Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

Article abstract of DOI:10.1002/adsc.201600914

The enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. We have increased the spectrum of carboxylate-reducing enzymes (CARs) with the sequence of a fungal CAR from Neurospora crassa OR74A (NcCAR). NcCAR was efficiently expressed in E. coli using an autoinduction protocol at low temperature. It was purified and characterized in vitro, revealing a broad substrate acceptance, a pH optimum at pH 5.5–6.0, a T_mof 45 °C and inhibition by the co-product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of NcCAR was demonstrated in a whole-cell biotransformation using the Escherichia coli K-12 MG1655 RARE strain in order to suppress overreduction to undesired alcohol. The fragrance compound piperonal was prepared from piperonylic acid (30 mM) on gram scale in 92 % isolated yield in >98% purity. This corresponds to a productivity of 1.5 g/L/h. (Figure presented.).

Full text of DOI:10.1002/adsc.201600914

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DOI: 10.1002/adsc.201600914
Selective Enzymatic Transformation to Aldehydes in vivo by
Fungal Carboxylate Reductase from Neurospora crassa
Daniel Schwendenwein,a Giuseppe Fiume,b Hansjçrg Weber,c Florian Rudroff,d
and Margit Winklera,b,*
a
acib GmbH, Petersgasse 14, 8010 Graz, Austria
Fax : (+43)-316-873-9302; phone: (+43)-316-873-9333; e-mail: margit.winkler@acib.at
Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, Petersgasse 14, 8010 Graz, Austria
Institute of Organic Chemistry, Graz University of Technology, NAWI Graz, Stremayrgasse 9, 8010 Graz, Austria
Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9/OCÀ163, 1060 Vienna, Austria
b
c
d
Supporting information for this article can be found under: http://dx.doi.org/10.1002/adsc.201600914.
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA. This is an open access article under
the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in
any medium provided the original work is properly cited.
Abstract: The enzymatic reduction of carboxylic thetic utility of NcCAR was demonstrated in
acids is in its infancy with only a handful of biocata- a whole-cell biotransformation using the Escherichia
lysts available to this end. We have increased the coli K-12 MG1655 RARE strain in order to suppress
spectrum of carboxylate-reducing enzymes (CARs) overreduction to undesired alcohol. The fragrance
with the sequence of a fungal CAR from Neurospora compound piperonal was prepared from piperonylic
crassa OR74A (NcCAR). NcCAR was efficiently ex- acid (30 mM) on gram scale in 92% isolated yield in
pressed in E. coli using an autoinduction protocol at >98% purity. This corresponds to a productivity of
low temperature. It was purified and characterized in 1.5 g/L/h.
vitro, revealing a broad substrate acceptance, a pH
optimum at pH 5.5–6.0, a Tm of 458C and inhibition
by the co-product pyrophosphate which can be alle- Keywords: aldehydes; biocatalysis; carboxylate re-
viated by the addition of pyrophosphatase. The syn- ductase; carboxylic acids; flavours and fragrances
Introduction
ed from the fungus and characterized.[20,22] The clas-
sification of this enzyme as aryl-aldehyde:NADP oxi-
Carboxylic acids show little reactivity and require doreductase[21] was based on its substrate scope which
a high level of activation to participate in chemical re- appeared to be restricted to compounds with aromatic
actions. Particularly their direct reduction is a strongly ring structures. Aliphatic acids and amino acids were
endergonic process. A mild and selective alternative not reduced by this enzyme.[23] The carboxylate reduc-
to chemical reduction protocols[1–3] is the enzymatic tion is dependent on ATP, NADPH and magnesium
reduction of carboxylic acids. Carboxylate reductase ions (Scheme 1), leading eventually to its classifica-
(CAR) enzymes exhibit a broad substrate tolerance tion as an E.C. 1.2.1.30 enzyme. These E.C. 1.2.1.30
for the conversion of organic acids to the respective
aldehydes.[4] However, only few CAR enzyme sequen-
ces have been elucidated[5–12] and are available for
biocatalysis to date, although Nature provides a great
versatility of organisms with the capability to catalyze
this reaction.[4,13–18] The reduction of salicylic acid,[19]
benzoic acid and derivatives[20] as well as cinnamic
acid and derivatives[21] has been observed in the asco-
mycete Neurospora crassa (Nc). The enzyme that was
able to reduce aromatic carboxylates has been isolat- dehydes by Neurospora crassa SY7A CAR.
