Biosynthesis of Stereoisomers of Dill Ether and Wine Lactone by Pleurotus sapidus

Article abstract of DOI:10.1021/acs.jafc.8b07263

The white-rot fungus Pleurotus sapidus (PSA) biosynthesizes the bicyclic monoterpenoids 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran (dill ether) (1) and 3,6-dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3H)-one (wine lactone) (2). Submerged cultures gro

Full text of DOI:10.1021/acs.jafc.8b07263

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Chemistry and Biology of Aroma and Taste
Biosynthesis of Stereoisomers of Dill Ether
and Wine Lactone by Pleurotus sapidus
Tobias Trapp, Tabea Kirchner, Florian Birk, Marco Alexander Fraatz, and Holger Zorn
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07263 • Publication Date (Web): 28 Feb 2019
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Journal of Agricultural and Food Chemistry
Biosynthesis of Stereoisomers of Dill Ether and Wine Lactone by Pleurotus sapidus
Tobias Trapp,† Tabea Kirchner,† Florian Birk,† Marco Alexander Fraatz,† Holger Zorn*,†,‡
†
Justus Liebig University Giessen, Institute of Food Chemistry and Food Biotechnology,
Heinrich-Buff-Ring 17, 35392 Giessen, Germany
‡
Fraunhofer Institute for Molecular Biology and Applied Ecology, Winchester Str. 2, 35394
Giessen, Germany
* Corresponding author (Tel: +49 641 99-34900; Fax: +49 641 99-34909; E-mail:
holger.zorn@uni-giessen.de)
ORCID
Tobias Trapp:
0000-0001-7059-0286
0000-0001-9353-3145
0000-0002-5028-9653
0000-0002-8383-8196
Florian Birk:
Marco Alexander Fraatz:
Holger Zorn:
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ABSTRACT: The white-rot fungus Pleurotus sapidus (PSA) biosynthesizes the bicyclic
monoterpenoids 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran (dill ether) 1 and 3,6-
dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3H)-one (wine lactone) 2. Submerged cultures
grown in different media were analyzed by gas chromatography-mass spectrometry (GC-MS).
The stereochemistry of the formed isomers was elucidated by comparing their retention
indices with those of reference compounds by enantioselective-multidimensional GC. The
basidiomycete produced the rare (3R,3aR,7aS)- and (3S,3aR,7aS)-stereoisomers of dill ether
and wine lactone. Kinetic analyses of the volatilome and bioprocess parameters revealed that
the biosynthesis of the bicyclic monoterpenoids correlated with the availability of the primary
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carbon source glucose. Spiking the media with C-labelled glucose demonstrated that the
compounds were produced de novo. Supplementation studies i.a. with isotopically labeled
substrates further identified limonene and p-menth-1-en-9-ol as intermediate compounds in
the fungal pathways. PSA was able to biotransform all enantiomeric forms of the latter
compounds to the respective isomers of dill ether and wine lactone.
KEYWORDS: Pleurotus sapidus, submerged cultivation, dill ether, wine lactone, p-menth-1-
en-9-ol, isotopic labeling studies, biotransformation
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INTRODUCTION
The basidiomycete Pleurotus sapidus (PSA) has been well investigated with respect to its
enzymatic activities and its specific capability to biosynthesize aroma compounds. Examples
include the biotransformation of myrcene, limonene, pinene, valencene, β-ionone, and
spirocyclic terpenoids.1–7 In a more recent study, the overall composition of odor-active
compounds of PSA cultivated submerged in a complex medium was analyzed for the first
time. For this purpose, a non-targeted approach of aroma dilution analyses by means of
headspace-solid phase microextraction (HS-SPME) and stir bar sorptive extraction (SBSE)
was performed.8 The aroma profile was strongly defined by arylic compounds, such as p-
anisaldehyde and its derivatives, which were previously described for other Pleurotus ssp.9,10
An interesting observation was the identification of several bicyclic monoterpenoid
compounds including odor-active isomers of 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran
(dill ether) 1 and 3,6-dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3H)-one (wine lactone) 2.
These compounds had not been described for PSA in previous publications, in which typically
chemically defined media and liquid-liquid extraction (LLE) were applied, nor for any other
basidiomycete. To the best of our knowledge, the only example of microbial formation of
these benzofuran derivatives was the bioconversion of nerol to isomers of 1 by the
ascomycete Botrytis cinerea.11
The structure of 1 comprises three chiral centers, resulting in a total theoretical number of
eight stereoisomers. The trans-isomers with an anti-ring connection are considered less
favorable from a stereochemical point of view12,13 and are thus not expected to be formed via
natural pathways. Among the cis-isomers, 1B–1D are assigned to rather unspecific fruity,
herbaceous, flowery or tarragon-like odor impressions, whereas 1A imparts a characteristic
dill-like odor.12,14–16 The latter isomer was also identified as a key aroma compound in dill,
herein occurring in enantiopure form.12,14 Other natural sources of dill ether isomers are e.g.
certain honey varieties.15,17 With respect to potential biosynthetic pathways, an acid-catalyzed
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degradation of 8-hydroxygeraniol resulting in isomers of 1 was demonstrated.18 However, this
reaction occurred only under harsh thermal and acidic conditions and did not proceed in a
stereoselective manner, so that this mechanism was considered highly unlikely to occur in
dill.19 Feeding experiments with stable isotopically labeled precursors later elucidated the
metabolic formation of enantiopure 1A in dill plants: (4R)-limonene 3A is hydrated to
(4R,8S)-p-menth-1-en-9-ol 4A, which was stereospecifically oxidized at the allylic position to
p-menth-1-en-3,9-diol. It was proposed that the latter compound cyclized spontaneously to
1A.20 A derivative of the mentioned diol, 9-hydroxy-piperitone α-D-glucopyranoside, was
isolated from dill, which further confirmed this pathway.19
The structural similarities of lactone 2 with the ether 1 are based on the same stereochemical
configuration. All eight stereoisomers of 2 were assigned to similar coconut-like smells,
whereof 2A possesses by far the lowest odor threshold.21 2A was identified in enantiopure
form in white wine varieties as key aroma compound.22 In grape berries, the intermediate
compound linalool is converted to (E)-8-carboxylinalool via multi-step catalysis by a
cytochrome P450 enzyme.23,24 The latter compound cyclizes in a slow, acid-catalyzed reaction
in a stereoselective manner to 2A during wine maturation.21,23–26
The chemical syntheses of stereoisomers of 1 and 2 comprise elaborate multistep reactions,
and some of these require toxic compounds such as pyridinium dichromate.13,16,21,27,28
A
biotechnological alternative based on an edible basidiomycete would thus be highly attractive.
