Selective oxygenation of ionones and damascones by fungal peroxygenases

Article abstract of DOI:10.1021/acs.jafc.0c01019

Apocarotenoids are among the most highly valued fragrance constituents, being also appreciated as synthetic building blocks. This work shows the ability of unspecific peroxygenases (UPOs, EC1.11.2.1) from several fungi, some of them being described recently, to catalyze the oxyfunctionalization of α- and β-ionones and α- and β-damascones. Enzymatic reactions yielded oxygenated products such as hydroxy, oxo, carboxy, and epoxy derivatives that are interesting compounds for the flavor and fragrance and pharmaceutical industries. Although variable regioselectivity was observed depending on the substrate and enzyme, oxygenation was preferentially produced at the allylic position in the ring, being especially evident in the reaction with α-ionone, forming 3-hydroxy-α-ionone and/or 3-oxo-α-ionone. Noteworthy were the reactions with damascones, in the course of which some UPOs oxygenated the terminal position of the side chain, forming oxygenated derivatives (i.e., the corresponding alcohol, aldehyde, and carboxylic acid) at C-10, which were predominant in the Agrocybe aegerita UPO reactions, and first reported here.

Full text of DOI:10.1021/acs.jafc.0c01019

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Biotechnology and Biological Transformations
Selective Oxygenation of Ionones and
Damascones by Fungal Peroxygenases
Esteban D Babot, Carmen Aranda, José C. del Río, René Ullrich, Jan Kiebist,
Katrin Scheibner, Martin Hofrichter, Angel T Martínez, and Ana Gutierrez
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c01019 • Publication Date (Web): 15 Apr 2020
Downloaded from pubs.acs.org on April 15, 2020
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Page 1 of 34
Journal of Agricultural and Food Chemistry
1 Selective Oxygenation of Ionones and Damascones by
2 Fungal Peroxygenases
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Esteban D. Babot,† Carmen Aranda,† José C. del Río,† René Ullrich,‡ Jan Kiebist,£
Katrin Scheibner,£ Martin Hofrichter, ‡ Angel T. Martínez,§ and Ana Gutiérrez *,†
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*Corresponding author: (Tel: +34 954624711; E-mail: anagu@irnase.csic.es)
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†Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Av. Reina Mercedes
10, E-41012 Seville, Spain.
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‡ TU Dresden, International Institute Zittau, Department of Bio- and Environmental
Sciences, Markt 23, 02763 Zittau, Germany
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£ JenaBios GmbH, Löbstedter Str. 80, 07749 Jena, Germany
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§ Centro de Investigaciones Biológicas Margarita Salas, CSIC, Ramiro de Maeztu 9, E-
28040 Madrid, Spain
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ABSTRACT: Apocarotenoids are among the most highly valued fragrance
constituents, being also appreciated as synthetic building blocks. This work shows the
ability of unspecific peroxygenases (UPOs, EC1.11.2.1) from several fungi, some of
them being described recently, to catalyze the oxyfunctionalization of - and -ionones
and damascones. Enzymatic reactions yielded oxygenated products such as hydroxy-,
oxo-, carboxy- and epoxy-derivatives that are interesting compounds for the flavor and
fragrance and pharmaceutical industries. Although a variable regioselectivity was
observed depending on the substrate and enzyme, oxygenation was preferentially
produced at the allylic position in the ring, being especially evident in the reaction with
-ionone, forming 3-hydroxy--ionone and/or 3-oxo--ionone. Noteworthy were the
reactions with damascones, in the course of which some UPOs oxygenated the terminal
position of the side chain, forming oxygenated derivatives (i.e. the corresponding
alcohol, aldehyde and carboxylic acid) at C-10, which were predominant in the
Agrocybe aegerita UPO reactions, and first reported here.
