl metabolites in B. adusta originate through phenylalanine, to attempt to determine how and when chlorination takes place and to identify any new metabolites. Two strains of B. adusta were studied in static liquid cultures: the BOS55 strain, well studied by others and a new strain, DAOM 215869 collected in Canada. The results of our initial experiments are presented in this paper, in which we have relied on stable isotope labelling, gas chromatographic/mass spectrometric (GC/ MS) analyses, and comparison with authentic materials, to identify metabolites. Several new B. adusta chlorometabolites have been identi®ed as not having some important precursors. Mechanistic aspects of the degradation/formation pathway are discussed. 2. Methods and materials 2.1. Chemicals L-Phenylalanine (13 C9 , 97±98%, CLM-2250), L-phenylalanine (ring-13 C6 , 99%, CLM-1055), Na 37 Cl (95%, CHLM-1225) and sodium pyruvate (2,3-13 C2 , 99%, CLM-3507) were obtained from Cambridge Isotope Laboratories (CIL), and used without further puri®cation in culture experiments. All other chemicals were either commercially available (Aldrich or Fluka) or were synthesized by structurally unambiguous routes as noted in the text or in the Yeast Biomimetic syntheses section involving production of mixtures of a-ketols (acyloins and 1-acyl ethanols) and diols. 2.2. Source of cultures 2.2.1. B. adusta DAOM 215869, isolated from a fruitbody on an Ulmus americana stump, Cantley, Quebec, Canada, 12 October, 1992, J.H. Ginns deposited in CCFC (Agriculture and Agri-Food Canada, Ottawa). Bjerkandera sp. BOS55 was obtained (source see Swarts et al., 1996) from Dr. J.A. Field, Wageningen, Netherlands. Cultures were maintained on sterile malt/agar/yeast extract slants at 4°C and sub-cultured onto malt/agar/ yeast extract (sterile) plates and incubated at 22°C. These sub-cultures were then used to inoculate liquid cultures for both species with a 5 mm agar plug aseptically taken from the edge of the fungal growth. 2.3. Culture media Several liquid cultures of each of the two strains were grown. The liquid media (for 1 l aqueous solutions) were

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as follows: 21.7 g D (+) glucose, 5.5 g peptone, 2.2 g yeast extract, 1:2 g KH2 SO4 ; 0:5 g MgSO4 ; 0.08 g NaCl [4] or 10.8 g D (+) glucose, 2.2 g peptone, 2.2 g yeast extract, 2:2 g KH2 PO4 ; 1:1 g MgSO4 , 1.1 mg thiamine hydrochloride (Tien and Kirk, 1988). Low-nitrogen media was 20 g glucose, 1:0 g KH2 SO4 ; 0:5 g MgSO4 and 0.07 g NaCl. Aliquots of 200 ml were placed in 500 ml erlenmeyer ¯asks and autoclaved (thiamine was ®ltersterilized). The media were inoculated and kept in the dark at ca. 25°C for up to 13.5 weeks without shaking. Na37 Cl-enriched cultures employed Field's medium (Field et al., 1995). L-Phenylalanine enriched cultures were grown in 150 ml tubes using Tien and Kirk (1988) medium and included 80 mg ring-13 C6 or 13 C9 -L-phenylalanine + 80 mg native L-phenylalanine in 100 ml of medium. Some cultures (DAOM 215869) were also grown in Tien and Kirk's medium containing sodium pyruvate (50 mg each of native and 2,3-13 C2 -pyruvate, sodium) in the presence of thiamine pyrophosphate (cocarboxylase) and L-phenylalanine and eight week cultures were extracted and analysed. 2.4. Extraction of fungal cultures Cultures were replicated (X2±X8) and harvested and extracted at several time intervals (as noted in results) up to 13.5 weeks. The mycelial mats were ®ltered from the extracellular ¯uid, the ®ltrate was either directly extracted or acidi®ed to either pH 4.0 or pH 2.0 with dilute sulphuric acid and then extracted with ethyl acetate. The extracts were dried over anhydrous Na2 SO4 , the solvent evaporated and the extract analysed by GC/MS directly or as derivatized extracts as noted. 2.5. Yeast biomimetic syntheses of a-ketols and diols Our discovery of new metabolites by the two strains of B. adusta involving a-ketols and 1-aryl-propane-1,2diols necessitated synthesis of these compounds as analytical standards for structural elucidation. We employed the biomimetic ability of fermenting Bakers' yeast, Saccharomyces cerevisiae to produce a-ketols (acyloins and 1-acylethanols) and diols from aromatic aldehydes (Ohta et al., 1986; Brambilla et al., 1995) with high diastereo/enantioselectivity. This reductive C2 -homologation of aldehydes gives mostly erythro isomers (diols) with 1R, 2S absolute con®gurations; the diols are likely produced from reduction of the a-ketols formed from the aromatic aldehyde and a C2 -unit (Brambilla et al., 1995). Recently, the enzyme involved in the C2 homologation has been shown to be pyruvate decarboxylase (PDC) having thiamine pyrophosphate as cofactor (PDC, 2-oxo-acid carboxyl lyase EC 4.1.1.1) (Crout et al., 1991). The aromatic aldehydes employed as reaction substrates in our work were benzaldehyde, p-anisaldehyde,

