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Applied and Environmental Microbiology, April 2001, p. 1551-1557, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1551-1557.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Biotransformation of Biphenyl by Paecilomyces
lilacinus and Characterization of Ring Cleavage Products
Manuela
Gesell,1,*
Elke
Hammer,1
Michael
Specht,2
Wittko
Francke,2 and
Frieder
Schauer1
Institut für Mikrobiologie und
Molekularbiologie, Ernst-Moritz-Arndt-Universität, Greifswald
D-17487,1 and Institut für
Organische Chemie, Universität Hamburg, Hamburg
D-20146,2 Germany
Received 30 August 2000/Accepted 25 January 2001
 |
ABSTRACT |
We examined the pathway by which the fungicide biphenyl is
metabolized in the imperfect fungus Paecilomyces lilacinus.
The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and
trihydroxylated metabolites. The fungus could cleave the aromatic
structures, resulting in the transformation of biphenyl via
ortho-substituted dihydroxybiphenyl to six-ring fission
products. All compounds were characterized by gas chromatography-mass
spectroscopy and proton nuclear magnetic resonance spectroscopy. These
compounds include 2-hydroxy-4-phenylmuconic acid and
2-hydroxy-4-(4'-hydroxyphenyl)-muconic acid, which were produced from
3,4-dihydroxybiphenyl and further transformed to the corresponding
lactones 4-phenyl-2-pyrone-6-carboxylic acid and
4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in
large amounts. Two additional ring cleavage products were identified as
(5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and
[5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We
found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.
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INTRODUCTION |
Biphenyl and 2- and
4-hydroxybiphenyl have been used to limit fungal growth on citrus
fruit. Toxic effects to humans after prolonged exposure to air
containing biphenyl and after cutaneous application are known
(14). Some biphenyl derivatives also are suspected
carcinogens and have estrogenic activity (21, 33, 39).
Most research on the degradation of biphenyls has been conducted with
bacteria (3, 11, 24, 36). Fungal transformation of
biphenyl has been studied to determine how this antifungal substance
may be inactivated but, more importantly, as a model of mammalian
biphenyl metabolism. Primary oxidation of biphenyl by yeasts of the
genera Candida and Debaryomyces (5, 22, 34,
44) and by filamentous fungi of the genera Absidia,
Aspergillus, Cunninghamella, Gliocladium, and
Helicostylum (7, 9, 12, 37), yields
monohydroxylated biphenyls, which can undergo a second hydroxylation
step or sugar conjugate formation (5, 7, 9, 12, 34, 37,
44). Usually, the initial hydroxylation favors the 4-position of
biphenyl (34, 37). Induction of biphenyl hydroxylase
activity in Aspergillus by several biphenyl substrates and
also by 4,4'-dihydroxybiphenyl has been reported (28).
Although fungi constitute the majority of microbial biomass in soil,
reports on fungal metabolism of biphenyl are limited to a few yeasts
and filamentous fungi and usually restricted to hydroxylation processes
(9, 34, 37). Since 1993 there have been two reports
suggesting that these organisms may transform hydroxylated biphenyls
and cleave the aromatic rings (22, 27). Each strain
appears to be able to form only a single ring cleavage product,
however, and the overall capacity for ring cleavage seemed low.
Our objective in this study was to determine how the filamentous fungus
Paecilomyces lilacinus transforms the fungicide biphenyl. The ability of this fungus to form a variety of oxidation products including ring cleavage products not previously described in fungi may
be one reason that this fungus appears resistant to high levels of biphenyl.
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MATERIALS AND METHODS |
Chemicals.
Biphenyl and 2-hydroxybiphenyl were purchased
from Merck (Darmstadt, Germany), and 3-hydroxy-, 4-hydroxy-, and
2,5-dihydroxybiphenyl as well as 4,4'-dihydroxybiphenyl were purchased
from Aldrich (Steinheim, Germany). 3,4-Dihydroxybiphenyl was obtained
from Promochem (Wesel, Germany), and 2,3-dihydroxybiphenyl was obtained from Wako Pure Chemical Industries (Neuss, Germany). All chemicals and
solvents were of the highest purity available.