Scheme 1. Reduction of carboxylic acids to the respective al-
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type CARs are comprised of three domains: an ade-
nylation domain (A-domain), a phosphopantetheinyl
binding domain, and a reductase domain (R-domain).
They require post-translational modification of the re-
spective domain by phosphopantetheinylation.[24]
Aldehydes such as vanillin and hexanal account for
flavour and fragrance sensing and are commercially
used in the food and perfumery industry. Typically, al-
dehydes are reactive moieties and can undergo many
chemical reactions such as reductive amination, aldol
reactions, reduction to alcohol, acetal formation,
Grignard reaction and many others. Therefore, they
are widely employed as building blocks and used as
precursors to pharmaceuticals and agrochemicals. Pi-
peronal (Heliotropin) is an aromatic aldehyde used in
many fragrances and also as a flavour component.[25]
It can be found in some essential oils, vanilla and
camphor,[25–27] and has been shown to have antibacte-
rial[28] and anxiolytic effects.[29] Moreover, some pi-
peronal derivatives show
a potential anticancer
effect,[30,31] antileishmanial activity,[32] and other phar-
macological effects.[33]
Figure 1. SDS 4–12% Bis-Tris-gel of NcCAR preparations
(1 h at 200 V in MOPS-buffer; staining: SimplyBlueTM Safe-
Stain solution). Lane 1: cell free extract. Lane 2: cell pellet
dissolved in 6 M urea. Lane 3: PageRulerTM Prestained Pro-
tein Ladder (10–140 kDa). Lane 4: NcCAR after Ni-affinity
chromatography. Lane 5: NcCAR after removal of the His-
Tag with TEV-protease and Ni-affinity chromatography.
Lane 6: NcCAR from lane 4 after gel filtration.
Results and Discussion
With the aim to identify the primary sequence of
Neurospora crassa CAR, four literature known CAR
enzyme
sequences
(NiCAR:[5]
Q6RKB1.1,
MmCAR:[6] B2HN69, SrCAR:[8] WP_013138593.1 and
AtCAR:[7] XP_001212808.1) were used as templates
for a blastp search against the Gen-Bank non-redun-
dant protein sequences of Neurospora crassa another fungal CAR from Trametes versicolor under
(taxid:5241). The hit with highest scores was a hypo- identical conditions.[10] Other published fungal CARs
thetical protein with NCBI accession code ATEG03630 (AnCAR) and StbB (SbCAR) have
XP_955820.1. On the sequence level, this protein been heterologously expressed in S. cerevisiae[7] and
showed low identities with the original search tem- Aspergillus oryzae,[12] respectively.
plates NiCAR, MmCAR, SrCAR and AtCAR
To confirm that XP_955820.1 codes for a carboxyl-
(17.3%, 17.5%, 18.5% and 22.5%, respectively), and ate reductase, a spectrophotometric NADPH deple-
also low identities with CARs that have been pub- tion assay was applied to screen for activity towards
lished during this study: MnCAR,[9] TvCAR,[10] the reduction of a panel of carboxylic acids (Table 1).
MsCAR,[11] NbCAR[11] and SbCAR[12] (16.4%, 26.1%, For certain substrates, however, the spectrophotomet-
17.7%, 17.3% and 22.4%, respectively). A codon opti- ric assay was not suitable due to absorption of the
mized synthetic gene coding for XP_955820.1 from N. acid or the aldehyde at the detection wavelength or
crassa OR74A was expressed with an N-terminal due to their low solubility in aqueous system. Vanillic
TEV cleavable HIS-tag from the multiple cloning site acid, e.g., is reduced to vanillin by NcCAR, as deter-
2 of the pETDUET1 vector. For the essential post- mined by HPLC analyses.
translational modification of CAR enzymes,[24] E. coli
The spectrophotometric assay was also used to de-
phosphopantetheinyl transferase (NCBI accession termine the biochemical characteristics of NcCAR in
code CAQ31055.1) was simultaneously expressed vitro. The highest initial rate activity was observed at
from multiple cloning site 1.[10] E. coli BL21 (DE3) 558C, however, stability measurements indicated com-
Star served as the host and the cells were cultivated plete loss of activity upon incubation of the enzyme
under autoinduction conditions.[34] The expected at this temperature for 90 min, whereas the residual
120 kDa protein was termed NcCAR and purified via activity after incubation at 358C was 84Æ17%. This is
Ni-affinity
(Figure 1). Notably, NcCAR was expressed very effi-
ciently in E. coli as a soluble protein, in contrast to from N. crassa was pH 7.7–8.1 as determined using
chromatography
and
gel-filtration consistent with the Tm of NcCAR of 45.38C.