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3-chloro-4-methoxy benzaldehyde and 3,5-dichloro-4methoxy benzaldehyde. The former two aldehydes were commercially available, while the latter two were synthesized from commercial 3-chloro- and 3,5-dichloro-4-hydroxy benzoic acids by methylation (diazomethane), lithium aluminum hydride reduction to the corresponding benzylic alcohol and oxidation to the aldehydes using pyridinium chlorochromate (PCC). After puri®cation each aldehyde was then subjected to whole yeast fermentation (Fleishmann's active dry yeast, Burns Philip Food, Ontario, Canada) with glucose to produce mixtures of the corresponding benzylic alcohol, a-ketols, and diols (ca. 10% yield) as shown in Scheme 1 (Ohta et al., 1986; Brambilla et al., 1995). Each resulting extract (ethyl acetate) was then subjected to GC/MS analysis either as direct extracts or as acetylated derivatives. 2.6. Derivatization techniques Acetylation was carried out by treatment with acetic anhydride (solvent)/pyridine at room temperature overnight followed by evaporation under dry nitrogen, dissolution in ethyl acetate followed by GC/MS analysis. Portions of acetylated extracts were also treated directly with ethereal diazomethane and re-analysed. Other extracts were treated with Sylon BTZ (Supelco) or directly with diazomethane prior to analysis. 2.7. Instrumental analyses All GC/MS analyses were performed on a Hewlett± Packard 5890 II GC/5971 MSD in the electron-impact (EI) mode at 70 eV. Injections were made in the splitless mode with helium as carrier gas. Analyses were performed either on a Supelcowax-10 (Supelco) or a HP-5MS capillary column (each 30 m, 0.25 mm id, 0:25 lm ®lm thickness). The column was temperature programmed from 50°C at the rate of 20°C= min to 250°C. Injector and detector temperatures were set at 250°C. Some extracts were analysed on a a-cyclodextrin column to e€ect some chiral separations (Supelco, 30 m, 0.25 mm ID, 0:25 lm ®lm thickness; a-dex 120, non-bonded; 20% permethylated a-cyclodextrin in SPB35 poly(35% phenyl/65% dimethylsiloxane)); this column was temperature programmed from 50°C, held for 5 min, and then at a rate of 5°C=min to 220°C and held for 10 min. Retention time data and mass fragmentation patterns of the fungal metabolites were compared to those for either authentic commercial compounds and/or their derivatives, mixtures from the yeast biomimetic syntheses or synthetic material prepared in our laboratory (as noted in the text) to give a high probability of the correct structural assignments.

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Scheme 1. Yeast biomimetic synthetic scheme for aryl propane diols and ketols prepared from aromatic aldehyde substrates.

3. Results Under the culture conditions described above, both strains of B. adusta produced an array of phenyl, anisyl, veratryl and chloroanisyl metabolites as previously reported (deJong and Field, 1997). There are obvious strain di€erences in the quantitative production of these compounds (Figs. 1 and 2, compounds 1±36), most of which occur at some level in the extracellular ¯uid of both strains even without exogenous supplements of Lphenylalanine. CAM production, in particular (structurally and quantitatively), by both fungal strains of B. adusta, in static liquid culture, con®rmed the literature evidence to date (deJong and Field, 1997) and also supported the enhanced production of CAM metabolites on addition of L-phenylalanine to the cultures (Mester et al., 1997). In low nitrogen media, both strains showed decreased growth and, after 13.5 weeks in culture, only trace quantities of some of the previously identi®ed compounds were detected (primarily aldehydes). Structures (Fig. 2) of phenyl and anisyl compounds and mono- and dichlorinated CAM's (3 and 3,5-chloroanisyl aldehydes, acids and alcohols) were con®rmed (data not shown) by comparison of retention times and EI-mass spectra with those of authentic materials. In the BOS55 strain, dichloro CAM's were more prevalent than their monochloro analogues. Monochloro CAM's