The objective of this study was to investigate the formation of the isomers of 1 and 2 detected
in submerged PSA cultures. First, the stereochemistry of the bicyclic monoterpenoids was
elucidated by means of enantioselective multidimensional gas chromatography (enantio-
MDGC). To elucidate the biochemical pathways towards the stereoisomers of 1 and 2,
supplementation studies using potential precursors and intermediate compounds in the
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pathways were performed. For this purpose, deuterium and C-labeled supplements were
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added to the culture media, and the incorporation of labeled atoms into the target compounds
was analyzed by mass spectrometry (MS).29
MATERIALS AND METHODS
Cultivation. Pleurotus sapidus (PSA) (DSM No. 8266) was obtained from the Leibniz
Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Brunswick,
Germany). Chemicals used for culture media were purchased from Alfa Aesar (Karlsruhe,
Germany), Applichem (Darmstadt, Germany), Carl Roth (Karlsruhe, Germany), Sigma-
Aldrich (Schnelldorf, Germany), and Th. Geyer (Renningen, Germany). Agar stock cultures
of PSA were kept on malt extract agar (per liter ddH2O, malt extract, 20 g, and agar, 15 g).
PSA was cultivated submerged in the following media (respective composition per liter
ddH2O): Standard nutrition solution 1 (SNS1): asparagine monohydrate (4.5 g), potassium
dihydrogen phosphate (1.5 g), magnesium sulfate hydrate (0.5 g), yeast extract (3 g), trace
element solution according to30 (1 mL), separately autoclaved D-glucose monohydrate (30 g).
SNS2: same as SNS1, except for separately autoclaved glucose monohydrate (20 g). SNS2*:
same as SNS2, except for separately autoclaved D-glucose monohydrate (10 g) and 2-13C-D-
glucose (10 g) (Isotec, Miamisburg, Ohio, USA). Yeast-peptone-dextrose-adenine medium
(YPDA): yeast extract (10 g), peptone from soybean meal (20 g), adenine sulfate (30 mg),
separately autoclaved D-glucose monohydrate (20 g). YPDA*: same as YPDA, except for
separately autoclaved 2-13C-D-glucose (20 g). Malt extract-peptone medium (MEP): malt
extract (30 g); peptone from soybean meal (3 g).
For cultivation, a cubic piece of a densely overgrown agar stock culture was transferred to an
Erlenmeyer flask (250 mL) containing autoclaved medium (filling volume 40% v/v) and
homogenized by means of an Ultra-Turrax T25 homogenizer (IKA, Staufen, Germany) for 30
s at 10,000 rpm. Pre-cultures of PSA were grown on a rotary shaker (150 rpm, 25 mm shaking
diameter; Orbitron, Infors, Einsbach, Germany) in darkness at 24 °C for 4 d (YPDA, MEP),
or 6 d (SNS). To inoculate the main culture, the pre-culture was homogenized under the same
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conditions as described above. The inoculum was added in a ratio of 1:10 to the main culture
containing the same medium, which was autoclaved before in an Erlenmeyer flask (filling
volume 40% v/v). Main cultivation was performed at 24 °C and 150 rpm in the dark for a
defined period as indicated in the respective section.
Synthesis of p-Menth-1-en-9-ol. 20 mmol (4R)-limonene 3A (Sigma-Aldrich) was
hydroborated with an equimolar amount of 9-borabicyclo[3.3.1]nonan (9-BBN) (0.5 M in
tetrahydrofuran) (Acros Organics, Geel, Belgium) according to a modified protocol of Brown
et al.21,31–33 The solution was stirred under nitrogen atmosphere at room temperature
overnight. For oxidation, 12 mL ethanol (Carl Roth), 4 mL sodium hydroxide (6 M) (Carl
Roth), and 8 mL hydrogen peroxide (30%) (Th. Geyer) were added successively prior to
heating at 50 °C for 2 h. The reaction was stopped by addition of 80 mL water. The solution
was extracted three times in a separatory funnel with 100 mL of an azeotropic mixture of n-
pentane and diethyl ether (both Th. Geyer) (P/D), which was distilled prior to use. The
organic phases were combined, dried over anhydrous sodium sulfate (Carl Roth) and
concentrated using a Vigreux column. The raw product was purified by means of preparative
column chromatography (stationary phase: silica gel, mobile phase: distilled P/D) to obtain a
1:1 mixture of the diastereomers (4R,8RS)-p-menth-1-en-9-ol 4A/4C. This procedure was
repeated with (4S)-limonene 3B (Alfa Aesar) to produce a diastereomeric mixture of
(4S,8RS)-p-menth-1-en-9-ol 4B/4D. The structures of the synthesized products were
confirmed by 1H NMR and 13C NMR spectroscopy. The mass spectra were in agreement with
those reported by Guth et al.21 Retention indices on two GC columns of different polarity
were in agreement with those from literature references (RIWAX = 1931 / 1934, RIDB5 = 1297 /
1298).34,35 The purity determined by GC-MS was 89.6% (4A/4C) and 91.4% (4B/4D),
respectively.