The compounds 2,3,4-trihydroxybiphenyl, 3,4,5-trihydroxybiphenyl,
3,4,4'-trihydroxybiphenyl, 4-phenyl-2-pyrone-6-carboxylic acid, and
2-hydroxy-4-phenylmuconic acid were not commercially available and were
synthesized as follows.
2,3,4-Trihydroxybiphenyl was synthesized by bromination of
1,2,3-trimethoxybenzene (
17). The Grignard reagent formed
from
the resulting 1-bromo-2,3,4-trimethoxy benzene was coupled with
cyclohexanone. The reaction products were dehydrated with oxalic
acid
and treated with
p-chloranil to produce
2,3,4-trimethoxybiphenyl.
Demethylation with AlCl
3 yielded
the corresponding 2,3,4-trihydroxybiphenyl
(
2,
23).
3,4,5-Trihydroxybiphenyl was synthesized by Ullmann coupling of
iodobenzene with 5-iodo-1,2,3-trimethoxybenzene (
10).
Separation
of the 3,4,5-trimethoxybiphenyl from the mixture of
biphenyls
followed by demethylation with AlI
3 yielded the
target product
3,4,5-trihydroxybiphenyl (
1).
3,4,4'-trihydroxybiphenyl was synthesized by coupling of veratrole with
N'-(4-nitrophenyl)-diazenium tetrafluoroborate and
transformation of the nitro group of the resulting compound into
the
corresponding amino derivative by hydrogenation with a PtO
2 catalyst. The
p-substituted aniline was transformed to the
corresponding
phenol via the diazenium salt following conventional
methods (
29).
The 4'-hydroxy-3,4-dimethoxybiphenyl
obtained was demethylated
with pyridine hydrochloride to
trihydroxylated biphenyl (
16,
29).
4-Phenyl-2-pyrone-6-carboxylic acid was synthesized starting from
trans-
-methyl cinnamic acid chloride as outlined by Rey
et al. (
32).
2-Hydroxy-4-phenylmuconic acid was produced photochemically from
4-phenyl-2-pyrone-6-carboxylic acid. Photochemical treatment
of
2-pyrones in methanol results in the formation of bicyclic
lactones
and/or ketenes, which add methanol under the reaction
conditions
(
19). Thus, treatment of 4-phenyl-2-pyrone-6-carboxylic
acid at 300 nm and 15°C yielded 2-hydroxy-4-phenylmuconic acid
in
small
amounts.
Growth and incubation conditions.
P. lilacinus
SBUG-M 1093 was isolated from wood chip piles (31) and has
been deposited in the strain collection of the Department Biology of
Greifswald University (SBUG) (DSMZ accession no. 14052). The strain was
maintained on malt agar slants (3% malt; Serva). For incubation
experiments, mycelium from an agar plate (1 cm2) was
transferred to a 500-ml Erlenmeyer flask with 100 ml of Sabouraud
glucose broth containing 40 g of D-glucose and 10 g of
peptone of casein (Merck) per liter. After growth for 72 h at
30°C and 180 rpm on a rotary shaker, 5 ml of homogenized mycelium (homogenized three times for 2 s each time at 3,000 U/min in an IKA Ultra-Turrax homogenizer [Janke & Kunkel, Stauffen, Germany]) was
used to inoculate 500-ml shake flasks containing 100 ml of a mineral
salts medium (MM) (20) and 1 g of
D-glucose as a carbon source. The slurry was cultivated as
before for another 72 h. Glucose-grown cells were harvested by
centrifugation (at 6000 × g for 5 min) and washed
twice with sterile MM. The pellet was resuspended in 100 ml of
sterilized MM. As substrates, biphenyl, 2-, 3-, or 4-hydroxybiphenyl,
3,4- or 4,4'-dihydroxybiphenyl, 3,4,4'- or 3,4,5-trihydroxybiphenyl,
and 4-phenyl-2-pyrone-6-carboxylic acid were added at concentrations
ranging from 0.05 or 0.1 to 1 mg/ml. Cells in MM without substrates and
biphenyl derivatives in MM were used as controls. All cultures were
tested for microbiological purity.
