Biocatalytic Oxidative Cascade for the Conversion of Fatty Acids into α-Ketoacids via Internal H2O2 Recycling

Article abstract of DOI:10.1002/anie.201710227

The functionalization of bio-based chemicals is essential to allow valorization of natural carbon sources. An atom-efficient biocatalytic oxidative cascade was developed for the conversion of saturated fatty acids to α-ketoacids. Employment of P450 monooxygenase in the peroxygenase mode for regioselective α-hydroxylation of fatty acids combined with enantioselective oxidation by α-hydroxyacid oxidase(s) resulted in internal recycling of the oxidant H₂O₂, thus minimizing degradation of ketoacid product and maximizing biocatalyst lifetime. The O₂-dependent cascade relies on catalytic amounts of H₂O₂ and releases water as sole by-product. Octanoic acid was converted under mild conditions in aqueous buffer to 2-oxooctanoic acid in a simultaneous one-pot two-step cascade in up to >99 % conversion without accumulation of hydroxyacid intermediate. Scale-up allowed isolation of final product in 91 % yield and the cascade was applied to fatty acids of various chain lengths (C6:0 to C10:0).

Full text of DOI:10.1002/anie.201710227

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Accepted Article
Title: Biocatalytic Oxidative Cascade for the Conversion of Fatty Acids
to alpha-Ketoacids via Internal H2O2 Recycling
Authors: Somayyeh Gandomkar, Alexander Dennig, Andela Dordic,
Lucas Hammerer, Mathias Pickl, Thomas Haas, Mélanie Hall,
and Kurt Faber
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201710227
Angew. Chem. 10.1002/ange.201710227
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10.1002/anie.201710227
Angewandte Chemie International Edition
COMMUNICATION
Biocatalytic Oxidative Cascade for the Conversion of Fatty Acids
to -Ketoacids via Internal H2O2 Recycling
Somayyeh Gandomkar,[a] Alexander Dennig,[a] Andela Dordic,[a,b] Lucas Hammerer,[a,b] Mathias Pickl,[a]
Thomas Haas,[c] Mélanie Hall,*[a] and Kurt Faber*[a]
[8]
Abstract: The functionalization of bio-based chemicals is essential
to allow valorization of natural carbon sources. An atom-efficient
biocatalytic oxidative cascade was developed for the conversion of
saturated fatty acids to -ketoacids. Employment of P450
monooxygenase in the peroxygenase mode for regioselective -
hydroxylation of fatty acids combined with enantioselective oxidation
by -hydroxyacid oxidase(s) resulted in internal recycling of the
oxidant H2O2, thus minimizing degradation of ketoacid product and
maximizing biocatalyst lifetime. The O2-dependent cascade relies on
catalytic amounts of H2O2 and releases water as sole by-product.
Octanoic acid was converted under mild conditions in aqueous
buffer to 2-oxooctanoic acid in a simultaneous one-pot two-step
cascade in up to >99% without accumulation of hydroxyacid
intermediate. Scale-up allowed isolation of final product in 91%
isolated yield and the cascade was applied to fatty acids of various
chain lengths (C6:0-C10:0).
P450CLA and OleTJE.[9] Due to the generally poor stability of
proteins (in particular the heme group itself) towards H2O2,[8,10]
stoichiometric use of this oxidant in enzymatic oxidative
processes is not desired. In situ H2O2 generation protocols
relying on the reduction of oxygen exist and can be cathodic[11]
or mediated by the coupled enzymatic[12] or flavin-mediated light-
driven[13] oxidation of a sacrificial electron donor. To the best of
our knowledge, no enzymatic internal H2O2 recycling protocol
has been described, in which H2O2 is consumed and
regenerated within a single enzymatic cascade that does not
rely on a sacrificial co-substrate.
The functionalization of fatty acids to -ketoacids is attractive
from a synthetic standpoint as follow-up chemistry can grant
access to a multitude of renewable-based synthons: amino acids,
aldehydes, amines, C1-truncaded (odd-membered) carboxylic
acids or even alkanes.[1b] To overcome above-mentioned
shortcomings of H2O2-mediated enzymatic oxidation protocols,
we envisioned a one-pot two-step enzymatic cascade[14] for the
conversion of saturated fatty acids to -ketoacids.
Regioselective -hydroxylation by P450 monooxygenase in the
peroxygenase mode was combined to enantioselective oxidation
of the hydroxyacid intermediate by -hydroxyacid oxidase. In
theory, only catalytic amounts of peroxide are necessary to kick-
start the reaction, which overall relies on the four-electron
reduction of oxygen, releasing water as sole by-product
(Scheme 1).