Kinetic Analyses. For kinetic studies, three PSA main cultures (SNS1, 1000 mL shake flasks)
were inoculated simultaneously from the same homogenized pre-culture. An aliquot (10.5
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mL) was taken under sterile conditions of each culture on the days 0 (after inoculation) to 9
and on day 11. The samples were centrifuged at 3000 x g and 4 °C for 10 min. The liquid
supernatant was used for analysis of pH, glucose content by means of a Biosen C-Line (Clinic
/ GP+) (EKF-diagnostic, Barleben, Germany), and volatiles by means of SBSE (see below).
The mycelial pellet was washed according to8, and then freeze-dried. The biomass was
estimated according to Equation 1:
g
weight of mycelium after freeze ― drying [g]
weight of culture aliqout before centrifugation [g]
132
(1)
Biomass
=
∙ 100
[
]
100 g
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Supplementation with 2-13C-D-Glucose and Potential Precursors. Main cultures of PSA in
SNS2* and in the reference medium SNS2 (both in 50 mL shake flasks) were inoculated from
the same homogenized pre-culture (PSA in SNS1) and cultivated for 4 d. Main cultures of
PSA in YPDA* and in the reference medium YPDA (pre-cultures: PSA in YPDA) were
cultivated under the same conditions.
Pre-cultures of PSA in SNS1 were combined and homogenized to inoculate the main cultures
in SNS1 (in 100 mL shake flasks). The following supplements (log Kow > 2.9) were dissolved
in ethanol and added to the main cultures under sterile conditions on the third culture day
(respective concentrations per culture volume): rac-limonene (1.0 mM), nerol (1.0 mM)
(both: TCI, Eschborn, Germany), rac-linalool (Sigma) (1.0 mM), (4R)-limonene 3A, (4S)-
limonene 3B (both: Alfa Aesar) (both: 0.2 mM, 0.4 mM, and 0.8 mM), rac-13C,D2-limonene
3* (Aromalab, Planegg, Germany) (0.5 mM, 1.0 mM) (Figure 1). The synthesized p-menth-1-
en-9-ol mixtures 4A/4C and 4B/4D (log Kow = 2.8) were supplemented without pre-
solubilization under otherwise same conditions (0.2 mM, 0.6 mM, 1.1 mM). Biological blanks
were prepared simultaneously by cultivating PSA under the same conditions by addition of
the same amount of ethanol, or without supplementation, respectively. Chemical blanks
without inoculation in SNS1 were treated and supplemented under the same conditions. The
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supplemented cultures as well as the blanks were analyzed after a total cultivation time of
seven days.
Sorptive Extraction. The submerged cultures were analyzed after removal of the mycelium
by means of centrifugation. Two mL (for HS-SPME) or 5 mL (for SBSE) of the liquid
samples were transferred into 20 mL vials with silicone/PTFE caps and stored at –20 ° until
extraction. For HS-SPME, a polydimethylsiloxane/divinylbenzene (PDMS/DVB, 65 μm)-
coated fiber (length 1 cm) (Supelco, Steinheim, Germany) was used. The extraction was
performed in a water bath at 40 °C under magnetic stirring at maximum speed for 30 min. For
SBSE, a stock solution of thymol (TCI) (1473 mg/L in acetonitrile (VWR, Darmstadt,
Germany), stored at –20 °C) was diluted with water 1:500 to prepare the internal standard
solution. The samples were thawed and 50 µL of the internal standard solution as well as a 10
mm Twister stir bar with 0.5 mm PDMS coating (GERSTEL, Mülheim an der Ruhr,
Germany) were added. The vials were extracted under magnetic stirring at 1000 rpm at 4 °C
for 60 min. For the screening for potential metabolites, the extraction time (90 min) and the
vial size (10 mL) differed. The further handling and thermodesorption parameters were
identical to those of a previous study.8
Liquid-liquid Extraction in Combination with Solvent-Assisted Flavor Evaporation
(LLE/SAFE). Submerged main cultures of PSA in YPDA and MEP as well as the reference
materials dried dill weed and linden blossom honey (bought from a local supermarket) were
extracted with distilled P/D in shaking flasks on a magnetic stirrer at maximum agitation rate.
The phases were separated by centrifugation in PTFE beakers. The extraction was repeated,
and the organic phases were combined. The specific extraction conditions were as follows:
3.5 g of dried dill weed was extracted twice with 75 mL P/D for 45 min each. 150 g of linden
honey dissolved in 250 mL aqueous sodium chloride (NaCl) (Carl Roth) solution (200 g/L)
was extracted thrice with 100 mL P/D for 30 min each. A submerged main culture of PSA in
YPDA (300 mL) was extracted directly after cultivation and addition of 30 g NaCl three times
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with 100 mL P/D for 30 min each. Supernatant (200 mL) of a submerged main culture of PSA
in MEP was stored at –20 °C until analysis. After addition of 20 g NaCl, it was extracted
thrice with 100 mL P/D for 30 min each.
The combined P/D fractions of each sample were dried over anhydrous sodium sulfate and
purified by means of solvent-assisted flavor evaporation (SAFE) using a BAENG distillation
unit (Bahr, Manching, Germany) connected to a high-vacuum pump (Pfeiffer Vacuum,
Asslar, Germany) under the following conditions: high vacuum, 1.9 x 10-4 mbar - 2.5 x 10-3
mbar; water bath temperature, 48 °C; head and leg temperature, 50 °C; distillation time, 2 –
4 h. The distillate was concentrated at 43 °C using a Vigreux column to a final volume of
approximately 1.5 mL.