Chemical analysis and identification of intermediates.
For
the detection and quantification of metabolites in the aqueous culture
supernatant, high-performance liquid chromatography (HPLC) was used. RP
(reversed-phase) analytical HPLC was performed on a Hewlett-Packard
(Bad Homburg, Germany) 1050 M HPLC apparatus equipped with a quaternary
pump system, a 1040 M series 1 diode array detector, and a
Hewlett-Packard Chemstation. Gradient elution mode was used for
separation in a mobile phase consisting of methanol and 0.1% (wt/vol)
(10.2 mM) phosphoric acid starting from an initial ratio of 20%
methanol and reaching 100% methanol in 14 min. The flow rate was 1 ml/min; detection wavelengths were 220 nm (UV) and 254 and 280 nm.
Data are reported as means for two separate experiments with replicated
batch cultures. Standard deviation was no more than
7%.
Purification of intermediates was carried out on a Merck-Hitachi HPLC
(Merck) system equipped with a model L 6200 A Intelligent
Pump, a
Rheodyne 7161 injection valve with a 100-µl loop, and
a model L-4250
absorbance detector operating at 254 nm. Gradient
elution mode was used
for separation in a mobile phase consisting
of methanol and 0.1%
(wt/vol) (16.7 mM) acetic acid starting from
an initial ratio of 20%
methanol and reaching 60% methanol in
18.5 min, at a flow rate of 1 ml/min. For both HPLC systems a
LiChroCart 125-4 RP-18 end-capped
(5-µm) column (Merck) was
used.
For gas chromatographic analysis, whole cultures were lyophilized using
an Alpha 1-4 apparatus (Christ, Osterode, Germany).
The dried samples
were resolved in 10 ml of methanol, centrifuged
(at 5,000 ×
g for 5 min), and evaporated under a vacuum at 40°C.
The
methanol extracts were examined by gas chromatography-mass
spectroscopy
(GC-MS). The GC-MS system consisted of a GC 8000
gas chromatograph
(Fisons, Mainz, Germany) and a Fisons MD 800
mass spectrometer
operating at 70 eV. Helium was used as the carrier
gas at a flow rate
of 1.9 ml/min. Separation was carried out on
a 30-m BPX 5 column
(0.25-mm by, 0.33-µm film; SGE, Weiterstadt,
Germany) at a
temperature program from 80 to 300°C (10°C/min).
High-resolution
mass spectra were recorded on a Vacuum Generators
(Manchester, United
Kingdom) analytical instrument, model 70-250
SE.
Derivatization was achieved by methylation with diazomethane as
described by De Boer and Backer (
8) in a microapparatus
(Z
10,100-1; Aldrich-Chemie).
Proton nuclear magnetic resonance (
1H NMR) spectroscopy was
carried out on a Bruker (Karlsruhe, Germany) ARX 300-MHz spectrometer
or on a Bruker DRX500 instrument at 500 MHz. Deuterated methanol
(99.99%) was used as a solvent and tetramethyl silane was used
as a
reference.
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RESULTS |
We identified 14 metabolites formed by P. lilacinus
SBUG-M 1093 from biphenyl and selected biphenyl derivatives in aqueous supernatants or in methanol extracts obtained from lyophilized whole
cultures (Table 1). After extraction of
supernatants with ethyl acetate, the same pattern of metabolites was
found. Seven mono- and dihydroxylated biphenyls were identified by
comparison of UV and mass spectra with those of authentic standards.
For some additional metabolites (products A through G), standards were
not available. These products were characterized by GC-MS, by
1H NMR, and (partially) by comparison with literature data
(products B and G).
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TABLE 1.
Formation of hydroxylated biphenyls and ring cleavage
products after incubation of P. lilacinus SBUG-M 1093 with
biphenyl, several hydroxylated biphenyls, and a synthesized ring
cleavage product
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Formation of hydroxylated biphenyls.
P. lilacinus
strain SBUG-M 1093 can tolerate biphenyl at concentrations up to 1 mg/ml, accumulating 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl.