The published pH optimum of the enzyme purified
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4.57 sÀ1 and vmax 2.33Æ0.06 mmolminÀ1 mgÀ1. The Km
value for 2a at 288C was 173Æ22 mM. The turnover
number kcat was 7.46 sÀ1 and vmax 3.70Æ0.12 mmol
minÀ1 mgÀ1.
Table 1. Selected results of spectrophotometric screening for
substrates of NcCAR.
Substrate
Specific activity [U/mg][a]
benzoic acid[b]
2.74Æ1.17
0.49Æ0.17
0.74Æ0.29
1.00Æ0.24
1.71Æ0.64
0.68Æ0.55
2.85Æ1.22
0.52Æ0.03
0.69Æ0.29
1.55Æ0.18
2.04Æ0.81
2.22Æ0.85
In vitro biotransformations coupled to HPLC analy-
ses showed that carboxylic acids were often not con-
verted quantitatively by NcCAR. Assuming a limita-
2-hydroxybenzoic acid[c]
2-methoxybenzoic acid[c]
3-hydroxybenzoic acid[c]
3-methoxybenzoic acid[c]
4-hydroxybenzoic acid[c]
4-methoxybenzoic acid[c]
benzyloxyacetic acid[b]
3-phenylpropionic acid[c]
pyridine-2-carboxylic acid[b]
1a cinnamic acid[b]
1
tion by ATP degradation, we performed H NMR in
combination with time-resolved 31P NMR analysis.
31P NMR had previously been used to aid the identifi-
cation of the acyl-adenylate intermediate in NiCAR-
mediated bioreduction[35] but not to monitor the reac-
tion or to investigate the nature of the phosphorus-
containing species.[36] First experiments towards the
elucidation of the co-products from ATP in N. crassa
CAR reductions indicated the formation of ADP and
phosphate,[21,37] whereas later studies by the same au-
thor(s) showed that AMP and pyrophosphate were
formed.[22,37] Figure 6 shows 31P NMR signals of the
reference measurements (top) and different time
points of the reaction in the presence of NcCAR. As
expected, 1a reduction resulted in AMP, pyrophos-
2a piperonylic acid[c]
[a]
One activity unit is defined as the amount of enzyme
preparation catalyzing the oxidation of 1 mmol NADPH
per minute.
Substrate dissolved in 0.1 M KOH.
Substrate dissolved in DMSO.
[b]
[c]
endpoint quantification of radiolabelled salicylic alde- phate and NADP+. Whereas the signals of ATP and
hyde formation.[20] The pH optimum of our recombi- NADPH decreased, AMP and pyrophosphate were
nant NcCAR appeared to be at pH 5.5–6.0 using the immediately formed after starting the reaction, how-
above mentioned spectrophotometric initial rate mea- ever, the reaction did not continue beyond a certain
surement with cinnamic acid (1a) as the substrate point when a white precipitate emerged, although 1a,
1
(Figure 2). This result was confirmed by an endpoint NADPH and ATP were still present according to H
measurement of cinnamaldehyde (1b) formation by and 13P NMR, respectively (Supporting Information,
HPLC (data not shown). These diverging results may Figure S5). Denaturation of the NcCAR was ruled
partly be a consequence of unequal reaction condi- out because it was stable in separate experiments
tions, additional post-translational modifications of under identical conditions. Controls with all potential
the native Neurospora crassa CAR or due to differen- by-products showed that the precipitate was pyro-
ces in the amino acid sequence as a result of different phosphate. Consistent with the observations from the
strain backgrounds (SY7A versus OR74A).