KEYWORDS: aromas, apocarotenoids, ionones, damascones, peroxygenases,
biocatalysis, UPO
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INTRODUCTION
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Apocarotenoids or norisoprenoids, which are usually formed by partial oxidative
degradation of carotenoids, include a wide range of compounds with different chemical
structures and biological activities. Among these compounds, those having thirteen
carbon atoms, such as ionones and damascones, are essential constituents of the aroma
of tea, grapes, roses, tobacco and wine, and are also relevant flavors or fragrances that
constitute an important economic resource for chemical industries.1 Moreover,
damascones and their derivatives have also been identified as a novel class of potential
cancer chemopreventive phytochemicals.2
The introduction of hydroxyl or keto functionalities in these compounds reduces
their volatility and increases the long lasting odor.3 Most of these derivatives (3-
hydroxy and 3-oxo--ionone, 4-hydroxy- and 4-oxo--ionone and hydroxy--
damascone isomers) are present in plants but in very small amounts and extraction is
not a viable process for their industrial use. For that reason, they are usually prepared by
chemical synthesis and, therefore, alternative methods for the bioproduction of these
compounds are of high industrial interest.1
Biotransformation of - and -ionones, and their respective - and -damascones
isomers, to a number of hydroxy and keto derivatives has been reported for several
fungi.3-7 However, microbial whole-cell biotransformations require long incubation
times, and often suffer from low conversion rates and substrate partial degradation. On
the other hand, biotransformations with isolated enzymes (enzymatic in vitro
conversion), such as some cytochrome P450 monooxygenases (P450s)8-10 including
engineered P450 BM-3 variants,11,12 have also been reported. Most P450s, however,
have the disadvantages of requiring expensive cofactors and auxiliary enzymes, and
their stability is usually low due to their intracellular nature.
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Unspecific peroxygenases (UPOs, EC.1.11.2.1) represent a relatively new and
appealing type of biocatalysts for organic synthesis that, unlike P450s, are extracellular
enzymes (therefore more stable) and only require H2O2 for activation.13 However, in
spite of all recent progresses in our understanding of UPO catalysis and application,14
some difficulties in UPO application are still to be solved. They include, in addition to
inactivation by hydrogen peroxide that affects enzyme reuse, the present limitations to
heterologously express UPOs in bacterial (and even in fungal systems), due to the more
recent discovery of these enzymes and their fungal origin. The latter aspects, to be
overcome in the future, currently limit enzyme engineering to tailor UPOs for specific
substrates and processes, as well as their production scale up. UPOs have been shown to
catalyze a diversity of interesting oxygenation reactions with aromatic substrates,15,16
aliphatic compounds such as fatty acids, alkanes, fatty alcohols,17-21 steroids and
secosteroids,22-24 and other flavor and fragrance compounds, such as isophorone.25 The
first UPO was described in the basidiomycetous fungus Agrocybe aegerita (AaeUPO)26
and since then, several other UPO enzymes have been purified from other
Basidiomycota and Ascomycota species such as Coprinellus radians,27 Marasmius
rotula (MroUPO)28 and Chaetomium globosum (CglUPO),29 which is indicative for
their widespread occurrence in the fungal kingdom. In addition to these wild-type (i.e.
non-recombinant) enzymes, there are other UPOs, e.g. from Coprinopsis cinerea
(rCciUPO)18 and Humicola insolens (rHinUPO),29 which are only known as
recombinant proteins heterologously expressed by Novozymes A/S (Bagsvaerd,
Denmark) in the mold Aspergillus oryzae.30 Very recently, a new UPO from the
ascomycetous mold Daldinia caldariorum has become available from Novozymes, after
gene expression in A. oryzae, being also expressible in Escherichia coli as a soluble and
active enzyme.31
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In the present work, the oxygenation of - and -ionones, and their respective
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isomers - and -damascones by several UPOs is shown for the first time. These are
new reactions of interest for the biotechnological synthesis of different natural flavors,
pharmaceuticals and synthetic building blocks to be added to the existing portfolio of
reactions catalyzed by these exciting enzymes.14
MATERIALS AND METHODS
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Enzymes. AaeUPO (isoform II), a wild UPO from cultures of A. aegerita TM-A1
(syn. Cyclocybe aegerita, DSM 22459) grown in soybean-peptone medium, was
purified as previously described.26 MroUPO is another wild-type UPO, from cultures of
M. rotula DSM-25031 (German Collection of Microorganisms and Cell Cultures,
Braunschweig), which was purified as described by Gröbe et al.28 CglUPO, the third
wild-type UPO originates from cultures of C. globosum DSM-62110, which was
purified as recently described.29 rCciUPO corresponds to the protein model 7249 from
the sequenced C. cinerea genome available at the JGI
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(http://genome.jgi.doe.gov/Copci1). The recombinant enzymes rCciUPO (44 kDa),
rHinUPO and rDcaUPO were provided by Novozymes A/S after expression in A.
