Chlorometabolite production by the ecologically important white rot fungus Bjerkandera adusta

Article abstract of DOI:10.1016/S0045-6535(00)00537-3

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 configurations. 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 ¹³C6₆- and ¹³C₉-L-phenylalanine showed that all identified aromatic compounds (with the exception of phenol) can be derived from L-phenylalanine. For the aryl propane diols, the ¹³C 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 Na³⁷Cl showed incorporation of ³⁷Cl in all identified chlorometabolites. Veratryl alcohol and the CAM alcohols, which occur in both strains and can be derived from L-phenylalanine (all ¹³C-labelled), have reported important physiological functions in this white rot fungus. Possible mechanisms for their formation through the newly discovered compounds are discussed.

Full text of DOI:10.1016/S0045-6535(00)00537-3

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Chemosphere 44 /2001) 1603±1616
www.elsevier.com/locate/chemosphere
Chlorometabolite production by the ecologically important
white rot fungus Bjerkandera adusta
a,
b
a
a
P.J. Silk *, C. Aubry , G.C. Lonergan , 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®gura-
tions. 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 13C6- and
13C9-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 13C 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 Na37Cl showed incorporation of 37Cl in all identi®ed chlorometabolites.
Veratryl alcohol and the CAM alcohols, which occur in both strains and can be derived from L-phenylalanine /all
13C-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
Gribble, 1988). Both natural and anthropogenic or-
ganohalogens 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 organochlo-
rine metabolites produced have important physiological
roles as methyl donors, antibiotics and as substrates for
H2O2-generating oxidases, for e.g., and are ``not bio-
logical accidents'' /deJong and Field, 1997). Halome-
thanes, halogenated aromatics and haloaliphatic
compounds have all been documented to occur in fungal
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 /Nei-
dleman and Geigert, 1986; Grimvall and de Leer, 1995;
* Corresponding author. Tel.: +1-506-452-1366; fax: +1-506-
452-1395.
E-mail address: psilk@rpc.unb.ca /P.J. Silk).
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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P.J. Silk et al. / Chemosphere 44 52001) 1603±1616
culture and in the environment /de Jong et al.,
1994a,b,c).
3-chloro-4-hydroxy benzoates stimulate aryl metabolite
production and lead to deuterated CAM metabolites
/Mester et al., 1997).
Of particular interest to us are the chlorinated aro-
matics of fungal origin such as chlorinated anisyl
/CAM's), chlorinated hydroquinone /CHM's) and
chlorinated orcinol /COM's) metabolites. These natu-
rally occurring chlorinated aromatic methyl ethers can
®nd their way into environmental compartments where
they can, on microbial demethylation, become incor-
porated into humus /deJong et al., 1994c) or by /oxi-
dative) coupling, potentially form chlorinated dioxins
and furans /Silk et al., 1997; Gribble, 1994, 1998).
Among the ecologically signi®cant group of organ-
isms 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 alco-
hols, aldehydes and acids /CAM's). Veratryl, anisyl, 3-
chloroanisyl 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 phenylala-
nine is added to cultures of Bjerkandera sp. /Mester et al.,
1997). Hydroxylation and methylation have been pro-
posed 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 en-
zymes have not been detected in the extracellular ¯uids
of basidiomycetes. It has, however, recently been sug-
gested that NADH-dependent halogenases are more
likely to be involved in halometabolite biosynthesis than
haloperoxidases /Hohaus et al., 1997).
The objectives of this present study were to determine
if aryl metabolites in B. adusta originate through phe-
nylalanine, to attempt to determine how and when
chlorination takes place and to identify any new me-
tabolites. 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 chlorom-
etabolites 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 /13C9, 97±98%, CLM-2250), L-phe-
nylalanine /ring-13C6, 99%, CLM-1055), Na 37Cl /95%,
CHLM-1225) and sodium pyruvate /2,3-13C2, 99%,
CLM-3507) were obtained from Cambridge Isotope
Laboratories /CIL), and used without further puri®ca-
tion 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 /Agri-
culture 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 asepti-
cally taken from the edge of the fungal growth.
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 benzal-
dehyde 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 phenylprop-
anoid pathway from aromatic amino acids since exoge-
nous tyrosine increases their production in culture
/Spinnler et al., 1994) and deuterated 4-hydroxy and
