Enzymatic One-Step Reduction of Carboxylates to Aldehydes with Cell-Free Regeneration of ATP and NADPH

Article abstract of DOI:10.1002/chem.201901147

The direct generation of aldehydes from carboxylic acids is often a challenging synthetic task but undoubtedly attractive in view of abundant supply of such feedstocks from nature. Though long known, biocatalytic carboxylate reductions are at an early stage of development, presumably because of their co-factor requirement. To establish an alternative to whole-cell-based carboxylate reductions which are limited by side reactions, we developed an in vitro multi-enzyme system that allows for quantitative reductions of various carboxylic acids with full recycling of all cofactors and prevention of undesired over-reductions. Regeneration of adenosine 5′-triphosphate is achieved through the simultaneous action of polyphosphate kinases from Meiothermus ruber and Sinorhizobium meliloti and β-nicotinamide adenine dinucleotide 2′-phosphate is reduced by a glucose dehydrogenase. Under these conditions and in the presence of the carboxylate reductases from Neurospora crassa or Nocardia iowensis, various aromatic, heterocyclic and aliphatic carboxylic acids were quantitatively reduced to the respective aldehydes.

Full text of DOI:10.1002/chem.201901147

A Journal of
Accepted Article
Title: Enzymatic one-step reduction of carboxylate to aldehyde with
cell-free regeneration of ATP and NADPH
Authors: Gernot Strohmeier, Inge C. Eiteljörg, Anna Schwarz, and
Margit Winkler
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To be cited as: Chem. Eur. J. 10.1002/chem.201901147
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Enzymatic one-step reduction of carboxylate to aldehyde
with cell-free regeneration of ATP and NADPH
Gernot A. Strohmeier, Inge C. Eiteljörg, Anna Schwarz and Margit Winkler*[a]
Abstract: The direct generation of aldehydes from carboxylic acids is
an often challenging synthetic task but undoubtedly attractive in view
of abundant supply of such feedstocks from nature. Though long
known, biocatalytic carboxylate reductions are at an early stage of
development, presumably because of their co-factor requirement. To
establish an alternative to whole-cell-based carboxylate reductions
which are limited by side reactions, we developed an in vitro multi-
enzyme system that allows for quantitative reductions of various
carboxylic acids with full recycling of all cofactors and prevention of
undesired over-reductions. Regeneration of adenosine 5’-
triphosphate is achieved through the simultaneous action of
polyphosphate kinases from Meiothermus ruber and Sinorhizobium
meliloti and β-nicotinamide adenine dinucleotide 2’-phosphate is
reduced by a glucose dehydrogenase. Under these conditions and in
the presence of the carboxylate reductases from Neurospora crassa
or Nocardia iowensis, various aromatic, heterocyclic and aliphatic
carboxylic acids were quantitatively reduced to the respective
aldehydes.
via chemoselective and mild activation under aqueous conditions.
CARs leave various functional groups and unsaturated moieties
untouched. These reductases are comprised of three domains
and possess the desirable feature of directly reducing carboxylic
acids into aldehydes without the need for any external pre-
activation. Mechanistically, the carboxylate is activated first in an
adenylation domain of the CAR as a mixed anhydride of the
carboxylic acid and adenosine monophosphate. Next, the
carboxylate is transferred to a 4’-phosphopantetheinyl residue in
the respective binding domain, thereby forming an enzyme-bound
thioester. Finally, NADPH-dependent reduction in the reductase
domain generates the corresponding aldehyde.[5] The overall
reduction of one equivalent of carboxylic acid consumes
equimolar amounts of NADPH and ATP and produces AMP,
NADP+, pyrophosphate and aldehyde. Whereas NADPH
recycling is well established, ATP recycling systems are still at the
proof of concept level.[6] Because of the obvious complexity
associated with the simultaneous recycling of NADPH and ATP,
the only economic way was to utilize CARs embedded in living
microbial cells and exploit their cellular metabolism for co-factor
regeneration. Processes involving microbial cells have many
advantages, but they also have drawbacks such as competing
metabolic pathways, kinetic restrictions due to physical barriers or
aldehyde-related toxicity to the biological system.[7] Host
background activities can annihilate perfect aldehyde yields by
detoxification mechanisms, leading e.g. to undesired over-
