Selective Epoxidation of Fatty Acids and Fatty Acid Methyl Esters by Fungal Peroxygenases

Article abstract of DOI:10.1002/cctc.201800849

Recently discovered fungal unspecific peroxygenases from Marasmius rotula and Chaetomium globosum catalyze the epoxidation of unsaturated fatty acids (FA) and FA methyl esters (FAME), unlike the well-known peroxygenases from Agrocybe aegerita and Coprinopsis cinerea. Reactions of a series of unsaturated FA and FAME with cis-configuration revealed high (up to 100 %) substrate conversion and selectivity towards epoxidation, although some significant differences were observed between enzymes and substrates with the best results being obtained with the C. globosum enzyme. This and the M. rotula peroxygenase appear as promising biocatalysts for the environmentally-friendly production of reactive FA epoxides given their self-sufficient monooxygenase activity and the high conversion rate and epoxidation selectivity.

Full text of DOI:10.1002/cctc.201800849

Accepted Article
Title: Selective Epoxidation of Fatty Acids and Fatty Acid Methyl Esters
by Fungal Peroxygenases
Authors: Carmen Aranda, Andrés Olmedo, Jan Kiebist, Katrin
Scheibner, José C del Río, Angel T Martínez, and Ana
Gutiérrez
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the VoR from the journal website shown below when it is published
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To be cited as: ChemCatChem 10.1002/cctc.201800849
Link to VoR: http://dx.doi.org/10.1002/cctc.201800849
A Journal of
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10.1002/cctc.201800849
ChemCatChem
COMMUNICATION
Selective Epoxidation of Fatty Acids and Fatty Acid Methyl Esters
by Fungal Peroxygenases
Carmen Aranda‡, Andrés Olmedo‡, Jan Kiebist, Katrin Scheibner, José C. del Río, Angel T. Martínez,
and Ana Gutiérrez*
Abstract: Recently discovered fungal unspecific peroxygenases
Conventional chemical route
from Marasmius rotula and Chaetomium globosum catalyze the
O
O
C
epoxidation of unsaturated fatty acids (FA) and FA methyl esters
H+
(a1)
(a2)
+
+
R1
C
OH
H2O2
+
R1
O
O
OH
H2O
(FAME), unlike the well-known peroxygenases from Agrocybe
aegerita and Coprinopsis cinerea. Reactions of
a series of
O
C
O
C
O
unsaturated FA and FAME with cis-configuration revealed high (up
to 100%) substrate conversion and selectivity towards epoxidation,
although some significant differences were observed between
enzymes and substrates with the best results being obtained with
the C. globosum enzyme. This and the M. rotula peroxygenase
appear as promising biocatalysts for the environmentally-friendly
production of reactive FA epoxides given their self-sufficient
monooxygenase activity and the high conversion rate and
epoxidation selectivity.
+
R3
R
C
C
H
R2
C
H
C
H
R3
R1
OH
R1
OH
2
H
This work: new enzymatic route
O
UPO
(b)
R2
C
H
C
H
+
R2
C
H
C
H
+
R3
H2O
R3
H2O2
Scheme 1. Chemical and enzymatic routes for the epoxidation of fatty acids.
Oils and fats are among the most important renewable feedstock
of the chemical industry, whose possibilities are still far from
being fully exploited.[1] By simple industrial operations, fatty
acids (FA) are available from vegetable oils in such purity that
they may be used for further chemical transformations. Their
known to catalyze epoxidation directly, such as cytochrome
P450 monooxygenases (P450), diiron-center oxygenases, and
plant peroxygenases.[5;6] However, they present some
drawbacks, such as their intracellular nature, and the
requirement for costly cosubstrates in the two former cases.
conversion to FA methyl esters (FAME) is
a well-known
Here, we show
a promising enzymatic technology to
application of fats and oils, largely investigated for biodiesel
production. Moreover, unsaturated FA and FAME can be further
epoxidized, and used in industrial syntheses of chemicals and
intermediates.
epoxidize unsaturated FA (Scheme 1b) under mild and
environmentally-friendly conditions, as potential alternative to
the above chemical and enzymatic epoxidations. This includes
the use of two recently discovered unspecific peroxygenases
(UPO, EC 1.11.2.1), from the fungi Marasmius rotula
(MroUPO)[7] and Chaetomium globosum (CglUPO).[8] These and
related fungal peroxygenases represent a new class of enzymes
that eludes some of the limitations of other monooxygenases
since they are secreted proteins, therefore far more stable, and
only require H2O2 for activation.