oryzae.30 All UPO proteins were purified by fast protein liquid chromatography (FPLC)
using a combination of size exclusion chromatography (SEC) and ion exchange
chromatography on different anion and cation exchangers. Purification was confirmed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and UV-
vis spectroscopy following the characteristic heme-maximum around 420 nm (Soret
band of resting-state UPOs). Enzyme concentration was estimated according to the
characteristic UV-vis band of the reduced UPO complex (Fe2+-heme) with carbon
monoxide.34
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Chemical compounds. Ionones, rac-(3E)-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-
buten-2-one (also known as -ionone) and (3E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-
3-buten-2-one (also known as -ionone), and damascones, rac-(2Z)-1-(2,6,6-trimethyl-
2-cyclohexen-1-yl)-2-buten-1-one (also known as -damascone) and (2E)-1-(2,6,6-
trimethyl-1-cyclohexen-1-yl)-2-buten-1-one (also known as -damascone) were tested
as substrates of the above UPOs. All the compounds were purchased from Sigma-
Aldrich except -ionone that was supplied by Fluka.
Enzymatic reactions. Reactions (1 mL volume) with ionones and damascones (0.5
mM) were performed at 30 C, in 50 mM phosphate buffer, pH 7.0 (pH 5.5 in MroUPO
reaction). The enzyme concentration was 0.5 M and the H2O2 was added every 6 min
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in doses of 0.5 mol to a final concentration of 2.5 mM.25 In control experiments,
substrates were treated under the same conditions (including H2O2) but without enzyme.
The blank experiments did not give any oxidation product. Samples, at 30 min reaction,
were extracted with ethyl acetate and directly analyzed by GC-MS. On the other hand, a
time course experiment with all substrates and enzymes was performed, and the
corresponding samples within several reaction times (5 min, 10 min, 15 min and 20
min) were analyzed by GC-MS. In addition, samples were dried under N2 to prepare
trimethylsilyl (TMS) derivatives with N,O-bis(trimethylsilyl)trifluoroacetamide
(Supelco) that were also analyzed by GC-MS. Reactions with higher substrate
concentration were also performed using 5 mM of β-ionone, 1 µM of enzyme and by
adding H2O2 with a syringe pump over 6 hours at 3 µmol h-1. Total turnover number
(TTN) (mol product x number of conversions/mol enzyme) as well as total turnover
frequency (TOF) (TTN/time) were calculated in these reactions.
GC-MS analyses. The analyses of samples (with and without silylation) were
performed in a Shimadzu GC-MS QP 2010 Ultra system, using a fused-silica DB-5HT
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capillary column (30 m × 0.25 mm internal diameter, 0.1 μm film thickness) from J&W
Scientific. The oven was heated from 50 °C (1.5 min) to 90 °C (2 min) at 30 °C min−1,
and then from 90 °C to 250 °C (15 min) at 8 °C min−1. The injection was performed at
250 °C and the transfer line was kept at 300 °C. Compounds were identified by mass
fragmentography and comparing their mass spectra with those of the Wiley and NIST
libraries, and those previously reported, 3,4,7,9,10,35-37 and relative quantification was
obtained from total-ion peak area, using response factors of the same (in the case of
substrates) or similar compounds. The mass spectra and chemical structures of
substrates and their reaction products (underivatized and as TMS derivatives) are
included in Supporting Information (Table S1).
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RESULTS AND DISCUSSION
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In the present work, several fungal UPOs namely AaeUPO, MroUPO, CglUPO,
rCciUPO, rHinUPO and rDcaUPO were tested for their ability to oxygenate
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apocarotenoids such as -ionone, -ionone, -damascone and -damascone (Figures 1-
4), using H2O2 as co-substrate and O-donor. The performance of enzymatic reactions
were evaluated by GC-MS, and the different activities and selectivities attained by the
UPOs are described and discussed in the following sections.
Reactions with -ionone. All UPOs were capable of completely transforming -
ionone within 30 min reaction time, except rCciUPO that only reached 21% conversion
under these conditions (Table 1). The time course of the reactions showed that
rHinUPO and rDcaUPO completely converted the substrate within 5 min, while
AaeUPO, MroUPO and CglUPO needed 15-30 min (Figure 5A).