were more prevalent than dichloro CAM's in the DAOM 215869 strain. The major strain di€erences noted were the trace level or the absence of p-anisaldehyde (4), methyl anisate (6) and p-anisyl alcohol (23) in the BOS55 strain compared to DAOM 215869 and the much larger benzoate (2)titre in BOS55 (Fig. 1). In addition to the CAM's, we have also found other important metabolites in both strains. Of particular interest is the identi®cation in early cultures of phenyl and anisyl non-chlorinated, monochlorinated and, in some cases, dichlorinated 1-aryl-propan-1,2-aketols (1-acyl ethanols) and 1-aryl-propan-1,2-diols (shown as the di-acetates 8,14,15,18). 1-anisyl-propan1,2-diol and 1-(3,5-dichloroanisyl)-propan-1,2-diol are new metabolites for which the names Bjerkanderol A and B, respectively, are proposed. Positive identi®cation of a-ketols and 1,2-diols was determined by comparison of retention time and EI-mass spectra of B. adusta underivatized (they occur as free ketols and diols in the culture ¯uid) and acetylated extracts with those from synthetic mixtures generated by Baker's yeast biomimetic syntheses in the presence of the corresponding aromatic aldehyde (Scheme 1). The postulated EI-mass spectral fragmentation patterns for all acetylated diols are shown in Fig. 3 with the characteristic ions B, C, D, and E shown in the proposed fragmentation pathway and noted in the lower table of Fig. 4.

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Fig. 1. Extract of B. adusta culture supplied with 13 C9 -L-phenylalanine, eight weeks in culture; Supelcolwax-10 column. (a) Untreated extract DAOM 215869 strain; (b) methylated extract DAOM 215869 strain; (c) acetylated then methylated extract DAOM 215869 strain; (d) acetylated then methylated extract BOS55 strain.

Fig. 2. Structures of compounds found in culture extracts.

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including the chlorinated aromatics and the aryl diols. The assigned structures of all identi®ed metabolites/degradation products are shown in Fig. 2 with the asterisk (*) denoting the presence of a 13 C-label at that side-chain carbon or, where the asterisk is in the centre of the phenyl ring, denoting that all ring-carbons were labelled. The compounds were identi®ed by comparison of retention time and EI-mass spectral data with authentic unlabelled synthetic materials, compounds synthesized by us or well-de®ned extracts (yeast). It is noteworthy that many compounds identi®ed are not visible in underivatized extracts (Fig. 1(a)) and the pH of the solution and the sequence of derivatization is important because hydroxyl and carboxyl functional groups are often present in the same molecule (e.g., mandelic and phenyllactic acids). Underivatized compounds with both hydroxyl and carboxyl functional groups will not readily elute through the GC column. Some trace level 13 C-labelled compounds were not identi®ed. Curiously, phenol, which did not appear to incorporate any 13 C-label was detected (con®rmed by acetylation) in moderate quantities, particularly in the low-nitrogen media. 3.3. Diols and a-ketol metabolites

Fig. 3. EI-mass spectra of phenyl, anisyl, chloroanisyl and dichloroanisyl propanediols(diacetates); 13-C-9-L-phenylalanine supplied cultures.

3.1. Incorporation of

37

Cl from Na37 Cl

In both Bjerkandera strains, cultures supplied with Na37 Cl showed incorporation of 37 Cl in all identi®ed chlorometabolites. Typical data are shown in Fig. 5, I for 3-chloroanisaldehyde and in Fig. 5, II for 1-(3, 5dichloroanisyl)-propane-1,2-diol diacetate. For ions containing Cl atoms, the mass spectra showed a distorted isotope pattern clearly indicating the speci®c incorporation of the 37 Cl label. This incorporation of label leads to the enhanced M ‡ 2 peaks (enhanced M ‡ 4 peaks are also observed in the case of Fig. 5, II). 3.2. Incorporation …13 C6 ; 13 C9 †

of

13

C

from

L-phenylalanine

Incorporation of L-phenylalanine (13 C6 -ring) or L-phenylalanine …13 C9 † in the cultures resulted in the labelling of all aromatic metabolites (except phenol)