Peroxygenases are structurally related to the P450s, as they
also contain the heme prosthetic group coordinated by a
cysteine ligand, but they do not depend on the reductive
activation of molecular oxygen and catalyze the transfer of an
oxygen atom from peroxide to substrates.[9] Initially, these UPO
were shown to catalyze oxygenation reactions on aromatic
compounds,[10] and their action on aliphatic compounds was
demonstrated later.[11] After the first UPO discovered in
Agrocybe aegerita (AaeUPO),[12] similar enzymes were found in
other basidiomycetes, such as M. rotula, and there are
indications for their widespread occurrence in the fungal
kingdom.[13] Over one-hundred peroxygenase-type genes have
been identified in the analysis of 24 basidiomycete genomes,[14]
including Coprinopsis cinerea.[15] One UPO from the latter
fungus is produced as a recombinant protein (rCciUPO) by
Novozymes (Bagsvaerd, Denmark) (Figure S1A).[16] Interestingly,
the recently described MroUPO presents differences with the
best studied fungal peroxygenases, such as the ability of
oxidizing bulkier substrates,[9;17] the terminal hydroxylation of n-
alkanes[18] and the chain-shortening of carboxylic acids.[19] On
The industrial-scale epoxidation of unsaturated FA
compounds is generally carried out by the Prileshajev[2] reaction
via percarboxylic acids (Scheme 1a2). However, this method,
which often includes strong mineral acids as catalysts for the "in
situ" generation of peracids (Scheme 1a1), suffers from several
drawbacks such as the relatively low selectivity for epoxides due
to oxirane ring opening in the acidic medium, the corrosive
nature of acids, and the unstable character of peracids.[3] Many
studies have been aimed at searching an alternative, such as
the chemo-enzymatic synthesis with lipases catalyzing the
carboxylic acid reaction with hydrogen peroxide.[4;5] However,
the latter reaction maintains most drawbacks of peracid-based
epoxidation. Therefore, direct enzymatic processes emerge as
an alternative solution for more selective and environmentally
friendly epoxidation of unsaturated lipids. Several enzymes are
[*] C. Aranda, A. Olmedo, Prof. J. C. del Río, Prof. A. Gutiérrez
Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC
Reina Mercedes 10, E-41012 Seville (Spain)
E-mail: anagu@irnase.csic.es
J. Kiebist, Prof. K. Scheibner
JenaBios GmbH
Orlaweg 2, D-00743 Jena (Germany)
Prof. A. T. Martínez
Centro de Investigaciones Biológicas, CSIC
Ramiro de Maeztu 9, E-28040 Madrid (Spain)
[‡] These authors contributed equally to the work
This article is protected by copyright. All rights reserved.

10.1002/cctc.201800849
ChemCatChem
COMMUNICATION
the other hand, CglUPO is the fourth wild-type described UPO,
and the first isolated from an ascomycete.[8]
A
E
The reactions of purified MroUPO and CglUPO (Figure
S1B,C) with a series of cis-monounsaturated FA (from C14:1 to
C22:1), showed that both enzymes are capable of oxygenating
these substrates, CglUPO being more active since it achieved
maximal substrate conversion with lower enzyme doses (Table
1). Interestingly, both peroxygenases generated the epoxidized
derivatives as main products (Figures 1A-B), unlike the other
well-known AaeUPO (not shown) and rCciUPO that were not
able to epoxidize the double bond and instead, produced the
hydroxyderivatives at the subterminal positions (Figure 1C).
Therefore, whereas AaeUPO and rCciUPO shows similar
regioselectivity towards saturated and unsaturated FA, MroUPO
and CglUPO behave differently, hydroxylating at the terminal
and/or subterminal positions the saturated FA and oxygenating
the double bonds of unsaturated ones. The hydroxylation of FA
at the subterminal positions by AaeUPO and rCciUPO was
previously described.[11;16] However, the epoxidation of a fatty
acid by a fungal peroxygenase is revealed here for the first time.
Moreover, whereas CglUPO was highly selective with all
unsaturated FA, over 90% epoxidation except with erucic
(C22:1) acid, epoxidation by MroUPO depended on FA chain
length, showing the highest value with myristoleic (C14:1) acid.