GC-MS analyses of enzymatic reactions revealed that all enzymes selectively (86-
98%) oxygenated -ionone at C-3 (allylic position) producing 3-hydroxy--ionone (3-
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OH--I) and 3-oxo--ionone (3-CO--I) (Table 1, Figures 1, S1). In addition to these
derivatives, the tautomer of the keto-derivative, 3-hydroxy-2,3-dedihydro--ionone (3-
OH-2,3-DH--I) (7%), was observed in rHinUPO reactions. Small amounts (1-14%) of
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4-epoxy--ionone (4-epoxy--I) were also detected in all reactions. The mass spectra of
these compounds (Table S1) were in agreement with those published in the NIST
library and in the literature.7,9,10
Different proportions of both cis-3-OH--I and trans-3-OH--I isomers were
observed in the reactions with the different UPOs, the trans-diastereoisomer being
generally the most abundant, except in AaeUPO reactions where the proportion of both
isomers was similar (Figures 6A, S1) after 30 min. The higher proportion of the trans-
diastereoisomer may be attributed to the faster further oxygenation of the cis-
diastereoisomer by these enzymes via a gem-diol intermediate that is in equilibrium
with the corresponding keto derivative (Figure 1),17 although an enzymatically
preference for hydroxylation of -ionone to the corresponding cis or trans
diastereomers may not be discarded.
Interestingly, all UPOs were able to over-oxygenate the 3-OH--I to form 3-CO--
I (via the gem-diol), except AaeUPO that only formed 5% of the latter compound
(Table 1). Low over-oxygenating activity of AaeUPO for hydroxy-derivatives was also
observed in the hydroxylation of the related -isophorone.25 The contrary was observed
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in rHinUPO reactions, in which 3-CO--I was the predominant product and the OH-
tautomer was also observed. The reason why the enol form was only found in rHinUPO
reactions may be due to the high amount of the keto-form in these reactions. With other
UPOs, the lower amount of the 3-CO--I formed may cause that the OH-tautomer is
below the detection limit.
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Similar oxygenated derivatives were reported for fungi-mediated biotransformation
of -ionone, although generally with lower substrate conversion rates3,5,7. On the other
hand, the biotransformation of -ionone catalyzed by P450 CYP109D1 showed the
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regioselective formation of 3-OH--I.9 The selectivity found here for most UPOs is
similar to that reported for cytochrome CYP101B1, where the trans diastereoisomer
was preferentially obtained (66%).10
Reactions with -ionone. All UPOs were able to completely convert β-ionone
under the same conditions within 30 min of reaction, except rCciUPO that only reached
41% conversion (Table 2). Time courses of the reactions show similar conversion
degrees as those observed with -ionone for the different UPOs, with the exception of
AaeUPO, which achieved almost complete substrate conversion within just 5 min
(Figure 5B). On the other hand, different regioselectivities were noticed for the
different UPOs tested, and oxygenation occurred at different positions (Table 2,
Figures 2, S2). CglUPO and rHinUPO were most selective (around 80%
regioselectivity) towards the C-4 position, resulting in the formation of 4-hydroxy--
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ionone (4-OH--I) and its over-oxygenated (via a gem-diol intermediate) derivative 4-
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oxo--ionone (4-CO--I). All UPOs oxygenated, in addition to C-4, other ring positions
(C-2 and C-3) although to lesser extent. These compounds were tentatively identified by
MS, and their mass spectra (Table S1) matched with those published in the NIST
library and literature.9,10,12,37,38 Moreover, other hydroxylated derivatives resulting from
a second and third oxygenation step (di-hydroxy and/or hydroxy-keto derivatives,
Figures S2-S3) were also detected in almost all reactions.
Only in the case of rCciUPO, the oxygenation of the terminal C-10 position in the
side chain (10-hydroxy--ionone, 10-OH--I) was observed (Figure S2C). The position
of the hydroxyl group was determined by the mass spectrum of the TMS derivative that
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showed a major peak at m/z 177 corresponding to the loss of the terminal hydroxyl
group (Figure S4A). This compound was only evidenced in the silylated sample. To our
best knowledge, the occurrence of this oxygenated derivative has not been reported so
far. The terminal oxygenation of side chains of other aliphatic cyclic compounds by
UPOs has been described for steroids, secosteroids and trans--methylstyrene.22,23,39
On the other hand, the presence of 7,11-epoxymegastigma-5(6)-en-9-one (EME)
and its hydroxylated derivative 4-hydroxy-7,11-epoxymegastigma-5(6)-en-9-one (4-
OH-EME) was only observed in the reaction with rDcaUPO (Figures 2, S2F), and to
our best knowledge , not reported so far for P450s or microbial cultures. This unusual
bicyclic ionone derivative would be formed by the oxygenation of the C-13 followed by
ring closure between position 7 and 13 of the ionone framework as reported for the
chemical synthesis of EME (in several steps) starting from racemic -ionone
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epoxidation.35 The presence of the hydroxylated derivative of -ionone at C-13
(Figures S2F, S4B) supports this mechanism. Moreover, the absence of the
dihydroxylated derivative of -ionone at C-4 and C-13 suggests that 4-OH-EME is not
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formed from 4-hydroxy--ionone although this could not be absolutely discarded.