The stable isotope labelling through 13 C9 -phenylalanine revealed that only seven of the nine labelled carbons remained in each of the diols (diacetates) with the 13 C6 -ring and the benzylic-carbon containing the label. This was determined from the fragmentation patterns (Fig. 4 and corresponding table) and is shown clearly in Fig. 3 with the characteristic B, C, D and E fragments (Fig. 4) identi®ed on the mass spectra. When present in the extract, the erythro and threo diastereomers of the corresponding acetylated diols were separated on the Supelcowax-10 column and the approximate ratios observed as depicted in the table corresponding to Fig. 4 for both strains; the erythro diastereomer predominates in each case with the highersubstituted ring diols showing the greatest erythro contribution. The relative stereochemistry of these diastereomers was deduced by comparison of retention time and EI-mass spectral data of erythro/threo diastereomers from the yeast biomimetic syntheses (Brambilla et al., 1995), which were well resolved on the Supelcowax10 column, with the erythro diastereomer eluting ®rst. A comparison of acetylated 13 C9 -phenylalanine culture extracts with acetylated yeast extracts was carried out by GC/MS using a a-cyclodextrin column. This column separated both diastereomeric and enantiomeric pairs (1S, 2R eluting before 1R, 2S). The data indicated that all diols (diacetates) had a predominant erthyro con®guration with a 1R, 2S …ca: > 90%ee:† absolute con®guration as reported by (Brambilla et al., 1995) for Trametol (Scheme 1). This compound is formed in Trametes versicolor cultures and the absolute stereo-

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Fig. 4. Proposed EI-mass spectral fragmentation pathway of aryl propanediols (diacetates); 13-C-9-L-phenylalanine supplied cultures.

Fig. 5. Typical EI-mass spectra of 37-Cl-labelled compounds. I ± 3-chloroanisaldehyde, II ± 1-(3,5-dichloro-anisyl)-propan1,2-diol (diacetate).

chemistry had previously been established by comparison with compounds produced by whole yeast biomimetic synthesis in which all diols had erythro 1R, 2S

con®gurations (Ohta et al., 1986). The enantiomeric separation by the a-cyclodextrin column is illustrated in Fig. 6 which shows the chromatographic resolution of the diacetate of anisyl-propan-1,2-diol. Fig. 6, A shows a whole yeast preparation of this compound by biomimetic synthesis with p-anisaldehyde which was left in culture for several weeks and had racemized, presumably through the (precursor) a-ketol tautomerism. Fig. 6, B illustrates a fresh preparation clearly showing > 95% ee of the 1R, 2S enantiomer (Brambilla et al., 1995). Fig. 6, C shows the enantiomer produced from the B. adusta culture. The structures are illustrated in the lower part of Fig. 6. The assignment of the relative stereochemistry of the threo enantiomers (1S, 2S and 1R, 2R) to the peaks in Fig. 6, A is unknown. Formation of the a-ketol was apparent only in early cultures (®ve weeks) of B. adusta in which the 1-acylethanol structure (Scheme 1, B, R1 ˆ OMe; R2 ˆ Cl; R3 ˆ H) was assigned (Fig. 7, I) based on the EI-mass fragmentation pattern and con®rmed by comparison with the GC/MS data of the compound from the whole yeast biomimetic synthesis with 3-chloro-anisaldehyde as substrate. The a-ketol appeared only as traces in 13 C9 experiments with phenylalanine. In addition, a vinyl substituted chloro-acyl product with only the ring and

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the benzylic carbon 13 C-labelled (data not shown) was also observed in some early cultures and assigned the structure shown in Fig. 7, II. This may, however, be a dehydration product formed from the 1-acyl-ethanol (aketol), during the acidic work-up of the extracts. 3.4. Incorporation of

Fig. 6. Alpha-cyclodextrin column separation of the diacetates of 1-anisyl-propan-1,2-diols. (a) Diols produced from whole yeast fermentation with anisaldehyde, allowed to racemise for two weeks prior to acetylation; (b) diols produced from whole yeast fermentation with anisaldehyde but extracted and acetylated after 4 h; (c) diols produced from B. adusta (DAOM 215869) eight weeks in culture and acetylated. The assigned stereochemistries are noted in the ®gure (assignment of the relative stereochemistry of the threo enantiomers is unknown).

Fig. 7. EI-mass spectra: I ± 1-(3-chloro-4-methoxyphenyl)-2hydroxy-propan-1-one; II ± 1-(3-chloro-4-methoxyphenyl)2-propen-1-one. DAOM 215869: ®ve weeks in culture supelcowax-10 column.