Gas Chromatography-Mass spectrometry. Gas chromatography was performed with an
Agilent 7890A gas chromatograph connected to an Agilent 7000B triple quadrupole mass
spectrometer (GC-MS/MS) or an Agilent 7890B gas chromatograph connected to a 5977B
mass spectrometer (GC-MS) (Agilent Technologies, Waldbronn, Germany). The systems
were optionally equipped with a Thermal Desorption Unit (TDU) and a Cooled Injection
System 4 (both: GERSTEL).8 Either polar Agilent J&W VF-WAXms (30 m × 0.25 mm i.d.,
0.25 μm film thickness), polar Agilent J&W DB-WAXms (30 m × 0.25 mm i.d., 0.25 μm), or
non-polar Agilent J&W DB-5ms columns (30 m × 0.25 mm i.d., 0.25 μm) were installed. The
GC oven temperature was held at 40 °C for 3 min, increased by 5 °C/min to 240 °C and held
for 12 min for polar columns. For non-polar columns, the final temperature was extended to
290 °C. Helium (5.0, Praxair, Duesseldorf, Germany) was used as carrier gas with a constant
column flow rate of 1.2 mL/min. If a system was additionally equipped with an Olfactory
Detection Port 3 (ODP 3) (GERSTEL), a constant column flow rate of 1.56 mL/min was split
1:1 into the ODP and the MS detector. Split ratios were adjusted to the sample matrices and
were in the range of 6:1–10:1 mL/min after SBSE or 5:1–150:1 mL/min for liquid injections.
The MS detectors were operated in electron-impact (EI) mode (70 eV) and in scan mode
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(TIC). Further GC-MS/MS conditions were identical to those reported in8 and further GC-MS
conditions were identical to those in36.
Liquid injection was performed with GC-MS and in split/splitless inlets at 250 °C. Retention
indices on the non-polar column (RIDB5) and isotopic labeling studies were measured by
SBSE followed by TDU-GC-MS-ODP. The screening for intermediate metabolites was
performed by SBSE-TDU-GC-MS. All other SBSE analyses were performed with the TDU-
GC-MS/MS-ODP system. Data were analyzed using the Agilent MassHunter Quantitative
Analysis and Qualitative Analysis software.
Enantioselective-Multidimensional Gas Chromatography-Mass Spectrometry (enantio-
MDGC-MS). An enantio-MDGC-MS system (Shimadzu Europa, Duisburg, Germany)
composed of two Shimadzu GC-2010 Plus gas chromatographs (GC 1 and GC 2) was used
for chiral separation. A Shimadzu AOC-20i autoinjector and a split/splitless inlet were used
for liquid injections. After SPME, the fibers were desorbed in an SPME liner (Shimadzu) for
at least 10 min. Injections were performed at 250 °C in splitless mode or with a split ratio
(range: 5:1–125:1) depending on the sample. The pre-separation in GC 1 was performed on an
Agilent J&W VF-WAXms column with helium as carrier gas and a linear velocity of 25 cm/s.
A multi Dean’s switch (MDS) allowed a selective transfer of the analytes to either GC 2 or to
an FID (250 °C; H2 flow, 40 mL/min; air flow, 400 mL/min; makeup gas N2 (5.0), 30
mL/min). The transfer line temperature between GC 1 and GC 2 was set to 220 °C. GC 2 was
equipped with a Shimadzu QP2010 Ultra mass spectrometer (scan mode, TIC; scan range, m/z
33−300; EI, 70 eV; source temperature, 200 °C; quadrupole temperature, 150 °C; transfer line
temperature, 220 °C).
To separate 1A-1D and 2A-2D, the conditions were as follows: GC 1: constant inlet pressure,
193.3 kPa; switching pressure, 118.8 kPa; temperature program, 40 °C (3 min), 5 °C/min, 220
°C (48.50 min); GC 2: Hydrodex β-TBDAc column (heptakis-(2,3-di-O-acetyl-6-O-tert-
butyldimethylsilyl)-β-cyclodextrin; 25 m × 0.25 mm i.d.) (Macherey-Nagel, Düren,
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Germany); initial linear velocity, 45 cm/s; temperature program, 40 °C (39.50 min), 5 °C/min,
220 °C (5 min).
For the isomers of p-menth-1-en-9-ol (4A-4D), the following conditions were applied: GC 1:
constant inlet pressure, 193.3 kPa; switching pressure, 146.1 kPa; temperature program, 40 °C
(3 min), 5 °C/min, 205 °C (12 min), 5 °C/min, 220 °C (15 min); GC 2: Astec Chiraldex B-
DM column ((2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin; 30 m × 0.25 mm
i.d., 0.12 μm) (Sigma-Aldrich); initial linear velocity, 45 cm/s; temperature program, 40 °C
(34.5 min), 5 °C/min, 160 °C (0 min), 20 °C/min, 220 °C (5 min). The same conditions were
used for separation of the enantiomers of limonene 3A and 3B, except for shorter isothermal
times in the GC temperature programs. The data were analyzed using Shimadzu MDGC
solution software 1.01. Extracted ion chromatograms (EIC) were used for the determination
of the enantiomeric purities of the following compounds: 1A-1D (m/z 137), 2A-2D (m/z 151),
3A-3B (m/z 68), 4A-4D (m/z 94).
Compound Identification. For the identification of volatile compounds, the retention indices
(RI) and mass spectra were compared to published data on two columns of different polarities
and the NIST 2011 MS library, respectively, and verified either with synthesized or
commercially available authentic standards: (-)-carveol (mixture of cis and trans), (-)-
dihydrocarveol (mixture of isomers), (+)-dihydrocarvone (mixture of isomers) (all from
Sigma-Aldrich), 4-acetyl-1-methyl-cyclohexen (Fluorochem), (+)-carvone (Alfa Aesar), (-)-
perillaldehyde (TCI). Synthesized references of wine lactones 2A–2D were provided by Nils
H. Schebb, University of Wuppertal.21
Quantitation. The concentrations of the target analytes in the fungal samples were estimated
via internal standard calibration by means of SBSE in combination with TDU-GC-MS/MS-
ODP. Standard solution mixtures of the analytes were prepared in autoclaved SNS1 without
glucose monohydrate and trace element solution and were diluted accordingly to obtain at
least nine calibration points. The internal standard thymol was added as described above to
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the calibration samples, which were analyzed under the same conditions as the fungal
samples. The ratios of the measured peak areas (A) were plotted against the ratios of the used
concentrations (c) (µg/L) (Equation 2), so that the response factors (RF) equaled the slopes of
the calibration lines:
cAn
= RF ∙ AAn
258
(2),
cIS
AIS
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
where An = analyte, IS = internal standard (thymol).