Prather group with NiCAR,[11] these results indicate
Apparent kinetic parameters were determined with the inhibition of NcCAR by the co-product pyrophos-
NcCAR after cleavage of the HIS-tag, using the spec- phate. An experiment including pyrophosphatase
trophotometric assay. The Km value for 1a at 288C showed that the concentration of 1b can indeed be in-
was 445Æ50 mM. The turnover number kcat was creased-two fold in vitro (Supporting Information,
Figured S4).
Currently, the most economical way of utilizing
CAR enzymes on a preparative scale is their use via
whole cell biocatalysts that allows cost effective re-
generation of the required co-factors (ATP,
NADPH).[8,38–40] At the same time, the enzymatic
background of bacterial cells rapidly reduces alde-
hydes (b) to the corresponding alcohols (c) which
minimizes the oxidative and electrophilic stress re-
sponses induced by the reactive carbonyl group in al-
dehydes.[39,41] To circumvent b reduction, we opted for
E. coli K-12 MG1655 RARE[42] – a knock-out strain
with reduced aromatic aldehyde reduction – as a host
for whole cell mediated aldehyde preparation. De-
spite this deliberate choice, initial experiments to-
wards the reduction of the model substrate cinnamic
acid 1a to 1b showed significant amounts of the unde-
sired alcohol 1c. First whole cell biotransformations
Figure 2. Relative activities of NcCAR at different pH
~
^
values. : citrate buffer, &: MES buffer, : Tris-HCl buffer,
ꢀ: glycine buffer.
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Figure 3. Optimization of whole cell biotransformation con-
ditions. black: 1b; striped: 1a; white: 1c; grey: not recov-
ered. The standard conditions were 20 mg wet cells in 1 mL
final volume, supplemented with 60 mM glucose, 10 mM
sodium citrate, 2 mM Mg2+ and 5 mM 1a in 15-mL Pyrex
tubes, which were incubated for 3 h. These standard condi-
tions were modified as follows A: 70 mM sodium citrate, in-
cubation time 20 h; B: 70 mM sodium citrate; C: 20% of n-
hexane; D: 50% of n-hexane; E: 50% n-hexane, 10 mM 1a,
60 mg wet cells.
Figure 4. Whole cell biotransformation of increasing
amounts of 2a with NcCAR expressed in E. coli K-12
MG1655 RARE. black: 2b; striped: 2a; white: 2c; grey: not
recovered. The standard conditions were 500 mg wet cells
in 1 mL final volume, supplemented with 120 mM glucose,
20 mM sodium citrate and 4 mM Mg2+ in 100 mL baffled
flasks. A: 20 mM 2a; B: 30 mM 2a; C: 40 mM 2a; D: 50 mM
2a.
yielded only 14% of 1b after 20 h and also 14% of 1c.
Reduction of the incubation time and co-factor recy-
cling components improved the formation of 1b and
decreased the amounts of 1c. Further increase of 1b
was achieved by using hexane as a second phase, in
order to circumvent product inhibition or toxicity, to
finally 6.8 mM of 1b (Figure 3).
Our goal was to demonstrate the synthetic utility of
NcCAR for the selective preparation of valuable al-
dehydes such as piperonal (2b).When we applied con-
ditions B (Figure 3) to the preparation of 2b using
NcCAR expressed in E. coli RARE, we were delight-
ed to find that formation of 2c was completely sup-
pressed and 2b was produced very efficiently even
without the need for n-hexane to increase product
titers. Within 3 h, 30 mM of 2a were fully converted
to 2b (Figure 4, B 3 h) and 40 mM, corresponding to
6.6 gLÀ1 were quantitatively reduced within 5 h
(Figure 4, C 5 h). Further increase of the substrate
concentration to 50 mM led to full conversion in less
than 24 h and the maximal observed concentration of
2b was 49.8 mM (data not shown), corresponding to
7.5 gLÀ1. In comparison, E. coli BL21 (DE3) Star ex-
pressing NcCAR used under conditions identical to
those described in Figure 4, B, gave predominantly
the alcohol 2c (Figure 5). Finally, piperonal 2b was
synthesized as follows: 1.99 g (30 mM) of 2a was
quantitatively reduced in two parallel batches exclu-
sively to 2b within 3 h (Scheme 2).