13

C from sodium pyruvate

13

C2

Extracts from cultures (DAOM 215869; Tien and Kirk (1988) medium) supplied with sodium pyruvate (native + 13 C-labelled) showed that pyruvate was metabolized with some of the label appearing in phenyl and anisyl diols. The EI-mass spectral fragments B and D (Fig. 4) of the acetylated diols each showed a second peak shifted upwards by 2 amu. However, incorporation was low and was not discernible in the chloro diols since the e€ect was masked by the ``M+2'' fragment originating from the natural 37Cl-contribution to the fragment ions. Some of the pyruvate label appeared in recovered pyruvate (est. >30%), acetoin, and other unidenti®ed low molecular weight compounds (data not shown), indicating a number of metabolic destinations for pyruvate under these conditions. Acetoin (con®rmed against authentic material) appeared in trace quantities in many of the B. adusta extracts (M‡ 88 amu; acetate M‡ 130 amu). 3.5. Other metabolites In addition to the diols, two other compounds were identi®ed in the 13 C9 -L-phenylalanine culture extracts as the 2-oxygenated-3-aryl propanoic acids, phenylpyruvic and phenyllactic acid. The EI-mass spectral evidence for phenylpyruvic acid is shown in Fig. 8. It is noteworthy that phenylpyruvic acid readily dimethylated (28) on addition of diazomethane (con®rmed with authentic phenylpyruvic acid derivative mass spectra, NMR and retention time data) with the (M ‡ 9) peak indicating that all nine prederivatization carbons had retained the 13 C label (Fig. 8, I). Further, con®rmation was obtained from the acetylated, then methylated, extract (Fig. 8, II) in which the enol acetate methyl ester (19) was evident, showing that all prederivatization carbon atoms were 13 C labelled. Phenyllactic acid was also identi®ed by retention time/EI-mass spectral data (against authentic material). Fig. 9, I clearly shows the methyl ester (32) with all nine prederivatization carbons labelled; con®rmed by the presence of the acetylated methyl ester (33) (Fig. 9, II). Evidence was also found for small amounts of trans-cinnamic acid (as the methyl ester derivative (5)) in methylated 13 C9 -L-phenylalanine culture extracts (Fig. 10,I). All nine prederivatization carbons retained the 13 C-label indicating that this acid could be derived

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Fig. 8. EI-mass spectra: I ± Enolmethyl ether methyl, ester(methylated extract); II ± Enolacetate, methyl ester (acetylated then methylated extract) of 3-phenulpyruvic acid from B. adusta strain DAOM 215869 supplied with 13-C-9-phenylalanine; Supelcowax-10 column.

Fig. 10. EI-mass spectra; extract of B. adusta supplied with 13C-9-L-phenylalanine nine weeks in culture Supelcowax-BOS55 Strain. I ± Trans-cinamic acid, methyl ester (Supelcowax-10); II ± Acetylated 4-hydroxy methylbenzoate (alpha-cyclodextrin); III ± Phenylacetaldehyde (Supelcowax-10).

Fig. 9. EI-mass spectra, B. adusta supplied with 13-C-9-Lphenylalanine eight weeks in culture Supelcowax-10 culture; Supelcowax-10 column. I ± Phenyllactic acid, methyl ester (BOS55); II ± Acetylated phenyllactic acid, methyl ester (DAOM 215869).

from L-phenylalanine. Con®rmation was by comparison to authentic trans-cinnamic acid derivatized and analysed similarly. Phenylacetic acid was also identi®ed in the extracts (as methyl ester derivative (Fig. 2, 3)) with all eight prederivatization carbons 13 C-labelled (data not shown). Con®rmation was by comparison to

authentic phenylacetic acid derivatized and analysed similarly. Phenylacetaldehyde (Fig. 2, 31) was detected in untreated BOS55 extracts (not in DAOM 215869 extracts) with the ring and the two side chain carbons 13 C-labelled (Fig. 10, III) and con®rmed by comparison to authentic material. Acetylated 4-hydroxy methyl benzoate (con®rmed against authentic material) was also found in the 13 C9 -L-phenylalanine culture extracts (Fig. 2, 34) with the ring and the benzylic carbon 13 C-labelled in the acetylated, then methylated, extracts and in traces in the acetylated extracts, suggesting that the compound existed, at least partly, in the culture ¯uid as 4-hydroxy and/or acetylated 4-hydroxyl methyl benzoate. The acetylated derivative was poorly resolved on the Supelcowax-10 column but was well separated in the a-cyclodextrin chromatography yielding the mass spectrum shown in Fig. 10, II. Partial mass spectra suggested the presence of 4-hydroxy-3-chloro methyl benzoate in early cultures at trace level but this could not be con®rmed (data not shown). Mandelic acid (con®rmed by comparison to authentic material) was also found and Fig. 11, I depicts the EImass spectra of the methyl ester (22) and the acetylated methyl ester (9) is shown in Fig. 11, II. All ring and the

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Fig. 11. EI-mass spectra: I ± Mandelic acid, methyl ester; II ± Acetylated mandelic acid, methyl ester; extract of B. adusta supplied with 13-C-9-L-phenyalanine. Strain DAOM 215869; eight weeks in culture; Supelcowax-10 column.