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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P.J. Silk et al. / Chemosphere 44 52001) 1603±1616
1605
as follows: 21.7 g
yeast extract, 1:2 g KH2SO4; 0:5 g MgSO4; 0.08 g NaCl
[4] or 10.8 g /+) glucose, 2.2 g peptone, 2.2 g yeast
D
/+) glucose, 5.5 g peptone, 2.2 g
3-chloro-4-methoxy benzaldehyde and 3,5-dichloro-4-
methoxy benzaldehyde. The former two aldehydes
were commercially available, while the latter two were
synthesized from commercial 3-chloro- and 3,5-di-
chloro-4-hydroxy benzoic acids by methylation /dia-
zomethane), 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 glu-
cose 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 sub-
jected to GC/MS analysis either as direct extracts or as
acetylated derivatives.
D
extract, 2:2 g KH2PO4; 1:1 g MgSO4, 1.1 mg thiamine
hydrochloride /Tien and Kirk, 1988). Low-nitrogen
media was 20 g glucose, 1:0 g KH2SO4; 0:5 g MgSO4
and 0.07 g NaCl. Aliquots of 200 ml were placed in 500
ml erlenmeyer ¯asks and autoclaved /thiamine was ®lter-
sterilized). The media were inoculated and kept in the
dark at ca. 25°C for up to 13.5 weeks without shaking.
Na37Cl-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-13C6 or 13C9-L-phe-
nylalanine + 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 2,3-13C2-pyruvate,
sodium) in the presence of thiamine pyrophosphate /co-
carboxylase) and L-phenylalanine and eight week cul-
tures were extracted and analysed.
2.6. Derivatization techniques
Acetylation was carried out by treatment with acetic
anhydride /solvent)/pyridine at room temperature over-
night followed by evaporation under dry nitrogen, dis-
solution in ethyl acetate followed by GC/MS analysis.
Portions of acetylated extracts were also treated directly
with ethereal diazomethane and re-analysed. Other ex-
tracts were treated with Sylon BTZ /Supelco) or directly
with diazomethane prior to analysis.
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 ex-
tracted 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 Na2SO4, the solvent
evaporated and the extract analysed by GC/MS directly
or as derivatized extracts as noted.
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-cyclodex-
trin 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 SPB-
35 poly/35% phenyl/65% dimethylsiloxane)); this col-
umn 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.
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-1,2-
diols necessitated synthesis of these compounds as ana-
lytical standards for structural elucidation. We
employed the biomimetic ability of fermenting Bakers'
yeast, Saccharomyces cerevisiae to produce a-ketols /a-
cyloins and 1-acylethanols) and diols from aromatic al-
dehydes /Ohta et al., 1986; Brambilla et al., 1995) with
high diastereo/enantioselectivity. This reductive C2-ho-
mologation 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 decar-
boxylase /PDC) having thiamine pyrophosphate as
cofactor /PDC, 2-oxo-acid carboxyl lyase EC 4.1.1.1)
/Crout et al., 1991).
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 synthe-
ses or synthetic material prepared in our laboratory /as
noted in the text) to give a high probability of the correct
structural assignments.
The aromatic aldehydes employed as reaction sub-
strates in our work were benzaldehyde, p-anisaldehyde,

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P.J. Silk et al. / Chemosphere 44 52001) 1603±1616
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
were more prevalent than dichloro CAM's in the
DAOM 215869 strain. The major strain di€erences no-
ted 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).
strains of B. adusta produced an array of phenyl, anisyl,
veratryl and chloroanisyl metabolites as previously re-
ported /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 L-
phenylalanine. CAM production, in particular /struc-
turally and quantitatively), by both fungal strains of
B. adusta, in static liquid culture, con®rmed the litera-
ture evidence to date /deJong and Field, 1997) and also
supported the enhanced production of CAM metabo-
lites 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 cul-
ture, only trace quantities of some of the previously
identi®ed compounds were detected /primarily aldehy-
des).
In addition to the CAM's, we have also found
other important metabolites in both strains. Of par-
ticular 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-a-
ketols /1-acyl ethanols) and 1-aryl-propan-1,2-diols
/shown as the di-acetates 8,14,15,18). 1-anisyl-propan-
1,2-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®ca-
tion 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 pro-
posed fragmentation pathway and noted in the lower
table of Fig. 4.
Structures /Fig. 2) of phenyl and anisyl compounds
and mono- and dichlorinated CAM's /3 and 3,5-chlo-
roanisyl 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