During incubations with a lower concentration of biphenyl (0.1 mg/ml),
4,4'-dihydroxybiphenyl was formed as the major metabolite (Fig.
1). 2- and 4-Hydroxybiphenyl and an additional product (product A) were detected in smaller amounts by
HPLC. After 24 to 96 h of incubation, concentrations of
4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl increased. In contrast,
the concentration of 2-hydroxybiphenyl increased slowly and at a
constant rate (Fig. 1).
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FIG. 1.
Time-dependent formation of hydroxylated products from
biphenyl by glucose-grown cells of P. lilacinus SBUG-M 1093 during incubation with biphenyl (0.1 mg/ml). Symbols: *,
2-hydroxybiphenyl;  , 4-hydroxybiphenyl;  ,4,4'-dihydroxybiphenyl;
×, product A.
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Initial oxidation of biphenyl leading to 4-hydroxybiphenyl,
2-hydroxybiphenyl, and traces of 3-hydroxybiphenyl was monitored
by
HPLC (for 4-hydroxybiphenyl,
max = 200, 262 nm; for
2-hydroxybiphenyl;
max = 248, 276 nm; for
3-hydroxybiphenyl,
max = 206, 252, 290
nm) and
GC-MS analysis [
m/z 170 (C
12H
10O
+, M
+), 141 (C
11H
9+, M
+-CHO), 139 (C
11H
7+,
M
+-CH
3O), 115 (C
9H
7+)] in comparison with
synthetic standards. Mass spectral data
[
m/z 186 (C
12H
10O
2+,
M
+) 157 (C
11H
9O
+,
M
+-CHO), 128 (C
10H
8+, M
+-2CHO), 115 (C
9H
7+), 77 (C
6H
5+)] of the main product
after incubation with biphenyl (0.1 mg/ml
[Fig.
1]) were consistent
with those for an authentic standard
of 4,4'-dihydroxybiphenyl.
When
P. lilacinus SBUG-M 1093 was incubated with
2-hydroxybiphenyl, 3-hydroxybiphenyl, and 4-hydroxybiphenyl, the
cultures
accumulated dihydroxylated derivatives of biphenyl, recognized
by the fragmentation pattern in their mass spectra after GC
separation
[
m/z 186 (C
12H
10O
2+,
M
+), 157 (C
11H
9O
+,
M
+-CHO), 128 (C
10H
8+,
M
+-C
2H
2O
2), 115 (C
9H
7+,
M
+-C
3H
3O
2), 77 (C
6H
5+,
M
+-C
6H
5O
2)].
Incubation of 2-hydroxybiphenyl led to the formation
of 2,3- and
2,5-dihydroxybiphenyl; 3-hydroxybiphenyl was transformed
to
3,4-dihydroxybiphenyl; and 4-hydroxybiphenyl was transformed
to
4,4'-dihydroxybiphenyl. Structural determinations were made
following
comparison of
Rf-values (HPLC), UV spectra, and
GC-MS
retention times with the data of authentic
standards.
GC-MS analyses of a methylated methanol extract of the lyophilized
culture supernatant suggest the presence of an additional
hydroxylated
product (named product A) in cultures incubated with
biphenyl (Fig.
1),
3,4-dihydroxybiphenyl (Fig.
2), and
4,4'-dihydroxybiphenyl.
The molecular ion peak of the methyl derivative
of this compound
at
m/z 244 and those of the main fragments
at
m/z 229 (C
14H
13O
3+,
M
+-CH
3), 201 (C
13H
13O
2+,
M
+-COCH
3), and 115 (C
9H
7+) were consistent with data
for trihydroxylated biphenyl. In accordance
with this assumption,
high-resolution mass spectrometry showed
the molecular formula to be
C
15H
16O
3 (mass found, 244.109138;
mass calculated, 244.109945). By comparing the GC-MS data with
those of
the synthesized 2,3,4-, 3,4,5-, and 3,4,4'-trihydroxybiphenyls
and by
coinjection of the methylated standards, product A was
identified as
3,4,4'-trihydroxybiphenyl.
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FIG. 2.