Finally, reactions with higher substrate (5 mM) and enzyme (1 µM) loading were
also performed, in which the co-substrate H2O2 was continuously supplied over 6 hours
with a syringe pump to preserve the enzyme stability (that is decreased by high local
concentrations of peroxide). Under these conditions, nearly complete substrate
conversion (except with rDcaUPO) and slightly increased selectivity were achieved,
taking into account that five times less enzyme/substrate ratio was used. That way, total
turnover numbers of 1,700-6,200 were reached (Table 3).
Reactions with -damascone. All UPOs tested transformed the substrate to high
extents (88-100%) within 30 min, except rCciUPO that only accomplished 11%
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conversion under the same conditions (Table 4). The time course of the reaction
showed that rHinUPO and rDcaUPO were more efficient than the other UPOs, since
they completely transformed the substrate within 5 min of the reaction (Figure 5C).
All UPOs (except rCciUPO) oxygenated the C-3 position to produce 3-hydroxy--
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damascone (3-OH--D) (Table 4, Figures 3, S5). In a similar way as in the reactions
with -ionone, the hydroxylation of -damascone by several UPOs yielded both
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diastereoisomers, cis-3-OH--D and trans-3-OH--D, whose mass spectra were in
accordance with those previously reported.4 MroUPO, CglUPO, rDcaUPO and
rHinUPO were more selective giving principally the trans-diastereoisomer, while
AaeUPO mainly produced the cis-diastereoisomer (Figure 6B). In addition, CglUPO,
rHinUPO and rDcaUPO over-oxygenated 3-OH--D to the 3-oxo derivative, and most
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UPOs (MroUPO, CglUPO, rDcaUPO and rHinUPO) formed 4-epoxy--damascone (4-
epoxy--D) as well, although in minor amounts. Again, similar products were obtained
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in fungi-mediated biotransformation of -damascone but with lower conversion rates,
maybe due to the toxicity of these compounds to fungal cells/hyphae.3
Interestingly, in addition to ring oxygenation, AaeUPO, MroUPO and rCciUPO
oxygenated the terminal position of the side chain (Figure 3) being the predominant
reaction (80% of total products) for the former UPO (Table 4, Figure S5). In these
reactions, the formation of the terminal alcohol (10-hydroxy--damascone, 10-OH--
D) was followed by its over-oxygenation producing the corresponding aldehyde (4-oxo-
4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-2-enal, 10-CHO--D) and the carboxylic acid
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(4-oxo-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-2-enoic acid, 10-COOH--D). These
compounds were tentatively identified by the mass spectra of their TMS derivatives
(Figure S6). The spectra show the molecular ions at m/z 280 (10-OH-α-D), m/z 206
(10-CHO-α-D) and m/z 294 (10-COOH-α-D) as well as the fragments corresponding to
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the loss of a methyl group [M–CH3]+ at m/z 265, 191 and 279, respectively, and the
fragment at m/z 123 from the intact ring moiety. Additionally, diagnostic fragments at
m/z 157 corresponding to the side chain butenoyl group in the 10-OH-α-D (Figure
S6A), and at m/z 177 due to the loss of the aldehyde group [M-CHO]+ (Figure S6B)
are also observed. To our best knowledge, such damascone derivatives at C-10 have not
been reported so far. This may be due to the fact that most authors analyzed these
compounds without derivatization, and we found that they can only be detected after
derivatization (silylation).
Reactions with -damascone. Among all UPOs tested, only CglUPO and rHinUPO
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completely converted -damascone within 30 min, while AaeUPO, MroUPO and
rDcaUPO) attained conversions of 81%, 72% and 34%, respectively, under the same
conditions; rCciUPO was practically incapable of oxidizing it (Table 5, Figures 4, S7).
Noteworthy is the low conversion of this compound by rDcaUPO compared to the other
substrates tested. The time course of the reaction showed that rHinUPO was more
efficient than the other UPOs, since it completely transformed the substrate within 5
min (Figure 5D).