reduction.[8] In vitro systems on the other hand require the
imitation of all relevant reaction steps by applying appropriate
enzymes.[9] They provide advantages in testing and optimizing the
reaction conditions, ideally allowing reducing - if not eliminating -
side reactions of the starting materials and aldehyde products. On
the other hand, possible incompatibilities with reagents required
to regenerate the co-factors and pH shifts during the reaction
Carboxylic acids represent a class of compounds that are
produced in large quantities in nature in the form of fatty acids,
citric acid, amino acids and many more. Consequently, they serve
as attractive starting materials to promote the implementation of
bio-based pathways in chemistry and related sciences. Their
applications as free acids or - after transformation to derivatives
with unchanged oxidation state - corresponding esters and
amides are well established. By contrast, selective reductions of
the carboxylate group are often not amenable in a direct way
towards aldehydes. The most frequent way to obtain aldehydes
from carboxylic acids is to perform a sequence consisting of ester
formation, reduction to the alcohol and selective re-oxidation to
the desired product. Poor atom economy and the use of reagents
like LiAlH4 for the reduction,[1] and chromine(VI) reagents, Dess-
Martin periodinane or Swern conditions for the re-oxidation[2] rise
concerns when considering the compatibility with functional
groups and applications on larger scales. However, apart from
chemical methods, there is a biocatalytic alternative available
through the application of carboxylate reductases (CARs).[3] This
class of enzymes was first described decades ago,[4] and is
gaining increased attention within the last few years. Carboxylate
reductases exhibit the intrinsic advantage of processing reactions
course that would have played no role in
transformation need to be considered.
a whole-cell
Herein, we focused on the selective preparation of aldehydes
as final product in a cell-free system. To make this process viable
for synthesis, the key is to implement a balanced, catalytic
cofactor recycling system for the activation and reduction of
thermodynamically nearly inert carboxylate moieties.
[a]
Dr. G. A. Strohmeier, Dr. I. C. Eiteljörg, A. Schwarz, Priv.-Doz. Dr.
M. Winkler
acib - Austrian Centre of Industrial Biotechnology, Petersgasse 14,
A-8010 Graz
Supporting information for this article is given via a link at the end of
the document.
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80
70
60
50
40
30
20
10
0
Conv.
to ATP
after
1h / %
10 mM
20 mM
30 mM
40 mM
Mg2+ concentration
Scheme 1. Reaction scheme for the in vitro reduction of carboxylic acids with
Figure 1. Regeneration of ATP from AMP (2.0 mM) in the presence of MrPPK
(20 µg/mL) and SmPPK (4 µg/mL) at pH 7.5, 30°C in buffers (100 mM)
containing NaCl (50 mM) and sodium polyP (medium chain length: 25, 60 mM
related to monophosphate units). The buffer systems are as follows: white bar:
HEPES, grey shaded: imidazole-HCl, hatched: MOPS, horizontal lines: PIPES,
black: potassium phosphate and vertical lines: Tris-HCl. An average standard
deviation of <5% conversion was observed.
full recycling of all cofactors. CAR
= carboxylate reductase, PPK =
polyphosphate kinase, GDH = glucose dehydrogenase, PolyP = polyphosphate,
PP = pyrophosphate, PPase = pyrophosphatase, P = ortho-phosphate. For
compound names see Table 2.
In initial test series, we identified polyphosphate kinases
(PPK)[10] as the optimal enzymes to perform the generation of ATP
from AMP in view of the reaction system to be realized (Scheme
1).[11] Full regeneration of ATP was found most efficient when
combining two PPK enzymes, i.e. MrPPK and SmPPK from
Meiothermus ruber[12] and Sinorhizobium meliloti,[13] respectively,
with an excess of MrPPK to obtain similar conversion rates in
each of the two phosphorylation steps (Supporting information
Table S3). Mg2+-ions are essential to adenylate forming
enzymes[14] such as PPKs and CARs but their complexation or
precipitation by several phosphate species in the system must be
assumed, necessitating a determination of the optimal Mg2+ input.
Therefore, the formation of ATP from AMP was monitored at a
defined polyphosphate (polyP) concentration by varying the Mg2+
concentration. Additionally, six different buffer systems were
investigated with the aim to stabilize a pH of 7.5 – a value
compatible to all enzymes and chemical components present in
the designed in vitro carboxylate reduction.