Formation of products by glucose-grown cells of P. lilacinus SBUG-M 1093 during incubation with 3,4-dihydroxybiphenyl
(0.05 mg/ml). Symbols:  , product A; *, product B;  , product C;
, product F;  , product G;  ,3,4-dihydroxybiphenyl
(3,4-DiOHBP).
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Formation of ring cleavage products.
In addition to the
hydroxylated biphenyl derivatives, further products (products B and C)
were enriched in the supernatant after incubation with biphenyl (0.1 mg/ml). When fungal cells were incubated with hydroxylated
intermediates (Table 1), it became clear that products B and C were
formed from 3- and 4-hydroxybiphenyl as well as from
3,4-dihydroxybiphenyl. When 3,4-dihydroxybiphenyl was used as the
substrate, additional metabolites (products D through G) were detected
by HPLC (Fig. 3) or GC-MS analyses.
Metabolites B, C, F, and G have HPLC retention values that suggested
these compounds were acids. Consequently, methylation of the extracted compounds was necessary for GC-MS analysis.
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FIG. 3.
HPLC elution profile of the aqueous supernatant after
incubation of P. lilacinus SBUG-M 1093 with
3,4-dihydroxybiphenyl (tR = 8.57 min; 0.1 mg/ml; 7 days) and UV absorption spectra of products B
(tR = 7.86 min), C
(tR = 6.28 min), F
(tR = 5.52 min), and G
(tR = 6.93 min).
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Metabolite B was identified as 4-phenyl-2-pyrone-6-carboxylic acid by
GC-MS and
1H NMR analysis. The methyl derivative of this
metabolite showed
a molecular ion peak at
m/z 230 (C
13H
10O
4) and main fragment ions
at
m/z 202 (C
12H
10O
3+,
M
+-CO), 171 (C
11H
7O
2+,
M
+-COOCH
3), and 115 (C
9H
7+,
M
+-COOCH
3-2CO) in GC-MS. The mass spectrum
(Fig.
4) and
1H NMR data
[6.83 (d, J
3,5 = 1.7 Hz, 1H, 5-H), 7.54 (m,
J
2'4',4'6' = 2.1, J
3'4',4'5' = 8.6 Hz, 1H,
4'-H)
7.55 (m, J
3'4',4'5' = 8.6 Hz, 2H, 3',
5'-H),
7.57 (d, J
3,5 = 1.7 Hz, 1H, 3-H), 7.78 (dd,
J
2'4',4'6' = 2.1, 2H, 2', 6'-H) ppm] were consistent with
those of a sample
of 4-phenyl-2-pyrone-6-carboxylic acid, synthesized
for reference.
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FIG. 4.
Mass spectra of methylated products B
(4-phenyl-2-pyrone-6-carboxylic acid) (top) and C
[4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid] (bottom).
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Metabolite C, the main product resulting from transformation of
3,4-dihydroxybiphenyl (Fig.
2), also was accumulated during
incubation
of
P. lilacinus with 4,4'-dihydroxybiphenyl,
3,4,5-trihydroxybiphenyl,
3,4,4'-trihydroxybiphenyl, and
4-phenyl-2-pyrone-6-carboxylic
acid (Fig.
5). On the basis of the MS and
1H NMR data, the formation of this compound from different
p,p'-hydroxylated
biphenyls, and its direct formation by
hydroxylation of 4-phenyl-2-pyrone-6-carboxylic
acid (Table
1), the
structure of metabolite C was determined
to be
4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. The mass
spectral data
of the methylated compound (Fig.
4) showed a molecular
ion at
m/z 260 and fragment ions at
m/z 232 (C
13H
12O
4, M
+-CO), 201 (C
12H
9O
3,
M
+-COOCH
3), and 145 (C
10H
9O,
M
+-COOCH
3-2CO). High-resolution mass
spectrometry indicated that
the molecular formula of product C was
C
14H
12O
5 after methylation.