Besides epoxides, minor amounts of other products were
found with CglUPO and especially with MroUPO, such as
oxygenated derivatives of epoxidized FA (ED) and hydroxylated
C18:1 cis-Δ9
18
20
22
24
26
28
30
Retention time (min)
B
C
E
ED
(-7)-OH
(-1)-OH
C18:1 cis-Δ9
COOH
-OH
18
20
22
24
26
28
30
Retention time (min)
HD
(-2)-OH
(-1)-OH
Table 1. Monounsaturated FA (R=H) and FAME (R=CH3) reactions with
CglUPO and MroUPO doses yielding maximal conversion into epoxides,
together with other oxygenated (hydroxyl, keto and carboxyl) derivatives at
different positions (arrows)
C18:1 cis-Δ9
O
O
O
O
O
O
UPO
+
OR
+
OR
( )m
( )m
( )m
( )m
18
20
22
24
26
28
30
( )n
( )n
( )n
( )n
OR
OR
H2O2 H2O
Retention time (min)
E
ED
HD
Products (%)
Figure 1. GC-MS of reactions of oleic acid (underlined) at 30 min with 60 nM
CglUPO (A), 200 nM MroUPO (B), and 100 nM rCciUPO (C), showing the
epoxide (E), epoxide derivatives (ED) and the hydroxylated derivatives (HD) of
oleic acid.
H2O2
(mM)
Tot
(µM)
Substrate
R=H
Enzyme dose
E
ED
HD
14:1 cis-Δ9
(n=5, m=1)
CglUPO
MroUPO
CglUPO
MroUPO
CglUPO
MroUPO
CglUPO
MroUPO
CglUPO
MroUPO
60 nM
5
99
86
91
65
91
38
95
62
50
67
-
1
98
99
99
99
99
96
94
99
77*
91
derivatives of FA (HD) mainly at terminal or subterminal
positions of the carbon chain, and at the allylic positions (Table 1,
Figure 1B). The higher efficiency and selectivity of CglUPO than
MroUPO for epoxidation of most FA is shown in reactions with
oleic (C18:1) acid (Figures S5-S6). Curiously, erucic acid, an
abundant fatty acid in rapeseed and mustard oils, was
transformed and epoxidized at a larger extent by MroUPO. The
high selectivity of these UPOs epoxidizing oleic and palmitoleic
(C16:1) acids (up to 100%) differs from that of P450 (BM3)
where hydroxylation (> 97% and 65%, respectively)
predominated over epoxidation (< 3% and 35%).[6]
200 nM
60 nM
5
8
6
16:1 cis-Δ9
(n=5, m=3)
5
4
5
200 nM
60 nM
2.5
2.5
5
17
2
18
7
18:1 cis-Δ9
(n=5, m=5)
200 nM
61
4
1
20:1 cis-Δ11
(n=7, m=5)
60 nM
5
1
400 nMa
250 nM
400 nMc
2.5
2.5
5
7
31
39
23
22:1 cis-Δ13
(n=9, m=5)
12
10
R=CH3
14:1 cis-Δ9
CglUPO
MroUPO
CglUPO
MroUPO
60 nM
2.5
2.5
5
100
68
-
-
94
100
75*
93
In addition to monounsaturated FA, some polyunsaturated
FA (linoleic and α- and γ-linolenic acids) abundant in vegetable
oils were tested as substrates. Although both UPOs transformed
almost completely linoleic acid at the highest enzyme doses
(Table 2), CglUPO was more selective producing the diepoxide
(both syn and anti-enantiomers) in very high yield (92% of total
products) (Figure 2A). MroUPO, besides the epoxides also
generated hydroxylated derivatives of the monoepoxides and
oxygenated derivatives at allylic positions (mainly at -7) (Figure
200 nM
1 µMb
200 nMc
13
-
19
2
18:1 cis-Δ9
98
2.5
73
7
21
Substrates (100 µM), enzyme doses, and estimated total products (µM), relative
abundance (% of total products) of epoxide (E), epoxide derivatives (ED),
hydroxylated derivatives (HD) are shown. Reactions were performed in 20%
acetone, 30 min at 30ºC except for reactions a,b and c that were performed in 40%
acetone at 40ºC, for 30 min, 60 min and 120 min, respectively. *Higher enzyme
concentration (up to 500 nM) did not improve conversion. See Figure S2 for GC-
MS of authentic standards and Figures S3A,S4 for mass spectra of E and ED.