To evaluate the effect of polyP, piperonylic acid 1a reduction
by Neurospora crassa carboxylate reductase (NcCAR) was
studied.[17] All conversions in reactions without polyP were similar
after 2 h at 30°C and almost independent of the buffer type used
(Table 1). Adding sodium polyP (100 mM; Merck type
1.065.291.000, medium chain length n = 25) to identical reaction
mixtures caused a significant drop of all conversions. This effect
was especially strong in case of phosphate buffer. We rationalized
this observation by the enforced complexation of Mg2+ ions by
polyP which, in addition to the ortho-phosphate (P) in the buffer
lowers the amount of freely accessible divalent ions. In the case
of all other buffers tested, this reduction in activity is substantially
lower and especially imidazole-HCl and HEPES seemed to be
most suitable for the carboxylate reduction with concurrent
cofactor regeneration.
In line with reports in the literature,[15] we found increasing
conversions with increasing concentrations of MgCl2 (Figure 1
and Supporting Information Table S4). Beyond about 30 mM Mg2+,
which corresponds to half the concentration of monophosphate
units in polyP, no significant further increase of ATP formation
was found. Consequently, this ratio was used for all following
investigations. Notably, at 30 mM and slightly higher
concentrations, no precipitation of magnesium phosphates
leading to undesired loss of the free divalent ions was visible at
pH 7.5. Tris-HCl was found as overall best and phosphate buffer
the least suitable for ATP recycling.
Table 1. Influence of polyphosphate and different buffer systems on NcCAR-
catalyzed carboxylate reductions of piperonylic acid 1a (5 mM).
Buffer[a]
Conv. / %
no polyP[b]
Conv. / %
100 mM polyP[c]
HEPES
95
94
94
89
94
94
60
64
48
42
14
39
Imidazole-HCl
MOPS
PIPES
One of the challenges in developing a multi-enzyme system
is the identification of the operational window that is dependent
on the requirements of each reaction factor (i.e. pH, T, buffer and
preference of each enzyme, solubility and inhibition, etc.). An
important aspect to be considered is potential cross inhibition of
CAR enzymes by substrates required for the cofactor
regeneration, especially the different phosphate species.[16]
K-phosphate
Tris-HCl
[a] 100 mM buffer containing 50 mM NaCl. [b] Conversion to piperonal 2a
after 2h at 30°C in the presence of NADPH (7.5 mM), ATP (10.0 mM), MgCl2
(10 mM) and NcCAR (50 µg/mL). [c] as described in [b] supplemented with
sodium polyP (100 mM).
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Having investigated in vitro ATP recycling, we went on to
select a suitable reduction system for NADP+. Ideally, this system
operates well at pH 7.5, tolerates moderate to high salt
concentrations and forms only non-interfering co-products. In
view of these requirements, the glucose dehydrogenase (GDH)-
based NADPH formation was selected. Initial experiments with
GDH showed substantial over-reductions to the corresponding
alcohols 3 catalyzed by host protein activities in the tested
commercial GDH preparations. Eventually, the most active GDH
was used at limiting amounts in the complex reaction system
(Scheme 1) to keep the steady-state concentration of NADPH low.
This measure minimized the formation of alcohols 3 to negligible
amounts in most of the investigated cases, allowing to exploit the
full selectivity advantage of CARs to form pure aldehydes.
as shown previously.[16-18] Expecting inhibitory effects of PP in our
investigations, PPase was added to standard reactions.
Under the conditions used, reactions in the presence of
Nocardia iowensis CAR (NiCAR)[19] showed the overall best
performance in converting a set of diverse carboxylic acids 1a-o
comprising substituted benzoic, heterocyclic, α,β-unsaturated
and aliphatic carboxylic acids. Apart from o-substituted benzoic,
cinnamic and aliphatic acids bearing longer chains than C8 were
fully or almost fully converted to the corresponding aldehydes. In
general, the amounts of alcohols formed were below 2% in most
cases (Table 2). NcCAR provided complete conversions only with
a few carboxylic derivatives under these conditions (1a, b, e and
j). One reason for lower conversions with NcCAR in this system
may be the reaction pH, which better overlaps with the optimal
pH-range of NiCAR. In this setup, no significant difference
between the outcome of reactions of 1a with or without PPase
was observed as determined by kinetic and endpoint
comparisons. We hypothesize the high Mg2+ concentration being
the main factor for complexing PP and thereby its inhibitory effect
on CAR is alleviated, again emphasizing on the critical role of
Mg2+ concentrations in this context.