Figure 5. Time course of whole cell biotransformation of
30 mM 2a using NcCAR expressed in E. coli BL21 (DE3)
Star. black: 2b; striped: 2a; white: 2c; grey: not recovered.
For the reaction scheme, see Figure 4.
Scheme 2. Preparative scale reduction of 2a to piperonal 2b
by NcCAR expressed in engineered E. coli.
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changed for 50 mM MES buffer, pH 7.5, containing 10 mM
MgCl2, 1 mM EDTA, and 1 mM DTT and aliquots of the re-
sultant protein solution were shock frozen in liquid nitrogen
and stored at À808C. Protein concentrations were deter-
mined with a Nanodrop 2000c spectrophotometer (Thermo
Scientific). Reactions were performed on a Thermomixer
comfort (Eppendorf). HPLC/MS analysis was carried out on
an Agilent Technologies 1200 Series equipped with G1379B
degasser, G1312B binary pump SL, G1367C HiP-ALS SL
autosampler, a G1314C VWD SL UV detector, G1316B
TCC SL column oven and a G1956B MSD mass selective
detector. For HPLC/UV analysis an Agilent Technologies
1100 Series equipped with a G1379A degasser, 1200 Series
Quaternary pump G1379A, G1367A autosampler, G1330B
autosampler thermostat, G1316A thermostatted column
compartment and a G1315B Diode Array Detector was
used.
The product was extracted into n-hexane and crys-
tallized in >98% purity to give 1.66 g of 2b in 92%
isolated yield. This corresponds to an excellent pro-
ductivity for enzymatic carboxylate reductions of
>1.5 gLÀ1 hÀ1. The only report of comparable produc-
tivity concerned the Nocardia iowensis CAR-mediat-
ed preparation of vanillin after extensive optimiza-
tion. Despite co-expression of GDH and the addition
of XAD-2 resin as a product scavenger, the mass bal-
ance did not attain 100% and vanillyl alcohol forma-
tion reduced the vanillin yield.[43] The co-expression
of GDH may also improve piperonal productivity.
Conclusions
The family of E.C.1.2.1.30 carboxylate reductase en-
zymes (CARs) was extended by a distinct enzyme se-
quence from Neurospora crassa OR74A (NcCAR).
Except for its optimal pH, NcCAR seems to resemble
aryl-aldehyde:NADP oxidoreductase which was puri-
fied from Neurospora crassa SY7A in the 1970s.
NcCAR is well expressed in E. coli, which represents
an excellent basis for biocatalytic applications. Herein
we have designed a whole cell biocatalyst for the se-
lective preparation of aldehydes and investigated the
critical parameters for its use, such as the cell density,
nature and amounts of additives, and substrate con-
centrations. NcCAR was most efficiently used for the
selective preparation of the flavour/fragrance alde-
hyde piperonal, reaching a productivity of approxi-
mately 1.5 gLÀ1 hÀ1 of the desired product without re-
sidual piperonylic acid or contamination by over-re-
duction product.
Protein Expression
A codon optimized synthetic gene coding for N-terminally
HIS-tagged NCBI accession # XP_955820.1 was ordered
from GenScript custom cloned into the pETDUET1 vector
with phosphopantetheinyltransferase from E. coli (NCBI ac-
cession # CAQ31055.1) in the upstream multiple cloning
site. After sequencing, E. coli BL21 (DE3) Star or E. coli
K12 MG1655 RARE was transfected with the plasmid pET-
DUET1:EcPPTaseHTNcCAR1 and colonies selected on
LB/Amp. For protein expression, the autoinduction protocol
as described by Studier[34] was used. After 24 h at 208C, the
cells were harvested by centrifugation and stored at À208C
or used for biotransformations.
Protein Purification
Thawed cells were disrupted by sonication and the protein
purified by nickel affinity chromatography and concentrated
NcCAR protein preparations were stored at À808C in
50 mM MES buffer, pH 7.5, containing 10 mM MgCl2, 1 mM
EDTA and 1 mM DTT. For determination of the kinetic pa-
rameters, the NcCAR preparation obtained from Ni-affinity
purification was further purified by gel filtration on an ꢁK-
TAPurifier 100. A HighLoadTM 16/60 SuperdexTM 200 pg
column was used (GE Healthcare) with a flow rate of
1 mLminÀ1 with 50 mM MES buffer, pH 7.5, containing
10 mM MgCl2, 1 mM EDTA, and 1 mM DTT as the mobile
phase. Fractions of 1 mL were collected in a Frac 950 collec-
tor (GE Healthcare) and the protein containing fractions
were pooled and stored at À808C.