Linear regression analysis was performed, and the obtained RF values were used for
quantitation. The used reference compounds and conditions for the RF determination of dill
ether, wine lactone, and p-menth-1-en-9-ol isomers are summarized in Table 1.
Statistics. All cultivations were performed in triplicate, except for the screening for
intermediate metabolites and the supplementation of PSA with the highest concentrations of
4A/4C and 4B/4D (1.1 mM) which were performed in duplicate. The isotopic labeling studies
and the chemical blanks supplemented with different concentrations of 4A/4C and 4B/4D
were performed in single measurements. Quantitative data were compared for fermentation
batches with the same pre-cultures and otherwise identical cultivation conditions. One-tailed,
homoscedastic Student's t-tests (α = 5%) were performed to analyze statistical differences
between groups.
RESULTS AND DISCUSSION
Cultivation of PSA in Different Media. The volatilome of PSA was highly dependent on the
used culture media. However, stereoisomers of 1 and 2 were detected after cultivation in all of
the tested media, which confirmed the capability of PSA to biosynthesize them. The bicyclic
monoterpenoids were minor compounds in the volatilome of PSA grown in SNS, whereas
stereoisomers of 1 were among the most abundant volatiles of PSA grown in YPDA or MEP
(Figure 2). Further monoterpenes identified were limonene and linalool. Interestingly,
diastereomers of p-menth-1-en-9-ol, an intermediate metabolite towards dill ether formation
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in dill plants, were detected in PSA grown in YPDA, however in significantly lower
concentrations than the bicyclic monoterpenoids (< 10 µg/L). The mass spectra and retention
indices of the diastereomers were in agreement with those of the synthesized reference
compounds on two columns of different polarity.
Elucidation of the Stereochemistry of Monoterpenoids. The stereochemistry of the formed
wine lactone isomers was investigated by comparing the retention indices of authentic
reference compounds 2A–2D with those of solvent extracts of PSA on the enantio-MDGC-
MS system. For identification of dill ether isomers, solvent extracts of dill weed and linden
honey were used as sources to obtain natural references of enantiopure 1A and a mixture of
the four cis-dill ether isomers, respectively.12,14,15,17,37 The chiral separation of 1A–1D as well
as of 2A–2D was achieved with a Hydrodex β-TBDAc column (Figure 3). Comparison of the
chromatograms of PSA grown in MEP (LLE/SAFE) to those of the natural references clearly
identified the fungal metabolites as isomers 1B and 1D, both with an enantiomeric excess (ee)
of ≥ 99.9. Likewise, the wine lactone isomers 2B and 2D were detected in the fungal extracts.
These findings were further confirmed for PSA grown in YPDA (LLE/SAFE) and in SNS
(HS-SPME). Thus, the same configuration (3R,3aR,7aS) and (3S,3aR,7aS) was found for the
isomers of dill ether and wine lactone. Other stereoisomers than 1A and 2A are extremely rare
in natural sources, and only few examples have been reported in the literature.11,15,37
Furthermore, the stereochemistry of p-menth-1-en-9-ol 4 formed by PSA was analyzed using
a Chiraldex B-DM column. The system allowed for the separation of the synthesized (4R)-
enantiomers 4A/4C as well as of the (4S)-enantiomers 4B/4D. In the extracts of PSA grown in
YPDA (LLE/SAFE), only 4B and 4D were identified (ee > 98 %). The 4S-configuration (in p-
menthan nomenclature) of 4B/4D equaled the 3aR-configuration of 1B/1D and 2B/2D (in
benzofuran nomenclature). This further indicated the potential role of p-menth-1-en-9-ol as
intermediate metabolite in the fungal biosynthetic pathway towards dill ether and wine
lactone.
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306
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312
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323
Kinetic Studies. Biomass, pH, glucose content, and the concentrations of the bicyclic
monoterpenoids were analyzed throughout the cultivation of PSA in SNS1 (Figure 4). The
biomass increased until the 4th culture day and the glucose was exhausted in average on day 7.
The estimated concentration of the potential precursor limonene reached its maximum of
roughly 3 µg/L in average after one day of cultivation. After the primary carbon source
glucose was consumed, the concentrations of 1B and 1D decreased. The concentration of 2B
began to decline in average one day later, whereas 2D remained on a steady level. These
results indicated that the formation of the bicyclic monoterpenoids was related to the
metabolic activity of PSA. By contrast, their generation was independent from the pH of the
medium. The measured pH values were also significantly higher than in model solutions used
for the investigation of acid-catalyzed cyclization reactions resulting in bicyclic
monoterpenoids.18,19,21,23 Thus, non-enzymatic mechanisms similar to those of the formation
of 1A during wine maturation were considered to be highly unlikely. The diastereomeric
excess of 1B and 1D as well as of 2B and 2D varied strongly throughout the cultivation,
which suggested the involvement of several enzymes with different affinities for the
precursors.
De novo Synthesis of Bicyclic Monoterpenoids by PSA. To investigate further, whether
PSA was capable of producing stereoisomers of 1 and 2 de novo, feeding studies with labeled
2-13C-D-glucose were performed. The EI-MS-spectra of the target compounds formed by PSA
324 in SNS2* were compared to those from PSA grown in SNS2 (Tables 2 and 3). For all
325
326
327
328
329
330
stereoisomers of dill ether and wine lactone, shifts of mass fragments of +1, +2, and +3 units,
which represented the [13C1]-, [13C2]-, and [13C3]-isotopologues, respectively, were observed.