The
1H NMR data (500 MHz) obtained after separation of the
lyophilized
extract by preparative HPLC were as follows:
6.70 (s,
1H, 5-H),
6.95 (d, 2H, 3',5'-H), 7.57 (s, 1H, 3-H), 7.70 (d, 2H,
2',6'-H)
ppm. The
1H NMR spectrum showed two signals with
single intensity and two
signals with double intensity, pointing to six
protons. The doublet
signals at
= 6.95 and 7.70 ppm with a
coupling constant of J
2'3';5'6' = 8.7 Hz indicate a
phenyl ring with a hydroxyl group in the
para position due
to two pairs of protons in
ortho positions of the
aromatic
ring.
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FIG. 5.
Formation of product C
[4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid] after incubation of
glucose-grown cells of P. lilacinus SBUG-M 1093 with
different biphenyl derivatives (BP, biphenyl; OHBP, hydroxybiphenyl;
DiOHBP, dihydroxybiphenyl; TriOHBP, trihydroxybiphenyl; PPCA,
4-phenyl-2-pyrone-6-carboxylic acid).
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Metabolites D and E, formed from 3,4-dihydroxybiphenyl, were detected
only in the methanol extract by GC-MS. Product D was
identified as
2-hydroxy-4-phenylmuconic acid as follows. According
to high-resolution
mass spectrometry analysis, the molecular ion
peak
m/z 276 of the methylated intermediate D had the molecular
formula
C
15H
16O
5 (mass found, 276.101303;
mass calculated, 276.099774).
The base peak at
m/z 217, assigned to C
13H
13O
3, resulted in
the
loss of COOCH
3 from the molecular ion. Further fragment
ions at
m/z 261 (C
14H
13O
5+,
M
+-CH
3), 245 (C
14H
13O
4+,
M
+-OCH
3), 202 (C
12H
10O
3+,
M
+-COOCH
3-CH
3), 185 (C
12H
9O
2+,
M
+-COOCH
3-CH
4O), and 59 (COOCH
3+) correspond with a structure
containing one or two methyl esters
and one methoxy function,
indicating that intermediate D is a
hydroxylated (di)carboxylic acid.
This compound was identical
to a sample of 2-hydroxy-4-phenylmuconic
acid, derived from the
photochemical treatment of
4-phenyl-2-pyrone-6-carboxylic acid,
by GC-MS analysis. This structural
assignment was further corroborated
by the fact that products B and D
can be chemically
interconverted.
Product E, formed from 3,4-dihydroxybiphenyl and
4-phenyl-2-pyrone-6-carboxylic acid (Table
1), was identified as
2-hydroxy-4-(4'-hydroxyphenyl)-muconic
acid. After methylation, the
mass spectrum of this compound showed
a molecular ion peak at
m/z 306 and the main fragment ions at
m/z 275 (C
15H
15O
5+,
M
+-OCH
3), 247 (C
14H
15O
4+,
M
+-COOH), 232 (C
13H
12O
4+,
M
+-COOCH
3-CH
3), 145 (C
10H
9O
+), and 59 (COOCH
3+). The similarities in the
fragmentation pattern to that of metabolite
D and the difference of
m/z 30 between the distinctive fragments
of both metabolites
suggests, a doubly hydroxylated muconic acid
with one hydroxy group at
the
para position of the aromatic ring
system.
Metabolite F, formed after incubation with 3,4-dihydroxybiphenyl (Fig.
2 and
3), 4,4'-dihydroxybiphenyl, and 3,4,4'-trihydroxybiphenyl,
was
separated by HPLC and identified by GC-MS as well as by
1H
NMR as [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic
acid. The molecular ion peak of the methylated compound and the
fragment ions were detected at
m/z 262 (C
14H
14O
5, M
+), 234 (C
13H
14O
4+,
M
+-CO), 189 (C
11H
9O
3+,
M
+-CH
2COOCH
3), 161 (C
10H
9O
2+,
M
+-CH
2COOCH
3-CO), 133 (C
9H
9O
+,
M
+-CH
2COOCH
3-2CO), and 132 (C
9H
8O
+). The
1H NMR
data were as follows:
2.38 (dd, J
1,2 = 16.4 Hz,
J
1, 3 = 9.5 Hz, 1H, 1-H), 2.96 (dd, J
1,2 = 16.4 Hz, J
2,3 = 2.7 Hz, 1H,
2-H), 5.95 (ddd,
J
1,3 = 9.3 Hz, J
2,3 = 2.7 Hz,
J
3,4 = 1.4 Hz,
1H, 3-H), 6.27 (d, J
3,4 = 1.4 Hz, 1H, 4-H), 6.92 (m, 2H, 2',6-H),
7.52 (m, 2H, 3'5'-H)
ppm.