two side-chain carbons were 13 C-labelled. Re-analysis of this extract on the a-cyclodextrin column (isothermal, 150°C), which resolved both R- and S-mandelic acid methyl esters (S-eluting ®rst), indicated that the mandelic acid found in the culture extracts was predominately S-mandelic acid (> 95%ee). Although present only in small quantities, phenylglyoxylic (benzoyl formic) and 3-phenylpropanoic acid (methyl esters, 35 and 36, respectively) were also identi®ed (Fig. 12, I, II, respectively). Both showed incorporation of 13 C label in the ring and all side chain carbons. Curiously, chlorinated methyl (and traces of ethyl) ketone (Fig. 2, 24) occurred in both strains at low levels. In the 13 C9 -L-phenylalanine experiment, all ring and the benzylic carbon were labelled while the methyl (keto) group was unlabelled. Although this compound is formed in small amounts by addition of diazomethane to the corresponding aldehyde (one carbon homologation) this methyl ketone also appeared in untreated extracts (Fig. 1, 24) and, therefore, may not be an artifact. In older cultures (8±13.5 weeks) of both strains, two chlorinated hydroquinone methyl ethers were also detected in underivatized extracts (Fig. 2, 29 and 30), in agreement with previous work (deJong and Field, 1997). These compounds were identi®ed as 3-chloro- and 3,5dichloro-1,4-dimethoxy benzenes by comparing their retention time and EI-mass spectra with that of authentic material. In addition, the 13 C9 -L-phenylalanine experiment showed that for both of these compounds only the ring was labelled (data not shown) indicating a complete cleavage of all three propyl side-chain carbons from phenylalanine.

Fig. 12. EI-mass spectra: I ± Phenylglyoxylic acid, methyl ester (DAOM215869) II ± 3-Phenylpropanoic acid, methyl ester (BOS55); extract of B. adusta culture supplied with 13-C-9-Lphenylalanine. Both strains, eight weeks in culture; Supelcowax-10 column.

4. Discussion It would appear that many, if not all, aromatic compounds extracted from B. adusta cultures can be derived through the shikimate pathway from L-phenylalanine. The 13 C-labelled phenylalanine experiments described in this study clearly support the genesis of many previously reported metabolites (deJong and Field, 1997) in this fungus through this amino acid. This initial study was designed only to identify phenylalanine degradation products and not, at this time, to establish product/precursor relationships and the enzymes involved. However, combined with published information and considering the several newly identi®ed compounds, it does allow a number of alternative pathways to be hypothesized as to the genesis of many previously identi®ed metabolites. Many, presumably enzymatic, reactions occur with B. adusta metabolism/catabolism of L-phenylalanine which might include deamination, ring hydroxylation, methylation of (phenolic) hydroxyls, ring chlorination, a variety of oxidoreductive processes on the propyl side-chain of L-phenylalanine, and decarboxylation. The possible genesis of many of the compounds identi®ed in this study is summarized in Scheme 2. The initial step in L-phenylalanine metabolism/catabolism in B. adusta could involve the removal of the amino group. This can occur via either of two well-known processes. The ®rst is through phenylalanine ammonia lyase (PAL) producing trans-cinnamic acid, a phenylalanine metabolite identi®ed in the white rot fungus P. chrysosporium

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Scheme 2. Possible degradation pathway for L-phenylalanine by B. adusta. * ± Reverse Claisen; ** ± hydroxylation/methylation; PAL ± phenylalanine ammonia lyase; AT ± aminotransferase; ADH & PDC ± as in Scheme 1; CPO? ± chloroperoxidase or halogenase (Hohaus et al., 1997); AAO ± aryl alcohol oxidase; LiP ± lignin peroxidase; [ ] ± compounds in brackets were not identi®ed but postulated by Jensen et al. (1994) in P. chrysosporium; *** ± route reviewed in Fewson (1988).

(Jensen et al., 1994). The PAL enzyme has been identi®ed in a number of white rot fungi (de Jong et al., 1994a). The second possible deamination pathway is through an L-amino acid oxidase, or, more likely, an aminotransferase (AT) which would produce 3-phenylpyruvic acid. This compound has not been identi®ed in P. chrysosporium (Jensen et al., 1994) but has been shown to be present in the related basidiomycete Polyporus tuberaster (Kawabe and Morita, 1994). In B. adusta, both pathways could play a role since transcinnamic and 3-phenylpyruvic acids, derived from L-phenylalanine, were found in the extracts. Aminotransferases, which can transfer the a-amino group of aromatic amino acids to an a-keto acid receptor, have been studied in various micro-organisms and catalyse the reversible transamination reaction. They also play a major role in the metabolism of aromatic amino acids because they are involved in the last step of biosynthesis and take part in their catabolism (Yvon et al., 1997). In many white rot fungi, veratryl alcohol is considered an important factor in ligninolytic activity and