Table 2. Reduction of several carboxylic acids 1 to their corresponding
aldehydes 2 in vitro after 18h at 30°C.
Example
Carboxylic acid 1[a]
C / %
NcCAR[b]
C / %
NiCAR[b]
a
b
c
d
e
f
Piperonylic acid
>99
>99
21[c]
77
>99
>99
66
Benzoic acid
2-Methoxy-benzoic acid
3-Methoxy-benzoic acid
4-Methoxy-benzoic acid
2-Chloro-benzoic acid
3-Chloro-benzoic acid
4-Chloro-benzoic acid
3-Nitro-benzoic acid
2-Thiophenecarboxylic acid
trans-Cinnamic acid
3-Phenyl-propionic acid
Hexanoic acid
>99
>99
65
>99
48
g
h
i
55
>99
>99
>99[c]
>99
34[d]
99
48
16[c]
>99
41[d]
30
j
k
l
m
n
o
20
>99
80
Figure 2. In vitro reduction of 1a (10 mM) to 2a + 3a (minor amount) in the
presence of different concentration of polyP. Reaction conditions: ATP (1.0 mM),
NADPH (0.5 mM), NcCAR (50 µg/mL), MrPPK (100 µg/mL), SmPPK (40 µg/mL),
PPase (25 µg/mL), GDH (0.2 U/mL), 100 mM imidazole-HCl buffer pH 7.5, 30°C,
β-D-glucose (100 mM), MgCl2 (70 mM) and sodium polyP (medium chain length:
25, 0 - 100 mM related to monophosphate units). Circles: overall conversion of
1a, squares: formally consumed fraction of polyP calculated based on the
amount of polyP and ATP initially added and the assumption that polyP is only
consumed in the regeneration of ATP.
Octanoic acid
0
Dodecanoic acid
0
2
[a] Reaction conditions: 10 mM carboxylic acid, ATP (1.0 mM), NADPH
(0.5 mM), NcCAR or NiCAR (50 µg/mL), MrPPK (100 µg/mL), SmPPK
(40 µg/mL), PPase (25 µg/mL), GDH (0.2 U/mL), 100 mM imidazole-HCl
buffer pH 7.5, 30°C, β-D-glucose (100 mM), MgCl2 (70 mM) and sodium
polyP (140 mM related to ortho-phosphate units). [b] Determined by rp-HPLC
analysis; <2% alcohol formed. [c] 5-8% of alcohol formed. [d] Minor amounts
of aldehyde-derived by-products were formed via β-hydration and aldol
reactions.
To estimate the limits for ATP regeneration under the general
reaction conditions, further investigations with varying polyP
concentrations were made. As outlined before, polyP should be
used at lowest possible concentrations to minimize adverse
effects on the Mg2+ supply and, consequently, on the conversion
in CAR reactions. ATP recycling starting from AMP requires two
molecules of ortho-phosphate which are added stepwise by PPKs
from polyP sources, thereby defining the minimum amount of
Assembling both cofactor regeneration reactions and the
carboxylate reduction in vitro, the system was tested with a
number of carboxylic acid substrates (Table 2). Pyrophosphate
(PP) inhibits CARs but this can be circumvented by adding a
pyrophosphate-hydrolyzing enzyme, pyrophosphatase (PPase)
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active phosphate needed. PPKs recently reported to be also able
to transfer more than one phosphate unit in a single event were
not considered for use in this work.[20] In addition to the selection
of PPKs, shorter polyphosphate chains are expected to be no
efficient donors[15] and therefore, an appropriate excess of polyP
must be provided.
volumetric unit in the preparative scale reaction but still keep the
performance maintained.
In summary, we have developed a cell-free system for the
direct reduction of carboxylic acids to aldehydes, reaching full
conversion for the majority of cases investigated. Using three
compatible enzymes, the concurrent formation of ATP and
NADPH with only catalytic amounts of these expensive cofactors
fuels enzymatic reduction of carboxylic acids to up to 100 %
aldehyde. With the protocol described herein, we show in vitro
aldehyde synthesis in an aqueous system, which mimics the
opportunities provided by a whole-cell reduction but avoids their
inherent disadvantages in applications.