Fatty acids obtained from renewable resources constitute a
large pool of abundant homogeneous carbon source, which
upon functionalization can be incorporated in further synthetic
transformations towards bio-based chemicals.[ 1 ] The use of
biocatalytic tools combined with strategies to minimize
requirement for stoichiometric cofactors and/or reagents overall
contributes to improving the sustainability of such processes:
renewable resources as starting materials, mild reaction
conditions and high atom-efficiency. Cytochrome P450
enzymes[2] are effective in the oxyfunctionalization of C-H bonds
in fatty acids with terminal to -regioselectivity.[3] Their practical
applicability is however impaired by the dependence on complex
electron transport chains to mediate reductive oxygen activation
by the heme prosthetic group.[4] A few P450s known to act as
peroxygenases (CYP152 family)[5] utilize H2O2 as shortcut to
generate reactive oxoferryl species compound I via the so-called
Scheme 1. Enzymatic air-oxidation of fatty acids to -ketoacids via internal
H2O2 recycling in a one-pot two-step cascade. P450: P450 monooxygenase in
peroxygenase mode; -HAO: -hydroxyacid oxidase.
[3b]
[7]
peroxide shunt pathway,[6] and include P450SP
,
P450BS,
For the first oxidative step, P450 from Clostridium
acetobutylicum (P450CLA) was selected for its reported activity in
the regioselective -hydroxylation of fatty acids.[8] P450CLA was
prepared according to published procedure[ 15 ] and used in
purified form for the hydroxylation of octanoic acid (1) in the
presence of increasing H2O2 concentration. Regioselective -
hydroxylation was confirmed and 2-hydroxyoctanoic acid (2) was
detected as sole newly-formed product. The reaction was
particularly efficient when using sub-stoichiometric amounts of
peroxide (0.03-0.1 eq.), monitored by (up to) full incorporation of
oxygen (ratio [2]/[H2O2] ~ 1, Figure 1). Increasing the oxidant
concentration expectedly resulted in enhanced conversion level,
concomitant with decreasing [2]/[H2O2] ratio, indicating less
efficient hydroxylation, due to partial decomposition of
[a]
Dr. S. Gandomkar, Dr. A. Dennig, Dr. A. Dordic, L. Hammerer, M.
Pickl, Dr. M. Hall, Prof. Dr. K. Faber
Department of Chemistry
University of Graz
Heinrichstrasse 28, 8010 Graz (Austria)
E-mail: melanie.hall@uni-graz.at or kurt.faber@uni-graz.at
Dr. A. Dordic, L. Hammerer
Austrian Center of Industrial Biotechnology c/o
Department of Chemistry
University of Graz
Heinrichstrasse 28, 8010 Graz (Austria)
Dr. T. Haas
[b]
[c]
Creavis, Evonik Industries, Bau 1420
Paul Baumann Strasse 1, 45772 Marl (Germany)
Supporting information for this article is given via a link at the end of
the document.
16
]
peroxide[
and/or catalyst inactivation.[8] P450CLA
-
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10.1002/anie.201710227
Angewandte Chemie International Edition
COMMUNICATION
enantioselectivity was independent of conversion levels and
consistently furnished (S)-2 with ~ 36% ee. This set a key
requirement for successful quantitative conversion of 1 into 3 in
a cascade setup (Scheme 1), as formation of intermediate
product 2 with moderate ee values necessitates the employment
of two stereocomplementary oxidases in the second step of the
cascade. To that end, (S)-specific -hydroxyacid oxidase from
Aerococcus viridans [(S)--HAO][17] and a newly identified D-
lactate oxidase from Gluconobacter oxydans (GO-LOX)[ 18 ]
reported (R)-specific on several short-chain 2-hydroxyacids [(R)-
-HAO] were selected. Both -HAOs were tested for activity on
rac-2 in presence of a catalase and were found active and highly
enantioselective. Both (R)- and (S)-2 were obtained in
enantiomerically pure form upon action of (S)--HAO and GO-
LOX, respectively (Schemes S1-S2 and Figure S5).
furnishes C1-truncated fatty acids and requires keeping H2O2
concentration low (Table S1). Initial trials performed with 5-10
µM P450CLA combined with 0.5 mg/mL (S)--HAO (~ 12 µM) and
2.0 mg/mL GO-LOX (~ 31 µM) led to 80-89% conversion of 1 to
3 within 24 h without detectable amount of 2. This corresponds
to ~ 8 full cycles of the peroxide and a proof-of-principle for the
internal enzymatic H2O2 recycling (Table S4). Further tests
focused on improving both H2O2 turnover number and extent of
conversion by varying oxidant and enzyme concentrations.