Experimental Section
General
ATP was obtained from Roche Diagnostics. NADPH and
MES were purchased from Roth, IPTG from Serva, and
MgCl2 from Merck. HPLC-MS grade acetonitrile was pur-
chased from J.T.Baker/Avantor Performance Materials,
Deventer, The Netherlands. All other chemicals were ob-
tained from Sigma–Aldrich/Fluka or Roth and used without
further purification.
Spectrophotometric Assay
E. coli cells were cultivated in an RS 306 shaker (Infors,
Bottmingen, Switzerland) and Multitron shakers (Infors
AG), and the cells were harvested with an Avanti J-20 cen-
trifuge (Beckman Coulter). Cell pellets were disrupted by
a 102C converter with a Sonifier 250 (Branson, Danbury,
CT), and the cell-free extract was obtained by centrifugation
in an Ultracentrifuge Optima LE80K (Beckman). Enzymes
were purified in an ꢁKTAPure 100 with a fraction collector
F9-C (Unicorn 6.3 software; GE Healthcare) and desalted
with ꢁKTAPrime (PrimeView 5.0 software; GE Health-
care) or alternatively purified using a gravity flow protocol.
The protein-containing fractions were pooled, the buffer ex-
An NADPH depletion assay was used to screen for the sub-
strate scope and to determine physicochemical characteris-
tics of NcCAR. Therefore, a number of carboxylic acids or
carboxylate salts were dissolved in water, 0.1 M KOH or
DMSO. The assay composition was as follows: the substrate
(10 mL of 100 mM stock solution) was added to 160 mL of
Tris-HCl buffer (100 mM, pH 7.5, containing 10 mM
MgCl2). Subsequently, 10 mL of NADPH (10 mM in water),
10 mL of ATP (20 mM in water) and 10 mL of CAR enzyme
preparation from Ni-affinity chromatography (0.2–
0.7 mgmLÀ1) were added. The depletion of NADPH was
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followed on a Synergy Mx Platereader (BioTek) at 340 nm
and 288C for 10 min. Appropriate blank reactions were car-
ried out in parallel and each reaction was carried out at
least in triplicate.
For the determination of the pH optimum, the standard
assay as described above was used with cinnamic acid 1a
(100 mM in 0.1 M KOH) as the substrate and the following
buffers: sodium citrate (pH 4.0, pH 4.5, pH 5.0 and pH 5.5),
MES (pH 5.5 and pH 6.0), Tris-HCl (pH 7.0 and pH 7.5)
and glycine (pH 9.0, pH 9.5 and pH 10.0).
For the determination of kinetic parameters, the standard
assay as described above was used with 160 mL of MES
buffer (100 mM, containing 10 mM MgCl2, pH 6.0) and
CAR enzyme preparation after gel filtration (0.1–
0.3 mgmLÀ1). Cinnamic acid 1a dissolved in DMSO was
used as a substrate. The final DMSO content was 5% v/v
and 1a concentrations were 0.00625, 0.0125, 0.025, 0.05, 0.1,
0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 mM, respectively. 2a was
dissolved in water in concentrations of 0.005, 0.05, 0.075,
0.125, 0.2, 0.25, 0.3, 0.5, 2.0, 5.0, 10 and 15 mM containing
equimolar amounts of KOH, respectively. Km and vmax were
calculated on the basis of non-linear regression by using
Sigma Plot 11.0.