Metabolite G resulted from the degradation of 3,4-dihydroxybiphenyl
(Fig.
2 and
3) to (3-phenyl-5-oxo-2,5-dihydrofuran-2-yl)-acetic
acid.
The mass spectrum of the methylated compound showed the
molecular
ion peak at
m/z 232 (C
13H
12O
4) and the main fragment
ions at
m/z 204 (C
12H
12O
3+,
M
+-CO), 159 (C
10H
7O
2+,
M
+-CH
2COOCH
3), 131 (C
9H
7O
+,
M
+-CH
2COOCH
3-CO), 103 (C
8H
7+,
M
+-CH
2COOCH
3-2CO), and 102 (C
8H
6+). Four signals in the
1H NMR spectrum [
2.38 (dd, J
1,2 = 16.2 Hz, J
1,3 = 9.3 Hz, 1H,
1-H), 2.92 (dd,
J
1,2 = 16.2 Hz, J
2,3 = 2.8 Hz, 1H,
2-H), 6.04
(ddd, J
1,3 = 9.3 Hz, J
2,3 = 2.8 Hz, J
3,4 = 1.5 Hz, 1H, 3-H), 6.45
(d,
J
3,4 = 1,5 Hz, 1H, 4-H) ppm] showed a system of
nonaromatic
protons similar to that concluded from the
1H
NMR spectrum of compound F. In contrast, the aromatic proton
signals at
7.49 (d, 1H, 4'-H), 7.51 (m, 2H, 2',6-H), and 7.65
(m, 2H, 3'5'-H)
ppm indicated a monosubstituted and hence unhydroxylated
aromatic ring
system. HPLC, GC-MS, and
1H NMR spectra, corresponded to a
metabolite isolated and characterized
after incubation of
Trichosporon mucoides SBUG-M 801, a yeast
that accumulates
large amounts of intermediate G after incubation
with
3,4-dihydroxybiphenyl (R. Sietmann et al., unpublished
data).
| |
DISCUSSION |
P. lilacinus SBUG-M 1093 can metabolize biphenyl to
monohydroxylated biphenyls (2-, 3-, and 4-hydroxybiphenyl), suggesting the action of monooxygenases during the initial oxidation step. Similar
initial oxidation during transformation of biphenyl is known for yeasts
of the genera Candida (5) and
Debaryomyces (22) and for genera of the
filamentous fungi Absidia, Aspergillus, Cunninghamella,
Gliocladium, and Helicostylum (9, 12, 37). Secondary hydroxylation occurs on the first hydroxylated aromatic ring
to produce 2,3-, 2,5-, and 3,4-dihydroxybiphenyl (4, 22). Furthermore, Mobley et al. (27) have reported the
formation of 3,4-catechols of substituted biphenyls. As reported for
several fungi, P. lilacinus favors the initial hydroxylation
in the 4-position, yielding 4-hydroxybiphenyl, and the second
hydroxylation in the 4'-positions, yielding 4,4'-dihydroxybiphenyl.
This fungus also can perform a third hydroxylation of biphenyl to
produce 3,4,4'-trihydroxybiphenyl. These results suggest that primary
oxidation steps leading to monohydroxylated and dihydroxylated
biphenyls and to 3,4,4'-trihydroxybiphenyl are similar to the metabolic
conversion of biphenyl in mammals (25, 26, 38).
We detected two new ring fission products,
2-hydroxy-4-phenylmuconic acid and
2-hydroxy-4-(4'-hydroxyphenyl)muconic acid, as intermediates
during degradation of 3,4-dihydroxybiphenyl by P. lilacinus.