For 1B/1D, the molecular ions of the labeled compounds [M]+● with m/z 153, 154 were
detected next to m/z 152 for the non-labeled compound. Apart from that, the fragment ions
m/z 138, 139, and 140 occurred next to the base ion [M-CH3]+ m/z 137 (Table 2).
Additionally, smaller fragment ions with a mass shift of +1 (e.g. m/z 92, 94, 96, and 110)
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339
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349
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351
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356
were enhanced after cultivation in SNS2*. Comparable labeling patterns were observed for 2B
and 2D (Table 3). These results confirmed that dill ether and wine lactone isomers are
produced by PSA de novo from glucose. One glucose molecule labeled at the C2-position is
catabolized via pyruvate to one molecule of labeled and one molecule of non-labeled acetyl-
CoA. From the available pool of acetyl-CoA molecules, isopentenyl pyrophosphate (IPP) is
synthesized via the mevalonate-pathway as the only known pathway towards terpenoid
compounds in fungi. Theoretically, this results in up to two 13C-labels of the IPP molecules.38
IPP is then further converted via geranyl pyrophosphate (GPP) to dill ether and wine lactone
with theoretically up to four 13C-labels. These results were also reproduced with PSA
cultivated in YPDA*, in which 100% of the added D-glucose possessed a 2-13C-label.
Screening for Intermediate Metabolites. The fungal pathways were further investigated
with respect to intermediate compounds following the bioconversion of GPP. Therefore, main
cultures of PSA grown in SNS1 were supplemented with intermediates known to be involved
in the biosynthesis of 1 and 2 in other organisms, i.a. with rac-limonene,20 nerol,11 and rac-
linalool.23,24 Compared to the non-supplemented biological controls, an increased formation
of the bicyclic monoterpenoids was observed after supplementation with rac-limonene, which
led to a twelve- and threefold increase of the amounts of the enantiomeric pairs 1A/1B and
1C/1D, respectively. A bioconversion of nerol to isomers of 1 as reported for the
ascomycetous fungus B. cinerea was not detected.11
Supplementation with Limonene. To further confirm the role of limonene in the
hypothesized pathway, isotopically labeled rac-13C,D2-limonene 3* was added to submerged
cultures of PSA. In the obtained TDU-GC-MS chromatograms, double peaks were observed
for all enantiomeric pairs of 1 and 2. Due to isotopic effects, the heavier isotopologues eluted
slightly earlier than their non-labeled counterparts. The respective mass spectra of the peaks
showed mass shifts of +3 units because of the labeled isotopologues, which contained one 13C
and two D atoms originating from the supplementation of 3* (Tables 2 and 3). Examples were
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363
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369
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374
375
376
377
378
379
380
381
382
the detected labeled fragment ions [M]+● (m/z 155), [M-CH3]+ (m/z 140), and [M-C2H4]+ (m/z
127) in the mass spectra of 1, and [M]+● (m/z 169), [M-CH3]+ (m/z 154), [M-C2H4]+ (m/z 141),
and [M-C2H3O]+ (m/z 126) in the mass spectra of 2 (Tables 2 and 3). Based on the observed
mass shift of +3 in wine lactone, the labeled atoms of 3* are supposed to be located in the
methyl group attached to the C3-position of 1 and 2. During the EI-MS fragmentation of 2,
this methyl group is retained in the mentioned fragment ions, e.g. the base peak [M-CH3]+.23
Varying labelling patterns between the bicyclic monoterpenoids were observed. For 1A/1B,
the non-labeled and labeled ions, e.g. for [M]+● and [M-CH3]+, were detected in similar
intensities after supplementation with 3* (1.0 mM). Conversely, the intensities of the labeled
fragments were significantly lower than those of their non-labeled counterparts in the mass
spectra of 1C/1D, 2A/2B, and 2C/2D (Tables 2 and 3). These results corroborated the
increased formation of 1A/1B compared to the other bicyclic monoterpenoids after
supplementation with non-labeled rac-limonene, as described above. The coexistence of non-
labeled and labeled fragment ions in the mass spectra indicated that the de novo formation of
the bicyclic monoterpenoids occurred in parallel, and their concentrations were increased by
the biotransformation of 3*. Labeled fragment ions with mass shifts of +1 or +2 were detected
as well. This may be caused by inter- or intramolecular hydrogen–deuterium exchanges
during submerged cultivation. Apart from that, an additional pathway towards to bicyclic
monoterpenoids cannot be fully excluded.
To investigate qualitative and quantitative differences between the conversion of (4R)-
limonene 3A and (4S)-limonene 3B by PSA the enantiomers were supplemented separately in
different concentrations. The addition of both, 3A and 3B, led to increased concentrations of
1A/1B and 1C/1D compared to the biological controls (Figure 5). Minor effects were
observed for 2A/2B and 2C/2D, respectively. The mean concentrations of 1A/1B and 2A/2B
were slightly higher after supplementation with 3B compared to 3A. However, this difference
was statistically not significant.
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The cultures supplemented with 3A and 3B were additionally analyzed by means of SPME-
enantio-MDGC-MS for their enantiomeric composition (Figure 3). After the addition of 3A,
PSA formed primarily the isomers 1B (ee ≥ 25.6%), 1D (ee ≥ 52.9%), and 2B in varying
amounts. Interestingly, the respective enantiomers 1A and 1C, which were not produced de
novo by the fungi, were also identified. 1A and 1C show the same configuration at the
respective positions as 3A. This indicated the bioconversion of 3A, next to the parallel de
novo formation of 1B and 1D. The supplementation with 3B led to increased amounts of 1B
(ee ≥ 96.4%), 1D (ee ≥ 93.7%), and 2B, even though also trace amounts of 1A and 1C were
detected. This observation may be explained by enantiomeric impurities detected in the used
reference compound for 3B (ee = 81%). Small amounts of 3A present in the supplemented 3B
were likely converted to 1A and 1C. However, an enantiopure reference for 3B would be
required to confirm this conclusion. The quantitative and qualitative analyses therefore
demonstrated that PSA was capable of biotransforming both limonene enantiomers 3A/3B to
the respective bicyclic monoterpenoid stereoisomers.