The analysis of piperonylic acid (1,3-benzodioxole-5-car-
boxylic acid, 2a), piperonal (heliotropin, 1,3-benzodioxole-5-
carboxaldehyde, 2b) and piperonyl alcohol (1,3-benzodiox-
ole-5-methanol, 2c) was carried out with a Kinetex 2.6m Bi-
phenyl 100A HPLC column (Phenomenex) with a Phenyl-
hexyl Security Guard ULTRA cartridge (Phenomenex). The
mobile phases were ammonium acetate (5 mM) and 0.5% v/
v
acetic acid in water and ACN at a flow-rate of
0.26 mLminÀ1. A stepwise gradient was used: 25–55% ACN
(5 min), 55–70% ACN (5.0–7.2 min) 70–90% ACN (7.2–
7.5 min). After 90 s, the column was re-equilibrated to start-
ing conditions. The compounds were detected at 254 nm
(DAD). For 2a, 2b and 2c, calibration with authentic stand-
ards was determined at 254 nm and linear interpolation
used for their quantification.
Time-Resolved 31P NMR
31P NMR spectra were recorded at 202.354 MHz on an
INOVA NMR spectrometer (Varian/Agilent) equipped with
a direct detection multinuclear probe as described previous-
ly.[36] NADPH, NADP+, AMP, ADP, ATP, K2HPO4, Na4P2O7
and MgCl2 were dissolved in TrisHCl buffer (100 mM, pH
7.5). Reference samples were composed of 335 mL of Tris-
HCl buffer (100 mM, pH 7.5), 50 mL of MgCl2 (200 mM),
25 mL MES buffer (100 mM, pH 7.5, 10 mM MgCl2, 1 mM
EDTA, 1 mM DTT), 40 mL KOH (0.1 M in water), 50 mL
D2O, and 50 mL of NADPH, NADP+, AMP, ADP, ATP,
phosphate or pyrophosphate solution (100 mM), respective-
ly. The biotransformation sample at time 0 was composed of
285 mL of Tris-HCl buffer (100 mM, pH 7.5), 50 mL of MgCl2
(200 mM), 25 mL NcCAR preparation (3.85 mgmLÀ1), 50 mL
ATP (100 mM), 50 mL NADPH (100 mM), and 50 mL D2O.
The reaction was started by addition of 40 mL of cinnamic
acid 1a (100 mM in 0.1 M KOH) (Figure 6).
Protein Melting Points Analysis
The nanoDSF grade standard glass capillaries were filled
with NcCAR in 50 mM MES buffer, pH 7.5, containing
10 mM MgCl2, 1 mM EDTA, and 1 mM DTT. A tempera-
ture gradient of 18CminÀ1 was applied and the intrinsic fluo-
rescence analyzed at 350 nm in
a Prometheus NT.48
nanoDSF device (NanoTemper Technologies).
Chromatographic assays
The typical assay to determine directly the reaction products
was carried out as follows: to 334 mL of 50 mM MES buffer,
pH 7.5, containing 10 mM MgCl2, 1 mM EDTA, and 1 mM
DTT, 16 mL of substrate solution (100 mM in 0.1 M KOH or
DMSO) was added followed by 10 mL of NcCAR prepara-
tion (approximately 0.02 mg mLÀ1), 20 mL of NADPH
(80 mM in ddH2O) and 20 mL of ATP (80 mM in ddH2O).
The reactions proceeded at 288C and 300 rpm in Eppendorf
Thermomixers. The reactions were stopped by the addition
of MeOH and vortexing. After centrifugation of precipitat-
ed protein, the supernatants were analyzed.
The analysis of E-cinnamic acid (1a), E-cinnamaldehyde
(E-3-phenylprop-2-enal, 1b) and cinnamyl alcohol (3-
phenyl-2-propen-1-ol, 1c) was carried out with a Kinetex
2.6m Biphenyl 100A HPLC column (Phenomenex) with
a Phenylhexyl Security Guard ULTRA cartridge (Phenom-
enex). The mobile phases were ammonium acetate (5 mM)
and 0.5% v/v acetic acid in water and ACN at a flow-rate of
0.4 mLminÀ1. A stepwise gradient was used: 15–50% ACN
(5 min) and 50–90% ACN (5.0–9.0 min). After 30 s, the
column was re-equilibrated to starting conditions. The com-
pounds were detected at 254 nm (VWD) and negative scan
mode (API-ES) as well as single ion monitoring of the acid
(MÀ1 147), the aldehyde (M+1 133) and the alcohol (MÀ1
133). For 1a, 1b and 1c, calibration curves were determined
at 254 nm and linear interpolation used for their quantifica-
tion.