a co-factor for lignin peroxidase (LiP) to complete its cycle (Koduri and Tien, 1994). Veratryl alcohol is readily ring-oxidised to a radical cation by LiP, to form an important oxidant (Goodwin et al., 1995), while at the same time it protects LiP against H2 O2 -mediated inactivation (Cancel et al., 1993). In the presence of oxygen, p-anisylalcohol is involved in H2 O2 production (substrate for peroxidases) by oxidation to anisaldehyde by an aryl alcohol oxidase (AAO). AAO's are common in white rot fungi but not in P. chrysosporium (Muheim et al., 1990; Gutierrez et al., 1994). The chloro-alcohols (3-chloro- and 3,5-dichloro p-anisyl alcohols) are even better substrates (higher anity) for AAO and are less susceptible to oxidation by LiP. The electron-withdrawing characteristics of ring chlorine substitution is suggested to increase the oxidation potential of the methoxy benzyl ring (deJong and Field, 1997). The aldehydes produced are then reduced intracellularly to the corresponding alcohols resulting in a physiologically sustainable cycle (de Jong et al., 1994a).

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The study of Jensen et al. (1994) with P. chrysosporium using U-14 C-L-phenylalanine in pulse-labelling and isotope trapping experiments, showed that the metabolic pathway likely proceeds from phenylalanine, through cinnamate, to benzoate and/or benzaldehyde to veratryl alcohol. In our study of B. adusta and 13 C9 -L-phenylalanine, all of these intermediates were detected but, unlike P. chrysosporium, 3-phenylpyruvic, phenylacetic, mandelic and benzoyl formic acids and phenylacetaldehyde were also found as phenylalanine metabolites as shown by 13 C-labelling studies. It appears, therefore, that B. adusta metabolism may incorporate two di€erent routes to benzaldehyde/benzoic acid: one through cinnamate and the other through 3-phenylpyruvic acid, phenylacetaldehyde and phenylacetic/mandelic/benzoyl formic acids. Precedence for the latter route exists elsewhere (Tsou et al., 1990; Fewson, 1988). Jensen et al. (1994) also postulated that hydration of cinnamate to 3hydroxy-3-phenyl propanoic acid would lead, by oxidation and a reverse Claisen reaction, to benzaldehyde/ benzoic acid. In our study, however, no evidence for the 3-oxo-compounds was found in B. adusta. Thus, our data more strongly support the 3-phenyl pyruvic/phenylacetic/mandelic/benzoyl formic acid pathway to benzaldehyde/benzoic acid (Scheme 2) and subsequently to veratryl alcohol in B. adusta since the required 2-oxo intermediates were identi®ed. In P. chrysosporium the cleavage of the phenylalanine propyl side chain likely occurs before hydroxylation and methylation of the aromatic ring enroute to veratryl alcohol (Jensen et al., 1994). Our results indicate that this is also likely the case in B. adusta since no phenolic (methylated phenolic), 2or 3-carbon side chain, carboxylic acid type metabolites were identi®ed. A recent study with Bjerkandera sp. BOS55 (Mester et al., 1997) showed that the cultures supplemented with deuterated benzoate and 4-hydroxybenzoate resulted in formation of deuterated aryl metabolites. Deuterated CAM's, however, were produced only when cultures were supplemented with deuterated 3-chloro-4-hydroxybenzoate, suggesting it's intermediacy in CAM biosynthesis. Our data (Fig. 10, II) supports the intermediacy of 4-hydroxy benzoate in the degradation pathway, as well as 4-hydroxy-3-chlorobenzoate as recently reported (Swarts et al., 1996) for Bjerkandera sp. BOS55. Chlorination in the formation of CAM's in B. adusta likely takes place following hydroxylation, but prior to methylation, since it has been shown (Mester et al., 1997) that supplementation of cultures with 4hydroxy benzoate signi®cantly stimulated the early production of 3-chloro-p-anisaldehyde (supplementary p-anisate stimulated only the late production of the same metabolite). The formation of 1-phenyl and chloroanisyl-propan1,2 diols under the present culture conditions by B. adusta (both strains) is intriguing and not without