Formation of similar intermediates with a 2-hydroxy muconic acid
structure was reported for degradation of phloroglucinol by
Fusarium solani (43), of gallic acid by
Aspergillus flavus (13), of dibenzofuran by
T. mucoides (15), and of diphenyl ether by
Trametes versicolor (18).
Presumably ring cleavage of the aromatic structure can take place after
introduction of a third hydroxyl group into the dihydroxylated aromatic
ring (22). After incubation with 3,4,5-trihydroxybiphenyl, only the lactone structure 4-phenyl-2-pyrone-6-carboxylic acid was
detected. The formation of 2-hydroxy-4-phenylmuconic acid is possible
but not detectable after this incubation.
After incubation with 3,4-dihydroxybiphenyl, lactonic structures
were prominent products (Fig. 2). However,
4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid also was
formed as a result of further hydroxylation of
4-phenyl-2-pyrone-6-carboxylic acid. The enrichment of further lactonic
structures, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid and
[5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid,
was demonstrated. The latter product also was found after incubation of Aspergillus parasiticus with biphenyl and
4,4'-dihydroxybiphenyl (27). In this case the authors
suggested that each biphenyl ring must be para-hydroxylated
as a prerequisite for lactone formation. In contrast, in P. lilacinus a muconolactone structure,
(5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, was formed
from 3,4-dihydroxybiphenyl after hydroxylation of one of the aromatic
ring systems followed by ring cleavage. Lactonization of
substituted and unsubstituted muconic acids yielding corresponding muconolactone structures was reported by nonenzymatic isomerization under acidic conditions (30) as well as by
the action of muconate cycloisomerases from various bacteria (40, 41, 42).
The accumulation of 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid and
[5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid and the
formation of 2-hydroxy-4-(4'-hydroxyphenyl)muconic acid suggest that
P. lilacinus can further transform ring cleavage products B,
D, and G. On the other hand, the trihydroxylated intermediate (3,4,4'-trihydroxybiphenyl) was cleaved by this filamentous fungus, leading directly to the hydroxylated lactone structures (Fig. 6).
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|
FIG. 6.
Proposed pathway for the initial steps of biphenyl
degradation in P. lilacinus SBUG-M 1093.  , transformation
of P. lilacinus; -- proposed
transformation step, but intermediate not observed.
|
|
The accumulation of hydroxyphenyl lactones suggests that they are
deadend products in the pathway of the metabolism of biphenyl and
substituted biphenyls. Comparison of the toxicity of hydroxylated biphenyls and the lactonic structures formed, using the method of
Singer-Bohne et al. (35), pointed to detoxification of
hydroxylated biphenyls by ring cleavage accompanied by lactone formation.
The results show that P. lilacinus oxidizes biphenyl to a
broad variety of products that were not previously known from
filamentous fungi. Six different ring cleavage products were
identified. Their structures indicate a ring fission mechanism going
via di- and trihydroxylated intermediates to muconic acid structures
which can lactonize. Since the same ring fission mechanism had been described for Aspergillus (27) and
Debaryomyces (22)
genera that are not related
to Paecilomycesthe strain-specific ability of this feature
seems to be widely distributed among different fungal species. The
ability to produce di- or trihydroxylated biphenyls which can undergo
ring fission to nontoxic products may be the reason for the resistance
of several filamentous fungi to the fungicide biphenyl.
| |
ACKNOWLEDGMENTS |
This study was supported by the Deutsche Bundesstiftung Umwelt.
We thank M. Kindermann and S. Siegert (Institute of Chemistry and
Biochemistry, University of Greifswald) for recording NMR data, S. Franke (Institute of Organic Chemistry, University of Hamburg) for
acquiring high-resolution mass spectrometry data, and R. Jack
(Institute of Immunology, University of Greifswald) for revising the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Ernst-Moritz-Arndt-Universität Greifswald, Institut für
Mikrobiologie und Molekularbiologie, F.-L.-Jahn-Str. 15, D-17487
Greifswald, Germany. Phone: 49-3834-864225. Fax: 49-3834-864202. E-mail: gesell{at}biologie.uni-greifswald.de.
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Applied and Environmental Microbiology, April 2001, p. 1551-1557, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1551-1557.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