Figure 2 clearly shows that a minimum amount of 40 mM
polyP (calculation based on ortho-phosphate units) is required to
reach complete reduction of 10 mM of 1a in vitro. Overall, almost
50% of the polyP (Merck type no. 1.065.291.000) can act as
active phosphate donor when using a combination of MrPPK and
SmPPK for ATP regeneration. In other words, polyphosphate
chains longer than 12 on average can serve as donor.[12b] To
enable a full conversion of the carboxylic acid, an at least five-fold
excess of this specific polyP should be used in the ATP recycling
system. This observation of a practically restricted use of shorter
chain polyphosphates may partially explain incomplete
conversions obtained in an earlier study when sodium
polyphosphate crystals (Aldrich cat # 305553) with an average
chain length of 17, according to the manufacturers product
specification, were applied in a reduction of 10 mM of 4-
hydroxybutanoic acid.[11a] In this reaction system, different CARs
were tested with the formate/formate dehydrogenase (FDH)
system for NADPH regeneration as well as two PPKs for ATP
regeneration and a pyrophosphatase.
Experimental Section
General. ATP was obtained from Carl Roth, PN HN35.2, Lot 347262238;
98.1 % purity, 8.6 % water content, ADP and AMP were obtained from
Roche Diagnostics. NADP+, NADPH were purchased from Carl Roth and
MgCl2 from Merck. HPLC-MS grade acetonitrile was purchased from
J.T.Baker/Avantor Performance Materials (Deventer, The Netherlands)
and VWR (Vienna, Austria). Sodium polyphosphate was obtained from
Merck (Darmstadt, Germany; order no 1.065.291.000; medium chain
length n = 25) and concentrations mentioned in this work are based on
ortho-phosphate units. Glucose dehydrogenase GDH-105 was obtained
from Codexis (Redwood City, CA, USA). All other chemicals were obtained
from Sigma-Aldrich/Fluka or Carl Roth and used without further purification.
Enzymes were prepared as described in the supporting information. Small
scale reactions were performed on a Thermomixer comfort (Eppendorf) in
1.5 mL-polypropylene tubes (Eppendorf, Hamburg, Germany). Flash
column chromatography was performed on silica gel 0.035-0.070 mm, 60
Å (product no. 240360300; Acros Organics, Geel, Belgium). 1H and 13C
NMR spectra were recorded on a Bruker AVANCE III 300 spectrometer
(1H: 300.36 MHz; 13C: 75.53 MHz) and chemical shifts are referenced to
residual protonated solvent signals as internal standard.
The optimized reaction conditions (Supporting Information
section 1.4.4 and 1.4.5) for in vitro reduction of carboxylic acids
were applied at increased substrate concentration. Taking
advantage of the protocol already designed to convert 20 mM
carboxylic acid at the 1 mL scale without any need to increase the
enzyme and reagent concentrations, 25 mM of piperonylic acid
1a were reduced close to completion to piperonal 2a in a 20 mL
reaction. To obtain full conversion at increased carboxylic acid
concentrations in the shortest time possible, the replacement of
imidazole by MOPS buffer at the same pH and the concomitant
use of PPase were crucial.
Preparative scale synthesis of piperonal 2a. To MOPS buffer pH 7.70
(5.0 mL, 400 mM, 100 mM concentration in reaction) in a 50 mL-
polypropylene
tube
(CELLSTAR®,
Greiner
Bio-One
GmbH,
Kremsmünster, Austria) was added water (10.83 mL), piperonylic acid 1a
(83.1 mg, 500 µmol, 2.00 mL of a 250 mM stock solution in 250 mM NaOH),
NADP+ (8.5 mg, 10.0 µmol), ATP (12.3 mg, 20.0 µmol), MgCl2 (133.3 mg,
1.40 mmol), β-D-glucose (541mg, 3.00 mmol), sodium polyphosphate (288
mg, 2.80 mmol based on ortho-phosphate units) and bromothymol blue
solution (50 µL, 8.0 mM in 20 mM potassium phosphate buffer pH 7.50).