Supplementation of PSA cultures with 3A and 3B also led to the formation of a number of
other compounds which were not formed de novo. p-Mentha-1,8-dien-7-al (perilla aldehyde),
p-mentha-6,8-dien-2-one (carvone), (E/Z)-p-mentha-6,8-dien-2-ol (carveol), (E/Z)-p-menth-8-
en-2-one (dihydrocarvone), and four enantiomeric pairs of p-menth-8-en-2-ol
(dihydrocarveol) were confirmed by comparison of the retention indices and mass spectra to
those of authentic standards. p-Mentha-1,8-dien-3-one (isopiperitone), p-mentha-1,8-dien-3-ol
(isopiperitol), p-mentha-1,4(8)-dien-3-one (piperitenone), and p-menth-8-en-7-al (shisool)
were tentatively identified. In particular, the formation of carveols and carvones by PSA after
biotransformation of limonene has been described previously.1,2 After supplementation with
3*, the mass spectra of these compounds exhibited a shift of +3 units of the relevant
fragments, which confirmed limonene as their common precursor. The broad spectrum of
compounds indicated the susceptibility of the 1,2-double bond for allylic oxidation at different
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positions. As some of the compounds were also identified in the supplemented SNS1 blanks, it
was uncertain whether the respective conversion occurred by biotransformation or by
autoxidative processes. The previously identified p-menth-1-en-9-ol 4 occurred in larger
amounts in PSA cultures supplemented with both 3A and 3B than in the biological and
chemical blanks (Figure 5). The oxidation of limonene resulting in stereoisomers of 4 was
thus most likely catalyzed enzymatically.
Supplementation with p-Menth-1-en-9-ol. For further investigation of the fungal pathways,
the synthesized diastereomeric mixtures of 4A/4C as well as of 4B/4D were added separately
to submerged grown PSA in different concentrations. The quantitation revealed substantially
increased concentrations of dill ether and wine lactone isomers after addition of both 4A/4C
and 4B/4D (Figure 6). Compared to the simultaneously grown non-supplemented control
cultures, the concentrations of the enantiomeric pairs 1A/1B, 1C/1D, 2A/2B, and 2C/2D were
up to 1047-, 30-, 62-, and 50-fold higher, respectively. In all cases, the highest supplement
concentration (1.1 mM) led to the highest concentrations of wine lactone isomers, whereas the
highest concentrations of dill ether isomers were reached with lower supplement
concentrations (0.2 mM or 0.6 mM). Regarding the differences between the supplements at
the respective concentrations, 4B/4D led to higher concentrations of the pairs 1A/1B, 2A/2B,
and 2C/2D, whereas 4A/4C resulted in higher concentrations of 1C/1D. 1A–1D were also
identified in the supplemented SNS1 medium controls, but in significantly lower amounts.
The conversion of 4A–4D to the respective dill ether isomers did thus not only occur
enzymatically, but also less effectively as a result of chemical conversion. Isomers of wine
lactones were not detected in these chemical blanks.
Enantio-MDGC-MS analyses of the samples indicated comparable trends for the
supplementation with 4A/4C and 4B/4D as with 3A and 3B, respectively (Figure 3), but with
considerably higher intensities. The addition of 4A/4C resulted in the non-de novo formed
isomers 1A (ee ≥ 99.8%), 1C (ee ≥ 98.4%), and 2A (ee ≥ 72.7%) in almost enantiopure form.
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In contrast, the mixture of 4B/4D was primarily converted to 1B (ee ≥ 92.4%), 1D (ee ≥
63.0%), and 2B (ee ≥ 96.7%), even though minor amounts of 1A, 1C, and 2A were present.
The latter enantiomers were presumably the result of the conversion of enantiomeric
impurities in the synthesized mixture of 4B/4D, for which the same reference standard 3B was
used as starting material as for the supplementation studies described above. For 2C and 2D,
comparable tendencies were observed as for 2A and 2B, though the signal intensities were too
low for determining the ee values. The different enantiomeric composition also affected the
quality of the perceived odors of the target compounds with (non-chiral) GC-olfactometry:
After supplementation with 4A/4C, a characteristic “dill-like” odor was assigned to the peak
of 1A/1B. The same peak was perceived as non-specifically herbal, fruity after addition of
4B/4D. The peak of the pair 2A/2B was perceived significantly more intensively after
addition of 4A/4C compared to 4B/4D due to the low odor threshold of 2A.21
Proposed Pathways. The results demonstrated that the formation of the isomers of 1 and 2
occurred in PSA via a “limonene-pathway” (Figure 7). The proposed metabolites 3 and 4
were identified in non-supplemented PSA cultures, and all stereoisomers of these
monoterpenes could be biotransformed to the respective bicyclic monoterpenoids. This
indicated i.a. the presence of an oxidase in PSA, which was able to convert both 3A and 3B
non-stereoselectively to 4A/4C and 4B/4D, respectively. In contrast, the hydratase proposed
for dill has been described as substrate-specific and stereoselective, as solely 1A was
identified in dill. The mechanism of the further conversion of 4 still remains to be elucidated.