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10.1002/cctc.201800849
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COMMUNICATION
Table 2. Oxidation of linoleic acid and its methyl ester by CglUPO and MroUPO
Linolenic acids (α- and γ-) were also transformed by
MroUPO and CglUPO (Figure S9). Both, monoepoxides (located
in two different double bonds) and diepoxides were generated
from α-linolenic acid under the conditions tested. However, only
one epoxide was observed in the γ-linolenic acid reactions.
To compare the efficiency of MroUPO and CglUPO oxidizing
unsaturated FA, apparent kinetic constants were determined for
oleic acid oxidation (Table 3) in spite of the difficulties for GC-
MS estimation of initial reaction rates. Regarding the turnover
rate, CglUPO presented three-fold higher kcat values than
MroUPO. In addition, the Km value was four-fold higher for
MroUPO, which represented less affinity by this enzyme
compared to CglUPO. As a result, one order of magnitude
higher catalytic efficiency (kcat/Km) was observed with CglUPO.
In agreement with these results, CglUPO presented total
turnover numbers (TTN) up to 8000 in oleic acid reactions when
the substrate concentration was increased to 1 mM while this
number was half for MroUPO with 50 nm enzyme being used in
both cases. This is a promising value accompanied by a
significantly higher product amount (of about 0.5 mM)
considering the limited solubility and other difficulties for fatty
acid epoxidation. Likewise, the turnover frequency (TOF) was
double for CglUPO (2.2 s-1) than for MroUPO (1.1 s-1). Solubility
limitations prevented calculation of accurate kinetic constants for
linoleic acid oxidation, since saturation could not be estimated
especially for MroUPO (Figure S10), but higher activity than
found for oleic acid was observed at high linoleic acid
concentration.
O
O
O
O
O
O
O
+
+
OR
OR
OR
O
12-E
ED
di-E
UPO
OR
O
O
O
O
O
H2O2 H2O
+
+
OR
OR
OR
9-E
ED
HD
Products (%)
di-E
H2O2
(mM)
12-E
9-E
ED
HD
Total
(µM)
Enzyme dose
R=H
CglUPO
30 nM
2.5
5
2.5
2.5
39
-
37
-
57
-
25
-
2
-
8
25
59
2
-
3
2
76
98
75
95
125 nM
100 nM
400 nM
92
10
39
MroUPO
R=CH3
CglUPO
250 nM
1 µM
200 nM
1 µM
5
5
2.5
5
13
-
39
-
59
-
40
-
28
100
4
-
-
-
-
9
-
80
100
95
MroUPO
8
51*
49
98
Substrate (100 µM), enzyme doses, H2O2 conc., amount of estimated total
products (µM) and relative abundance (% of total products) of 12-epoxide (12-E),
9-epoxide (9-E), di-epoxide (di-E), epoxide derivatives (ED) and hydroxylated
derivatives (HD) are shown. Arrows indicate the main chain positions oxidized by
the enzymes in epoxidized derivatives (ED) and hydroxylated derivatives (HD).
Reactions conditions: 20% acetone at 30ºC, 30 min (R=H), 40% acetone at 40ºC,
60 min (CglUPO) and 120 min (MroUPO) (R=CH3).* Mono- and di-epoxide
derivative. See Figure S7 for GC-MS of authentic standards and Figures S3B, S8
for mass spectra of E and ED.
2B). When lower doses of enzymes were used, a predominance
of monoepoxides over diepoxides was observed (Table 2).
FAME, usually obtained from vegetable oils by
transesterification with methanol, were also tested as substrates
of MroUPO and CglUPO. Namely, the methyl esters of two
monounsaturated (myristoleic and oleic acids) and one
diunsaturated FA (linoleic acid) were selected. Both
peroxygenases were able to transform and epoxidize the
monounsaturated FAME (Figures 3A,C). CglUPO showed
similar selectivity towards the esters than with the free FA, but
differences in the case of oleic acid were observed with MroUPO
(Table 1). Regarding the methyl ester of linoleic acid, CglUPO
showed a strict selectivity towards epoxidation (generating the
diepoxide) while MroUPO was less selective towards diepoxide
formation (Table 2, Figures 3B,D). In contrast, P450 BM3, which
hydroxylate/epoxidize free fatty acids, was reported as unable to
hydroxylate FAME [20]. This seems related to the fact that the
free carboxyl group is required to fix the substrate at the
entrance of P450 active site [6]. Finally, it is interesting that
different patterns of oxygenation were observed with the cis
isomers of the substrates (compared to the trans isomers).