The reaction was started by adding sequentially GDH-105 (8.0 µL, 8 U,
1000 U/mL in 20 mM in potassium phosphate buffer pH 7.50), NiCAR (429
µL, 1.50 mg), MrPPK (151 µL, 1.00 mg), SmPPK (116 µL, 0.40 mg),
EcPPase (18.5 µL, 0.20 mg) and then gently stirred with a floating stirring
bar under temperature control at 30 °C. Through the stepwise addition of
NaOH (900 µL in total, 1.8 mmol, 2.0 M in water), the pH was kept between
7.0 and 7.5, as visualized by the bromothymol blue color. After 24 h, the
conversion had reached more than 90% as checked by rp-HPLC analysis
and the product was extracted with ethyl acetate (3× 20mL). The combined
extracts were dried over sodium sulfate, then concentrated under reduced
pressure and finally separated using flash chromatography (12.0 g silica
gel, 18 × 1.3 cm column size, cyclohexane/ethyl acetate = 3:1 (v/v)). All
fractions containing the product 2a (Rf = 0.51; cyclohexane/ethyl acetate
3:1 (v/v)) were pooled and concentrated under reduced pressure. The yield
was 57.0 mg (76 %) of 2a as colorless solid. Mp 37-38 °C (commercial 2a,
mp 38°C). 1H-NMR (300 MHz, CDCl3): δ = 6.08 (2H, s, -CH2-), 6.93 (1H,
d, 3J(H,H) = 7.9 Hz, C3-H), 7.33 (1H, d, 4J(H,H) = 1.3 Hz, C6-H), 7.41 (1H,
Scheme 2. Reaction scheme for the in vitro reduction of piperonylic acid 1a
(25 mM) in the presence of NADP+ (0.5 mM), ATP (1.0 mM), MgCl2 (70 mM),
polyP (140 mM) and β-D-glucose (150 mM) at 30°C.
In contrast to the reactions at 10 mM carboxylic acid
concentrations, the preparative scale variant required continuous
control and adjustment of the pH to keep within the optimal range
of 7.0-7.5. Moreover with a target time of one day for full
conversion, it was possible to reduce the enzyme amounts per
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dd, 3J(H,H) = 7.9 Hz, 4J(H,H) = 1.3 Hz, C4-H). 13C-NMR (75 MHz, CDCl3):
δ = 102.3, 107.1, 108.5, 128.8, 132.1, 148.9, 153.3, 190.4.
[8]
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Acknowledgements
This work has been supported by the Federal Ministry of Science,
Research and Economy (BMWFW), the Federal Ministry of Traffic,
Innovation and Technology (bmvit), the Styrian Business
Promotion Agency SFG, the Standortagentur Tirol, the
Government of Lower Austria and Business Agency Vienna
through the COMET-Funding Program managed by the Austrian
Research Promotion Agency FFG. A.S. received funding from the
Austrian Research Promotion Agency FFG (FEM-Tech stipend,
grant no. 870897). We thank Jun. Prof. Jennifer N. Andexer for
generously providing the genes coding for polyphosphate kinases
and Prof. Bernd Nidetzky for pyrophosphatase.
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Keywords: aldehyde • in vitro ATP generation • enzyme
cascade • carboxylate reductase • multi-enzyme catalysis
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10.1002/chem.201901147
Chemistry - A European Journal
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COMMUNICATION
Aldehydes are directly synthesized
from carboxylic acids by carboxylate
reductases operating in combination
with recycling systems for NADPH
and ATP in an in vitro fashion.
G. A. Strohmeier, I. C. Eiteljörg, A.
Schwarz, M. Winkler*
Page No. – Page No.
Enzymatic one-step reduction of
carboxylate to aldehyde with cell-free
regeneration of ATP and NADPH
((Insert TOC Graphic here))
Layout 2:
COMMUNICATION
G. A. Strohmeier, I. C. Eiteljörg, A.
Schwarz, M. Winkler*
Page No. – Page No.
Enzymatic one-step reduction of
carboxylate to aldehyde with cell-free
regeneration of ATP and NADPH
Text for Table of Contents
Aldehydes are directly synthesized from carboxylic acids by carboxylate reductases operating in combination with recycling systems
for NADPH and ATP in an in vitro fashion.
This article is protected by copyright. All rights reserved.