Under optimized conditions, 77% and 98% conversion levels
could be obtained using 0.1 and 0.3 eq. of H2O2, respectively
(Table 1, entries 4-5 and Tables S5-S6). Thus, the combination
of three enzymes in tandem allowed internal recycling of the
peroxide, leading to close to quantitative conversion of the fatty
acid. The recycling was more efficient at low peroxide
concentration (higher TONH2O2, Table 1, entries 1-3).
The cascade combining both oxidation steps in
a
simultaneous one-pot fashion was first tested with P450CLA and
(S)--HAO with sub-stoichiometric amounts of H2O2. Analysis of
the conversion of 1 revealed formation of final oxo-product 3
along with accumulation of intermediate product 2. Importantly,
the data confirmed that internal recycling of H2O2 is possible and
conversion to 3 is only limited by the poor enantioselectivity of
P450CLA (max. 36% ee towards (S)-2, i.e. 68/32 ratio of (S)/(R)-
enantiomers), furnishing max. 68% of 3 and 32% of 2 using 0.32
eq. of H2O2 (Table S3). Notably, the amount of accumulated (R)-
2 accurately matched with that of peroxide used, highlighting
(R)-2 as dead-end pathway when employing the two enzymes.
Table 1. Enzymatic oxidation of 1 to 3 via internal H2O2 recycling performed in a
three-enzyme (A) or two-enzyme (B) one-pot cascade.
100
80
60
40
20
0
1.4
1.2
1
Cascade
H2O2 [eq.]
0.01
A. Three enzymes[a]
B. Two enzymes[b]
Entry
Conv. [%]
TONH2
Conv. [%]
TONH2
O
2
O
2
0.8
0.6
0.4
0.2
0
1
2
3
4
5
20
30
57
77
98
20.0
15.0
11.4
7.7
n.d.
14
35
66
99
n.d.
7.0
7.0
6.6
3.3
0.02
0.5
0.1
0.3
3.3
0
0.2
0.4
0.6
0.8
1
H2O2 / molar eq. →
Reactions conditions: [a] 10 mM 1, H2O2 as indicated, 5 µM P450CLA, 0.1 mM
FMN, 12 µM (S)--HAO, 3 µM GO-LOX, Pi buffer (pH 7.4, 100 mM), 10%
EtOH (co-solvent), r.t., 24 h, 170 rpm. [b] 10 mM 1, H2O2 as indicated, 5 µM
P450SPα, 0.1 mM FMN, 24 µM (S)--HAO, Pi buffer (pH 7.4, 100 mM), 10%
EtOH (co-solvent), r.t., 24 h, 170 rpm. n.d. not determined. Conversion (%) to
final product (yield).
Figure 1. Oxidation of octanoic acid (1) to 2-hydroxyoctanoic acid (2) by
P450CLA. Reaction conditions: 5 µM P450CLA, 10 mM 1, H2O2 as indicated, Pi
buffer (pH 7.4, 100 mM), 10% EtOH (co-solvent), r.t., 24 h, 170 rpm.
X
conversion; ○ ee of (S)-2; ■ ratio [2]/[H2O2].
The addition of (R)--HAO to prevent accumulation of (R)-2
rendered a three-enzyme one-pot cascade, which was first
tested with 0.1 eq. of H2O2 (1 mM) and 10 mM octanoic acid.
The adjustment of all three enzyme concentrations was crucial
Initiation of the cascade by an enantioselective -
hydroxylation yielding enantiopure 2-hydroxyacid implies that
only a single oxidase is necessary in the second oxidative step.
In an attempt to simplify the system, P450SPwas selected in
place of P450CLA. The conversion of 1 was confirmed to proceed
with high enantioselectivity[19] towards formation of (S)-2 (>99%
ee, Table S2 and Figure S6) with comparable efficiency
compared to P450CLA (Figure 1). The two-enzyme one-pot
to avoid accumulation of 2-hydroxyacid by maintaining
a
constant supply of H2O2 (i.e. fast second oxidation step), while
the first step should be rapid enough to avoid decomposition[16]
of H2O2. An additional issue is the sensitivity of 2-oxoacids to
(non-enzymatic)
H2O2-mediated
decarboxylation,
which
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10.1002/anie.201710227
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COMMUNICATION
cascade was tested at various concentrations of H2O2, and full
conversion could be obtained using 0.3 eq. of peroxide (i.e.