precedence in white rot fungi. Phenyl and 1-(3-chloro4-methoxyphenyl)-propan-1,2-diols were reported in Coriolus ( ˆ Trametes) versicolor cultures with high diastereomeric and enantiomeric purity (Brambilla et al., 1995). In our laboratory, cultures of CAM-producing Hypholoma spp. (H. elongatum, H. subericaem, and H. sublateritium) studied under similar culture conditions, showed the presence of Bjerkanderol B (1(3, 5-dichloro-4-methoxyphenyl)-propan-1,2-diol) (unpublished results). Our data also indicated a correlation between a particular aldehyde and the corresponding propan-1,2-diol in B. adusta. For e.g., in the BOS55 cultures, 3,5-dichloro-anisaldehyde was present and so was the corresponding dichloro-p-anisyl-propan-diol (Bjerkanderol B). This latter diol was not found in the DAOM 215869 strain as would be expected since the postulated precursor aldehyde was either absent or in low quantities. 13 C9 -labelling, through L-phenylalanine, indicated a stereoselective re-synthesis of these diols since the two outer carbons in the propyl sidechain from phenylalanine had been cleaved. The 2,3-13 C2 pyruvate labelling experiment supported the thesis that pyruvate is a likely source of this C2 -unit, although incorporation of this label was low in B. adusta cultures. For this to occur, the involvement of a pyruvate decarboxylase-like enzyme would be implied since the C2 -unit is re-introduced stereoselectively. The early production of the a-ketol (Fig. 7, I) and acetoin in some cultures also supports this hypothesis since the diols are likely formed by reduction of the a-ketols (Brambilla et al., 1995). Interestingly, veratraldehyde, although present in our cultures, did not appear to readily form an analogous diol. This is likely due to the 3,4-dimethoxy substitution which may render the aldehyde function less susceptible to attack by the required acetyl anion equivalent generated from pyruvate and pyruvate decarboxylase. It was not surprising, therefore, that attempts to prepare veratryl propane-diols from veratraldehyde using yeast were either not successful or gave very low yields. Further work is required to elucidate the mechanism of diol formation in B. adusta and other white rot fungi. Interestingly, P. chrysosporium, which does not produce CAM's, has not been reported to produce aryl-propandiols either. What role these diols play in the physiology of B. adusta, if any, is obscure, but they may be substrates for the chlorinating and/or hydroxylating enzymes yet to be identi®ed in B. adusta, or may be involved in the re-cycling of CAM aldehydes and alcohols. Incorporation of 37 Cl into chlorometabolites of B. adusta provided direct evidence that this fungus has the required tools to generate chloro-compounds from chloride anion. This ®nding thus strongly supports the thesis that organochlorine compounds can be produced by natural processes.

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Results of the 37 Cl labelling experiments were suggestive of the existence of a chloroperoxidase-like enzyme in B. adusta, or an NADH-dependent halogenase (Hohaus et al., 1997). All chlorometabolites found in this study were labelled in the Na37 Cl experiments, including the interesting chlorohydroquinone methyl ethers, which were also derived through L-phenylalanine and in which all the propyl carbons were cleaved. This could occur by a hydroxylative decarbonylation of CAM compounds (deJong et al., 1994b). The mechanisms of formation of many of these compounds derived from L-phenylalanine (Scheme 2), including the enzymes involved, remain to be elucidated. Support for our mechanistic arguments emerged while our work was in review (Lapadatescu et al., 2000), although no mention was made by these authors of chlorometabolities or aryl-propan-diols. Given the published literature and the data in this study, Scheme 2 may provide a useful model for L-phenylalanine metabolism/catabolism in B. adusta and perhaps, in other white rot fungi. Acknowledgements Funding for this work was provided by the Canadian Chlorine Coordinating Committee (C4) and the Canadian Chemical Producers Association (CCPA). We also acknowledge support from NSERC Canada for an Industrial Post-Doctoral fellowship to one of us (C.A.). We thank Dr. Scott Redhead, Curator of the National Mycological Herbarium (DAOM), Agriculture and Agri-Food Canada, Ottawa, Ontario who provided valuable technical advice and Carolyn Babcock, Curator, CCFC, Agriculture and Agri-Food Canada, Ottawa, who expertly provided many of our cultures. Troy Smith of RPC provided excellent technical assistance. References Brambilla, U., Nasini, G., De Paua, O.V., 1995. Secondary mold metabolites, part 49. Isolation, structural elucidation, and biomimetic synthesis of trametol, a new 1-arylpropane1,2-diol produced by the fungus Trametes sp. J. Natural Products 58, 1251±1253. Cancel, A.M., Orth, A.B., Tien, M., 1993. Lignin and veratryl alcohol are not inducers of the ligninolytic system of Phanerochaete chrysosporium. Appl. Environ. Microbiol. 59, 2909±2913. Crout, D.H.G., Hutchinson, D.W., Miyagoshi, M., 1991. Studies on pyruvate decarboxylase: acyloin formation from aliphatic, aromatic and heterocyclic aldehydes. J. Chem. Soc. Perkin Trans. 1, 1329±1334. deJong, E., Cazemier, A.E., Field, J.A., deBont, J.A.M., 1994a. Physiological role of chlorinated aryl alcohols biosynthesized de novo by the white rot fungus Bjerkandera sp. Strain BOS55. Appl. Environ. Microbiol. 60, 271±279.

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