Resting Cell Biotransformations
Electrocompetent E. coli K-12 MG1655 RARE cells were
transfected with pETDUET1:EcPPTase-HTNcCAR1 and
the cells cultivated as described above. The initial biotrans-
formation conditions (Figure 3, A) were 50 mM potassium
phosphate buffer at pH 7.4 supplemented with 60 mM glu-
cose, 70 mM citrate, 2 mM Mg2+ and 5 mM cinnamic acid
(1a). 15 OD600 units (approximately 15 mg of cwwmLÀ1)
were used. The reaction was carried out in 24-well multidish
plates (Thermo Fisher Scientific) in a total volume of 1 mL.
For the biotransformation of cinnamic acid (1a) to cinna-
maldehyde (1b) different adjustments to the initial condi-
tions were made. The concentration of glucose and the con-
centration of Mg2+ seem to have little influence on the con-
version of 1a. The citrate concentration was lowered to
10 mM because a lower 1b yield and a higher amount of al-
cohol (1c) was detected in biotransformations with citrate
concentrations ꢀ20 mM. Different amounts of cells (15–
100 mg cwwmLÀ1) were tested and 20 mg cww mLÀ1 was the
optimal cell density. The addition of n-hexane up to a 50%
of the total volume showed the highest improvement on the
yield of 1b. The reaction with n-hexane was carried out in
15-mL Pyrex tubes and incubated at 288C in a tissue culture
tube rotator for 3 h. The reaction with a total volume of
1 mL contained 50 mM potassium phosphate buffer at pH
7.4 supplemented with 60 mM glucose, 10 mM citrate, 2 mM
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6
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Figure 6. 31P NMR reference spectra of all phosphorus-containing species potentially involved in ATP and NADPH depen-
dent NcCAR mediated 1a reduction and time resolved reaction.
Mg2+, 5 mM 1a and 500 mL of n-hexane. 20 OD600 units
Acknowledgements
(about 20 mg of mg cww mLÀ1) were used per tube.
Typical experiments towards 2a reduction were carried
out in 100-mL baffled flasks and contained 50 mM potassi-
The skillful technical assistance of Philippe Oberhemm, Ker-
stin Steiner and Gernot A. Strohmeier is gratefully acknowl-
um phosphate buffer at pH 7.4 supplemented with 120 mM
glucose, 20 mM citrate, 4 mM Mg2+ and 10–50 mM 2a in
a total volume of 10 mL. 500 OD600 units (about 50 mg
cwwmLÀ1) were used per flask. The flasks were agitated at
100 rpm and 288C in a Certomat BS-1.
edged. We thank Kristala L. J. Prather for generously provid-
ing Escherichia coli K-12 MG1655 RARE and Tobias Pflꢀg-
er (NanoTemper Technologies GmbH) for kindly providing
access to the Prometheus NT.48. The Austrian science fund
FWF (Elise-Richter fellowship V415-B21 and P 28477-B21)
is kindly acknowledged for financial support. This research
has also been supported by the Austrian BMWFW, BMVIT,
SFG, Standortagentur Tirol, Government of Lower Austria
and ZIT through the Austrian FFG-COMET-Funding Pro-
gram. We thank the COST action Systems Biocatalysis WG2
and TU Wien ABC-Top Anschubfinanzierung for financial
support.
Preparative Scale Biotransformations
Reactions were carried out in two baffled 2-L flasks in
a volume of 200 mL (0.99 g of 2a per flask). The flasks were
agitated at 100 rpm and 288C in a Certomat BS-1. The reac-
tions contained 50 mM potassium phosphate buffer pH 7.4,
120 mM glucose, 20 mM citrate, 4 mM Mg2+, 30 mM of 2a
and 50 g LÀ1 of fresh wet cells. Reduction of 2a to 2b was
complete before 3 h according to HPLC analysis. Piperonal
2b was extracted into n-hexane directly from the biotrans-
formation broth and crystallized as colourless needles in
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Selective Enzymatic Transformation to Aldehydes in vivo
by Fungal Carboxylate Reductase from Neurospora crassa
9
Adv. Synth. Catal. 2016, 358, 1 – 9
Daniel Schwendenwein, Giuseppe Fiume, Hansjçrg Weber,
Florian Rudroff, Margit Winkler*
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ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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