0.01% solution) after 24 h reaction time (Table 1, entry 5).
Detailed analysis of the reaction progression at lower peroxide
concentrations (Figure 2) revealed that the reaction, although
incomplete, ceased after a few hours (max. 66% conversion
(cascade B) seems more tolerant to elevated fatty acid
concentration, as improved conversion levels compared to
cascade A starting at 30 mM substrate concentration could be
reached with no accumulation of intermediate product 2.
Table 2. Single vs. double addition of H2O2 in the conversion of 1 to 3 in the
three-enzyme (A) or two-enzyme (B) cascade setup (24 h reaction time).
after
4 h using 0.1 eq. H2O2), which was attributed to
spontaneous disproportionation of H2O2,[16] thereby depleting the
reaction mixture of oxidant. A second portion of peroxide was
added in both cases after 5 h (0.5 and 1 mM H2O2, Table 2).
Comparison of conversion levels obtained after single and
double addition of peroxide clearly showed that both enzymes
were still active after 5 h and more importantly, H2O2 supply was
indeed limited since in both cases (0.5 and 1 mM), a significant
increase in conversion was observed after the second addition
of peroxide (Table 2, entries 1-2 and 3-4). Also, comparison of
peroxide turnover numbers reveals that at comparable total
H2O2 supply (1 mM in total), the recycling is more efficient in the
double addition mode, with ~ 25% more product formed (Table 2,
entries 2&3). Similarly, double peroxide addition boosted
conversion levels tested in the three-enzyme cascade (Table 2)
and led to more efficient oxidation at comparable final H2O2
concentration. Collectively, the data suggest that H2O2
decomposition is the major factor limiting the efficiency of the
internal recycling at low peroxide concentration.
Cascade
A. Three enzymes[b]
B. Two enzymes[c]
Entry
H2O2 [mM]
Conv. [%][b]
TONH2
Conv. [%][b]
TONH2
O
2
O
2
1
2
3
4
0.5
60
92
81
99
12.0
9.2
33
76
61
95
6.6
7.6
6.1
4.8
0.5 + 0.5[a]
1
8.1
1 + 1[a]
5.0
[a] Second H2O2 portion added after 5 h. Reaction conditions: [b] 10 mM 1,
H2O2 as indicated, 5 µM P450CLA, 0.1 mM FMN, 24 µM (S)--HAO, 15 µM GO-
LOX, Pi buffer (pH 7.4, 100 mM), 10% EtOH (co-solvent), r.t., 170 rpm. [c]
H2O2 as indicated, 5 µM P450SPα, 0.1 mM FMN, 24 µM (S)--HAO, Pi buffer
(pH 7.4, 100 mM), 10% EtOH (co-solvent), r.t., 170 rpm. Conversion (%) to
final product (yield).
100
80
60
40
20
0
Table 3. Conversion of 1 to 3 at various substrate concentrations in presence
of 1.0 mM H2O2 in both oxidative cascade setups.
Cascade
[1] [mM]
10
A. Three enzymes[a]
B. Two enzymes[b]
[c]
[c]
Entry
Conv. [%]
TONH2O2
Conv. [%]
TONH2O2
1
2
3
4
5
79
53
25
10
3[d]
7.9
66
38
30
20
11
6.6
7.6
8.9
8.0
5.7
20
10.6
7.5
0
5
10
15
20
30
Time [h] →
40
4.1
50
n.a.
Figure 2. Time profile of conversion of 1 to 3 in the two-enzyme cascade set-
up (B) at varying H2O2 concentration: (▲) 0.05. eq., (■) 0.1 eq., (●) 0.3 eq.
(only traces of 2 detected). Reaction conditions: 10 mM 1, 5 µM P450SPα, 0.1
mM FMN, 24 µM (S)--HAO, Pi buffer (pH 7.4, 100 mM), 10% EtOH (co-
solvent), r.t., 170 rpm.