In most cases, the hydroxylation at C-4 position was dominant over that at C-3
position with the exception of AaeUPO. On the other hand, rHinUPO, and to minor
extent CglUPO, were the only enzymes that over-oxygenated the 4-OH--D to 4-oxo--
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damascone (4-CO--D) (Figures S7D-E). The mass spectra of these compounds (Table
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S1) are in agreement with those previously reported.3,36,38 On the other hand, as in -
damascone reactions, oxygenated derivatives (alcohol, aldehyde and carboxylic acid) at
C-10 were also formed (Figure 4), preferentially by AaeUPO and MroUPO (Figure
S7). These compounds were identified by the mass spectra of their TMS derivatives
(Figure S8), which showed similar characteristic fragments to those of α-damascone
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derivatives, as the molecular ions at m/z 280 (10-OH-β-D), m/z 206 (10-CHO-β-D) and
m/z 294 (10-COOH-β-D) and the fragments corresponding to the loss of a methyl group
[M–CH3]+ at m/z 265, 191 and 279, respectively. Likewise, the fragment at m/z 177
originated by the loss of the aldehyde group [M-CHO]+ (Figure S8B) is also observed.
Comparison of oxygenation patterns by different UPOs. Generally, all UPOs
accomplished high conversion rates for all substrates tested, except rCciUPO. rHinUPO
and rDcaUPO were the most efficient ones (that converted more substrate in less time
under the same conditions) in oxidizing all substrates tested (with the exception of -
damascone that was only moderately converted by rDcaUPO) (Figure 5). On the other
hand, AaeUPO showed higher efficiency with -ionone than with any other substrate.
Regarding selectivity of the reactions with ionones, all UPOs showed higher
regioselectivities (up to 99%) with -ionone than with -ionone (Tables 1-2). Although
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- and -ionones differ only in the position of the cyclohexene double bond, UPOs
realized different reactivities towards them. Analyses of the different oxygenation
products formed by the UPOs revealed that the position of the double bond in the
cyclohexenyl ring had seemingly an effect on the regioselectivity of hydroxylation,
directing it towards the allylic position. In this sense, - and -ionone were mainly
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hydroxylated by most UPOs at C-3 and C-4 to form 3-hydroxy--ionone and 4-
hydroxy--ionone, respectively. However, this effect was more pronounced with -
ionone, since several oxygenated derivatives at other positions (C-2 and C-3) of the ring
(or in the side-chain) were formed with -ionone. The opposite was reported for P450
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BM-3 mutants that selectively produced 4-OH--I, while they contrarily oxidized -
ionone to a mixture of four products.12 Interestingly, rHinUPO was the UPO that most
pronouncedly oxygenated mono-hydroxyl derivatives further into the corresponding
keto derivatives (that are in equilibrium with the gem-diol counterparts formed first). On
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the other hand, most of the tested UPOs left unaffected the C-13 methyl group of both
- and -ionone. Only in the reaction with rDcaUPO the hydroxylation of the C-13
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317
carbon atom of -ionone was observed, producing the interesting EME and 4-OH-EME
products.
The preference for the hydroxylation of the allylic position in the cyclohexene ring
by UPOs was also observed in the reactions with - and -damascones. A strict
318
regioselectivity for this position was especially evident in the reaction of -damascone
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with rHinUPO and rDcaUPO, followed by CglUPO, and in the reaction of -damascone
with rHinUPO and CglUPO. Interestingly, in damascones reactions, a different
regioselectivity with respect to that observed with ionones was ascertained. Therefore,
in addition to the cyclohexenyl ring, hydroxylation was produced at the terminal
position of the butenoyl side chain, being predominant in AaeUPO reactions but
completely absent in rHinUPO, rDcaUPO and CglUPO reactions. Thus, the change in
the position of the carbonyl and alkene moieties in the butenoyl side chain caused a
drastic change in regioselectivity of AaeUPO towards damascones compared to ionones.
It can be concluded that the oxyfunctionalization of the ionone and damascone
isomers catalyzed by UPOs reveal clear advantages over P450 catalysis (e.g. engineered
P450 BM-3 variants) due to the higher conversion rates, enzyme stabilities and little
requirements concerning cofactors. These enzymes can therefore be of high interest for
the production of valuable compounds, of interest for the flavor and fragrance, cosmetic
and pharmaceutical industries. However, it is necessary to mention that some current
limitations of UPOs, related to their large-scale production and application, need to be
solved for their efficient industrial implementation.