The proposed intermediate compound in dill plants p-menth-1-en-3,9-diol was not detected in
PSA cultures. Nonetheless, an allylic oxidation at the C3-position of 4 is considered as very
likely, due to the broad spectrum of active oxidases in PSA39 and the various descriptions of
the fungus’ ability to catalyze this reaction type.1,2,5–7 The identification of p-mentha-1,8-dien-
3-one and its corresponding alcohol in PSA cultures supplemented with 3A/3B further
supported the hypothesized fungal ability to oxidize 4 at the respective position. Furthermore,
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p-menth-1-en-3,9-diol was described as highly instable, so that it might have been converted
further chemically or enzymatically with high efficacy.20
In non-supplemented cultures, only the enantiomers 1B/1D, 2B/2D, and 4B/4D were detected,
which possess the same configuration as 3B. Therefore, the lack of a (4R)-limonene synthase
might be the reason why the bicyclic monoterpenoids 1A/1C and 2A/2C are not formed de
novo in PSA. However, the amounts of limonene in the LLE/SAFE extracts were not
sufficient to determine the genuine enantiomeric ratio formed by PSA and to support the latter
conclusion.
Another particularity of the PSA metabolisms is that PSA produced both, ether 1 as well as
lactone 2. In the kinetic investigations as well as in the supplementation studies, the formation
of both bicyclic monoterpenoids followed similar principles. In addition, the same
stereoisomers of 1 and 2 were detected in all cultures. This suggested similar formation
mechanisms and linked pathways of the bicyclic monoterpenoids. In an earlier study of our
group, (2RS)-2-[(1R)-4-methylcyclohex-3-enyl]propanoic acid 5A/5C was chemically
synthesized from 4A/4C. The mixture of 5A/5C was biotransformed with lyophilized mycelia
of PSA to 2A/2C with a conversion rate of 90%.33 For this reason, it was hypothesized that
active cells of PSA are able to oxidize the alcohols 4A–4D to their corresponding acids 5A–
5D, which were further converted to 2A–2D based on the same principles as mentioned above
(Figure 7). Nevertheless, an enzymatic conversion of 1 to 2 remains a theoretical option, as
the transformation of cyclic ethers to lactones has been demonstrated for fungi.40
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ABBREVIATIONS
A, peak area; An, analyte; ee, enantiomeric excess; enantio-MDGC, enantioselective-multidimensional
GC; IS, internal standard; LLE/SAFE, liquid-liquid extraction in combination with solvent-assisted
flavor evaporation; log Kow, logarithmic partition coefficient of analyte between octanol and water;
MEP, malt extract-peptone medium; PSA, Pleurotus sapidus; P/D, azeotropic mixture of n-pentan and
diethylether; RF, response factor; SNS, standard nutritional solution; YPDA, yeast-peptone-dextrose-
adenine medium.
ACKNOWLEDGEMENTS
This project has partially been financed with funds of LOEWE – Landes-Offensive zur Entwicklung
Wissenschaftlich-ökonomischer Exzellenz-AromaPlus (State Offensive for the Development of
Scientific and Economic Excellence). We are grateful to Nils Helge Schebb for providing authentic
aroma standards from the former working group of Helmut Guth. Furthermore, we thank Dr. M. Gand,
A. Kremser, M. Marankoz, D. Röder, R. Strohmaier, and R. Walter for their support in the realization of
the syntheses, extractions, and supplementation studies.
SUPPORTING INFORMATION
NMR spectra of synthesized references 4A/4C (Figure S1) and 4B/4D (Figure S2); screening for
intermediate metabolites (Figure S3); proposed fragmentation mechanisms of 2 and 1 in EI-MS (Figures
S4 and S5); theoretical labeling patterns of 2 and 1 in de novo studies (Figure S6); mass spectra of target
compounds after isotopic labeling studies (Figure S7); chiral separation of p-menth-1-en-9-ol
stereoisomers (Figure S8); formation of other compounds after biotransformation of limonene
enantiomers (Figure S9); mass spectra of target compounds after biotransformation of rac-13C,D2-
limonene 3* (Figures S10–S13); proposed conversion of rac-13C,D2-limonene 3* by PSA resulting in
labeled isomers of 1 and 2 (Figure S14); PSA in SNS1 supplemented with 4A/4C and 4B/4D (extended)
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(Figure S15); enantiomeric ratios of dill ether and wine lactone isomers (Table S1); identified
compounds after biotransformation of limonene enantiomers (Table S2).
NOTES
The authors declare no competing financial interest.
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FIGURE CAPTIONS
Figure 1. Structures of used and identified compounds.
Figure 2. Chromatogram of PSA cultivated in MEP. LLE/SAFE extract of culture medium analyzed by
GC-MS (VF-WAXms, TIC).
Figure 3. Chiral separation of stereoisomers of dill ether (m/z 137) (A-D) and wine lactone (m/z 151) (E-
H) by means of enantio-MDGC-MS: natural dill ether references (A), synthesized wine lactone
references (E), PSA in MEP (LLE/SAFE) (B, F), PSA in SNS1 supplemented (SPME) (C, D, G, H).
Figure 4. Kinetic analysis of submerged main cultures of PSA in SNS1: comparison of bioprocess
parameters (A) and concentrations of bicyclic monoterpenoids determined by SBSE-TDU-GC-MS/MS-
ODP (B). Measurements in triplicate, standard deviations are shown as error bars.
Figure 5. PSA grown in SNS1 supplemented with limonene enantiomers 3A and 3B (dissolved in
ethanol) and analyzed by means of SBSE-TDU-GC-MS/MS-ODP. PSA SNS (biological blank):
supplemented only with same amount of ethanol. SNS (chemical blank): without inoculation,
supplemented with both 3A and 3B (each 0.2 mM) under same conditions. Standard deviations are
shown as error bars.
Figure 6. PSA grown in SNS1 and supplemented with synthesized diastereomeric mixtures of p-menth-
1-en-9-ol 4A/4C and 4B/4D; analysis was performed by means of SBSE-TDU-GC-MS/MS-ODP. PSA
SNS (biological blank): without supplementation. Standard deviations are shown as error bars.
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Figure 7. Proposed metabolic pathways occurring in submerged PSA. The hypothesized intermediate
compounds 2-[(1R)-4-methylcyclohex-3-enyl]propanoic acid 5A/5C and 5B/5D were not detected in
PSA.
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