While MroUPO converted predominantly myristelaidic,
palmitelaidic and elaidic acids (or their methyl esters) into the
hydroxyderivatives at the allylic positions, CglUPO generated
mainly the epoxides, with the only exception of elaidic acid (data
not shown).
di-E-syn
A
di-E-anti
ED
C18:2 cis,cis-Δ9,Δ12
18
20
22
24
26
28
30
Retention time (min)
B
di-E-syn
ED
di-E-anti
ED
(-7)-OH-9-E
(-7)-OH-12-E
C18:2 cis,cis-Δ9,Δ12
18
20
22
24
26
28
30
Table 3. Estimated kinetic parameters for oleic acid oxidation by
CglUPO and MroUPO. Data represent mean values of three
replicates with standard errors
Retention time (min)
kcat (s-1)
8.1 ± 0.9
2.6 ± 0.2
Km (µM)
10.7 ± 4.0
38.9 ± 6.1
kcat/ Km (M-1·s-1)
7.6 ± 3.0 x 105
6.7 ± 1.1 x 104
Figure 2. GC-MS of reactions of linoleic acid (underlined) at 30 min with 125
nM CglUPO (A) and 400 nM MroUPO (B), showing the diepoxides (di-E),
epoxide derivatives (ED) and the hydroxylated derivatives of linoleic acid (see
mass spectrum of the diepoxide in Figure S3B).
CglUPO
MroUPO
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10.1002/cctc.201800849
ChemCatChem
COMMUNICATION
Keywords: epoxidation • fatty acids • fatty acid methyl esters
fungal peroxygenases • oxidoreductases
E
di-E-syn
A
B
di-E-anti
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CglUPO at 60 min (A) and 200 nM MroUPO at 120 min and (C), showing the
epoxide (E), epoxide derivatives (ED); and the hydroxylated derivatives, and
methyl linoleate (right, underlined) with 1 µM CglUPO at 60 min (B) and 1 µM
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(ED) and the hydroxylated derivatives of methyl linoleate.
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The selective epoxidation of FA and FAME, a reaction of
great interest for the chemical industry,[21] must be added to the
repertoire
of
UPOs,
as
dream
biocatalysts
for
oxyfunctionalization chemistry.[9;22;23] The structural determinants
driving to selective epoxidations in MroUPO and CglUPO
(compared to AaeUPO and CciUPO) are difficult to be identified
with the information available on these new heme-thiolate
enzymes (note that only one UPO crystal structure has been
published to date). However, in related P450, epoxidation vs
hydroxylation rates have been related to the balance between
the iron hydroperoxo and oxenoid forms after the oxidative
activation of the enzyme, with an active site threonine being
involve in the transition as the proton donor.[24;25] Interestingly, a
threonine residue is present at the active sites of both AaeUPO
and CciUPO, and absent from those of MroUPO and CglUPO,
as shown by Aranda et al.[26] but its relevance in the FA
hydroxylation/epoxidation balance is still to be experimentally
investigated. This and other structural-functional studies with
UPOs will help to understand the reaction mechanisms of these
versatile enzymes, and to obtain ad-hoc variants for
biotechnological application.
Acknowledgements
This work was funded by the BIORENZYMERY (AGL2014-
53730-R) project of the Spanish MINECO (co-financed by
FEDER) and by the CSIC 201740E071 project. H. Lund
(Novozymes) is acknowledged for rCciUPO.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201800849
ChemCatChem
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Title
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Carmen Aranda‡, Andrés Olmedo‡, Jan
Kiebist, Katrin Scheibner, José C. del
Río, Angel T. Martínez, and Ana
Gutiérrez*
H2O2
H2O
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Selective Epoxidation of Fatty Acids
and Fatty Acid Methyl Esters by
Fungal Peroxygenases
OR
OR
Recently discovered fungal unspecific peroxygenases from Marasmius rotula and Chaetomium globosum catalyze the epoxidation of
unsaturated fatty acids (FA) and FA methyl esters (FAME), unlike the well-known peroxygenases from Agrocybe aegerita and
Coprinopsis cinerea. Reactions of a series of unsaturated FA and FAME revealed high substrate conversion (with up to 8000 total
turnover number in oleic acid reactions) and selectivity towards epoxidation (often >90%), the best results being obtained with the C.
globosum enzyme.
This article is protected by copyright. All rights reserved.