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ACKNOWLEDGMENTS
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We thank Novozymes A/S (Bagsvaerd, Denmark) for providing the recombinant
enzymes (rCciUPO, rHinUPO and rDcaUPO) used in this study and A. González-
Benjumea for helpful discussion.
FUNDING SOURCES
This work was supported by the EnzOx2 (H2020-BBI-PPP-2015-2-1-720297) EU-
project and the CSIC (201740E071) project.
Supporting Information
Chemical structures and mass fragmentations of ionones and damascones and their
oxygenated derivatives from enzymatic reactions with several UPOs (Table S1), GC-
MS analyses of ionones and damascones reactions with several UPOs (Figures S1, S2,
S5 and S7), Chemical structures of the dihydroxy and keto-hydroxy-derivatives from
UPO reactions with -ionone (Figure S3), and mass spectra and
formulae/fragmentations of several oxygenated derivatives of ionones and damascones
from reactions with several UPOs (Figures S4, S6 and S8).
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FIGURE LEGENDS
Figure 1. -Ionone (-I) oxygenation by UPOs, showing the 3-hydroxy--ionone (3-
OH--I), 3-oxo--ionone (3-CO--I) formed via a gem-diol intermediate, its tautomer
3-hydroxy-2,3-didehydro--ionone (3-OH-2,3-DH--I) and 4-epoxy--ionone (4-
epoxy--I).
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Figure 2. -ionone (-I) oxygenation by UPOs showing the different oxygenated
derivatives: 4-hydroxy--ionone (4-OH--I), 3-hydroxy--ionone (3-OH--I), 2-
hydroxy--ionone (2-OH--I), 10-hydroxy--ionone (10-OH--I), 13-hydroxy--
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498
ionone (13-OH--I), 4-oxo--ionone (4-CO--I), 2-oxo--ionone (2-CO--I), 7,11-
epoxymegastigma-5(6)-en-9-one (EME), and 4-hydroxy-7,11-epoxymegastigma-5(6)-
en-9-one (4-OH-EME).
Figure 3. -damascone (-D) oxygenation by UPOs, showing the, 3-hydroxy--
damascone (3-OH--D), 10-hydroxy--damascone (10-OH--D), 4-epoxy--
damascone (4-epoxy--D), 3-oxo--damascone (3-CO--D), 4-oxo-4-(2,6,6-
trimethylcyclohex-2-en-1-yl)but-2-enal (10-CHO--D), 4-oxo-4-(2,6,6-
trimethylcyclohex-2-en-1-yl)but-2-enoic acid (10-COOH--D) derivatives.
499
500
501
502
503
504
505
506
Figure 4. -damascone (-D) oxygenation by UPOs showing the 3-hydroxy--
damascone (3-OH--D), 4-hydroxy--damascone (4-OH--D), 10-hydroxy--
damascone (10-OH--D) and the over-oxygenated compounds 4-oxo--damascone (4-
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508
509
510
CO--D), 4-oxo-4-(2,6,6-trimethylcyclohex-1-en-1-yl)but-2-enal (10-CHO--D) and
4-oxo-4-(2,6,6-trimethylcyclohex-1-en-1-yl)but-2-enoic acid (10-COOH--D).
Figure 5. Time course of the transformation of 0.5 mM -ionone (A), -ionone (B), -
511
512
513
514
515
516
517
518
damascone (C) and -damascone (D) by 0.5 M AaeUPO (purple), MroUPO (yellow),
rCciUPO (light blue), CglUPO (green), rHinUPO (red) and rDcaUPO (blue). The
standard deviations (SD) calculated for duplicates (at 30 min) are indicated.
Figure 6. Abundance (relative percentage) and chemical structures (relative
configuration) of the different diastereoisomers formed in the reactions at 5 min and 30
min of -ionone (A) and -damascone (B) with several UPOs. The standard deviations
(SD) calculated for duplicates (at 30 min) are indicated.