Reaction conditions: [a] 1 as indicated, 10 µM P450CLA, 0.1 mM FMN, 12
µM (S)--HAO, 15 µM GO-LOX, Pi buffer (pH 7.4, 100 mM), 10% EtOH
(co-solvent), r.t., 24 h, 170 rpm. [b] 1 as indicated, 5 µM P450SPα, 0.1 mM
FMN, 24 µM (S)--HAO, Pi buffer (pH 7.4, 100 mM), 10% EtOH (co-
solvent), r.t., 24 h, 170 rpm. [c] Corresponds to amount of 3 formed (mM).
[d] Hydroxyproduct 2 as final product. n.a. not applicable. Conversion (%)
to final product (yield).
Finally, both oxidative cascades were implemented at higher
substrate concentrations, aiming at increasing the productivity of
the system (Table 3). The peroxide concentration was set to 1.0
mM in all cases to allow comparison of the recycling efficiency at
varying substrate concentration. Internal recycling of H2O2 was
most efficient on 20 mM substrate in the three-enzyme cascade
(Table 3, entry 2), furnishing highest amount of product 3 (10.6
mM, 1.7 g/L). In both cascades, conversion levels decreased as
substrate concentration increased. Importantly, while inhibition
can be confidently expected in the three-enzyme cascade at 50
mM of 1 (no formation of final product 3 observed along with low
amount of intermediate 2 detected), the two-enzyme system
Scale-up was performed and yielded the following: After 17 h,
cascade A (60 mL) led to 98% conversion of 1 resulting in 74%
isolated yield (70 mg of 3 in 90% purity) and cascade B (35 mL)
allowed recovery of 50 mg of 3 in 97% conversion level and 91%
isolated yield (92% purity, see ESI and Figures S13-S15).
Finally, fatty acids of varying chain lengths (C6:0, C7:0 and
C10:0) were tested in both set-ups and could be successfully
transformed to their 2-oxoacids. Decanoic acid was most
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10.1002/anie.201710227
Angewandte Chemie International Edition
COMMUNICATION
reactive and was converted in up to 93% to 2-oxodecanoic acid
in a 0.01% peroxide solution (Table S8).
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The internal recycling of H2O2 could be successfully
implemented in a three- and two-enzyme one-pot cascade for
the conversion of saturated fatty acids to -ketoacids. The
system displays attractive features, notably the use of peroxide
as 'catalytic' reagent in sub-stoichiometric amounts, as well as
high-atom efficiency and sustainable character of the reaction,
producing water as sole by product and using air as major
oxidant. With this setup, the use of P450 monooxygenases in
the peroxygenase mode at low H2O2 concentration (≤0.01%)
appears attractive for synthetic applications, owing to improved
enzyme lifetime. Additionally, non-enzymatic oxidative
decarboxylation of 2-oxoacid products is completely avoided and
no reaction engineering technique (e.g. in situ product removal)
is required. Altogether, the biocatalytic oxidative cascade
combining P450s in the peroxygenase mode with -hydroxyacid
oxidase(s) is a promising tool for functionalization of bio-based
chemicals and contributes to more sustainable synthetic routes.
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Experimental Section
Reactions were performed in duplicates in closed glass-vials in 1 mL
buffer (KPi, 100 mM, pH 7.4), as described in the text. Derivatization for
GC and GC-MS analysis was performed by silylation of the organic
phase (Figure S8-S12). Protocols for production of all biocatalysts,
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Acknowledgements
Prof. Xu and Dr. Gao (Shandong University, China) are thanked
for plasmid preparation of GO-LOX. Tamara Reiter is thanked
for her excellent assistance. Financial support by the Austrian
Science Fund (FWF) within the DK “Molecular Enzymology”
program (project no. W901) is gratefully acknowledged. Funding
by the Austrian BMWFW, BMVIT, SFG, Standortagentur Tirol,
Government of Lower Austria and ZIT through the Austrian
FFG-COMET-Funding Program is gratefully acknowledged.
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Keywords: P450 • 2-hydroxyacid oxidase • H2O2 recycling •
biocatalysis • bio-based chemicals
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10.1002/anie.201710227
Angewandte Chemie International Edition
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S. Gandomkar, A. Dennig, A. Dordic, L.
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Biocatalytic Oxidative Cascade for
the Conversion of Fatty Acids to -
Ketoacids via Internal H2O2 Recycling
An atom-efficient biocatalytic oxidative cascade that combines a P450
monooxygenase in the peroxygenase mode with -hydroxyacid oxidase(s) was
developed for the conversion of saturated fatty acids to -ketoacids and resulted in
internal recycling of the oxidant H2O2. Overall, the O2-dependent cascade relies on
catalytic amounts of H2O2 and releases water as sole by-product.
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