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Table 1. Conversion (percentage of substrate transformed) of -ionone (-I) by several
UPOs (30 min) and abundance (relative percentage) of the reaction products, including
hydroxy (OH), keto (CO) and epoxy derivatives
AaeUPO MroUPO rCciUPO
CglUPO
rHinUPO rDcaUPO
Conversion (%)
Products (%)
3-OH--I
3-CO--I
4-epoxy--I
>99
>99
21
>99
>99
>99
93
5
2
55
31
14
63
35
2
72
22
6
1
98*
1
45
53
2
* including the keto derivative (91%) and its tautomer 3-hydroxy-2,3-didehydro--ionone (7%)
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Table 2. Conversion (percentage of substrate transformed) of -ionone (-I) by several
UPOs (30 min) and abundance (relative percentage) of the reaction products, including
hydroxy (OH) and keto (CO) derivatives, and/or 7,11-epoxymegastigma-5(6)-en-9-one
(EME), and dihydroxy or hydroxy keto derivatives of -I (others)
AaeUPO MroUPO rCciUPO
CglUPO
rHinUPO rDcaUPO
Conversion (%)
Products (%)
4-OH--I
3-OH--I
2-OH--I
10-OH--I
13-OH--I
EME
2-CO--I
4-CO--I
4-OH-EME
Others
>99
>99
41
>99
>99
>99
55
22
13
-
-
-
-
3
-
7
55
-
7
-
-
-
18
1
-
19
61
19
2
14
-
-
-
2
-
64
-
1
-
-
-
-
20
-
21
-
-
-
-
-
-
58
-
21
53
19
3
-
7
11
-
-
5
2
2
15
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Table 3. Conversion (percentage of substrate transformed), abundance (relative percentage) of
reaction products - including hydroxy (OH), keto (CO) and dihydroxy or hydroxy keto
derivatives (others) - total turnover number (TTN) and total turnover frequency (TOF) in the
enzymatic transformation of 5 mM -ionone (-I)
AaeUPO
MroUPO rCciUPO
CglUPO
rHinUPO rDcaUPO
Conversion (%)
Products (%)
4-OH--I
4-CO--I
Others
95
93
22
98
91
52
87
-
13
5200
14
67
13
20
5900
16
83
-
17
1700
5
70
24
6
6200
17
57
24
19
6000
17
54
-
46
2800
8
TTN
TOF (min-1)
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Table 4. Conversion (percentage of substrate transformed) of -damascone (-D) by
several UPOs (30 min) and abundance (relative percentage) of the reaction products,
including hydroxy (OH), aldehyde (CHO), keto (CO), carboxy (COOH) and epoxy
derivatives
AaeUPO MroUPO
rCciUPO
CglUPO
rHinUPO rDcaUPO
Conversion (%)
Products (%)
3-OH--D
4-epoxy--D
10-OH--D
3-CO--D
>99
88
11
>99
>99
>99
18
-
-
-
2
55
6
6
-
14
19
-
-
41
-
52
7
68
16
-
16
-
27
3
-
70
-
-
83
4
-
13
-
-
10-CHO--D
10-COOH--D
80
-
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Page 27 of 34
Journal of Agricultural and Food Chemistry
Table 5. Conversion (percentage of substrate transformed) of -damascone (-D) by
several UPOs (30 min) and abundance (relative percentage) of the reaction products,
including hydroxy (OH), aldehyde (CHO), keto (CO), carboxy (COOH) and epoxy
derivatives
AaeUPO MroUPO rCciUPO CglUPO rHinUPO rDcaUPO
Conversion (%)
Products (%)
3-OH--D
4-OH--D
10-OH--D
4-CO--D
81
72
1
>99
>99
34
32
9
3
-
8
8
46
6
-
17
23
-
-
6
88
-
6
-
5
53
-
42
-
24
71
5
-
-
>99
-
-
-
10-CHO--D
10-COOH--D
48
-
-
-
27
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Journal of Agricultural and Food Chemistry
Page 28 of 34
Figure 1
11
12
6
O
1
9
7
2
3
10
8
5
13
4
-I
H2O2
H2O
UPO
O
O
+
HO
O
3-OH--I
H2O2
4-epoxy--I
UPO
H2O
O
O
O
H2O
HO
HO
O
HO
H2O
gem-diol
3-CO--I
3-OH-2,3-DH--I
28
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Page 29 of 34
Journal of Agricultural and Food Chemistry
Figure 2
O
-I
H2O2
H2O
UPO
O
O
O
O
O
OH
HO
+
+
+
+
OH
HO
OH
4-OH--I
3-OH--I
2-OH--I
H2O2
13-OH--I
10-OH--I
H2O2
H2O
UPO
UPO
H2O
O
O
O
O
O
UPO
O
O
H2O2
H2O
OH
O
4-CO--I
2-CO--I
EME
4-OH-EME
29
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