Characterization of Type IV Carboxylate Reductases (CARs) for Whole Cell-Mediated Preparation of 3-Hydroxytyrosol

Article abstract of DOI:10.1002/cctc.201900333

Fragrance and flavor industries could not imagine business without aldehydes. Processes for their commercial production raise environmental and ecological concerns. The chemical reduction of organic acids to aldehydes is challenging. To fulfill the demand of a mild and selective reduction of carboxylic acids to aldehydes, carboxylic acid reductases (CARs) are gaining importance. We identified two new subtype IV fungal CARs from Dichomitus squalens CAR (DsCAR) and Trametes versicolor CAR (Tv2CAR) in addition to literature known Trametes versicolor CAR (TvCAR). Expression levels were improved by the co-expression of GroEL-GroES with either the trigger factor or the DnaJ-DnaK-GrpE system. Investigation of the substrate scope of the three enzymes revealed overlapping substrate-specificities. Tv2CAR and DsCAR showed a preferred pH range of 7.0 to 8.0 in bicine buffer. TvCAR showed highest activity at pH 6.5 to 7.5 in MES buffer and slightly reduced activity at pH 6.0 or 8.0. TvCAR appeared to tolerate a wider pH range without significant loss of activity. Type IV fungal CARs optimal temperature was in the range of 25–35 °C. TvCAR showed a melting temperature (T_m) of 55 °C indicating higher stability compared to type III and the other type IV fungal CARs (T_m 51–52 °C). Finally, TvCAR was used as the key enzyme for the bioreduction of 3,4-dihydroxyphenylacetic acid to the antioxidant 3-hydroxytyrosol (3-HT) and gave 58 mM of 3-HT after 24 h, which correlates to a productivity of 0.37 g L^?1 h^?1.

Full text of DOI:10.1002/cctc.201900333

Accepted Article
Title: Characterization of type IV carboxylate reductases (CARs) for
whole cell mediated preparation of 3-hydroxytyrosol
Authors: Melissa Horvat, Susanne Fritsche, Robert Kourist, and Margit
Winkler
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Characterization of type IV carboxylate reductases (CARs) for
whole cell-mediated preparation of 3-hydroxytyrosol
Melissa Horvat,[a] Susanne Fritsche,[a] Robert Kourist,[b] and Margit Winkler*[a,b]
Abstract: Fragrance and flavor industries could not imagine
business without aldehydes. Processes for their commercial
production raise environmental and ecological concerns. The
chemical reduction of organic acids to aldehydes is challenging. To
fulfill the demand of a mild and selective reduction of carboxylic
acids to aldehydes, carboxylic acid reductases (CARs) are gaining
importance. We identified two new subtype IV fungal CARs from
Dichomitus squalens (DsCAR) and Trametes versicolor (Tv2CAR).
Expression levels were improved by the co-expression of GroEL-
GroES with either the trigger factor or the DnaJ-DnaK-GrpE system.
Investigation of the substrate scope of the three enzymes revealed
overlapping substrate-specificities. Tv2CAR and DsCAR showed a
preferred pH range of 7.0 to 8.0 in bicine buffer. TvCAR showed
highest activity at pH 6.5 to 7.5 in MES buffer and slightly reduced
activity at pH 6.0 or 8.0. TvCAR appeared to tolerate a wider pH
range without significant loss of activity. Type IV fungal CARs
optimal temperature was in the range of 25-35°C. TvCAR showed a
melting temperature (Tm) of 55°C indicating higher stability
e.g. to reduce the aldehyde to the corresponding alcohol,[7]–[9]
and CARs may become the limiting step in such reaction
sequences.[10] To create more variety for cascade applications, a
repertoire of characterized key enzymes is of most importance.
Within recent years, various bacterial and few fungal CARs have
been described in literature. A detailed list can be found in SI
Table S1.
A phylogenetic analysis revealed that all 30
experimentally confirmed bacterial CARs are categorized as
subtype I and seemed to be relatively conserved, whereas the 4
fungal CARs with very low sequence identities (<25%) fall into
three distinct subclasses.[11],[12] The activity of fungal CAR from
the ascomycete Neurospora crassa was described in 1968 by
Gross et al.[13] In 2016, the gene sequence was associated to
the protein and cloned into E. coli with an additional
phosphopantetheinyl transferase from E. coli.[14] Like other
CARs,[15] Neurospora crassa CAR (NcCAR) requires the co-
expression of a phosphopantetheinyl transferase. Biophysical
characterization of the type III NcCAR enzyme indicated a
compared to type III and the other type IV fungal CARs (Tm 51-52°C). preference for an acidic environment (pH 5.5-6.0) and a broad
Finally, TvCAR was used as the key enzyme for the bioreduction of
3,4-dihydroxyphenylacetic acid to the antioxidant 3-hydroxytyrosol
(3-HT) and gave 58 mM of 3-HT after 24 h, which correlates to a
productivity of 0.37 g L-1h-1.
substrate tolerance.[14],[16] Aspergillus terreus CAR (AtCAR)[17]
and Stachybotrys bisbyi CAR (SbCAR)[18] belong to the subtype
II and are the only CARs so far placed in this same subtype.[12]
The first and only CAR of subtype IV was described from
Trametes versicolor (TvCAR) and was functionally expressed in
rather low amounts. Partially purified enzyme preparations of the
TvCAR were used to explore its substrate scope. Surprisingly, it
showed similar specific activities for aromatic, aliphatic and
substituted aromatic carboxylic acids.[19] For the reduction of
aliphatic substrates the bacterial MmCAR from Mycobacterium
marinum was utilized almost exclusively so far.[8]
Introduction
Increasing sustainability of chemical processes is one of the key
goals in industrial biotechnology today.[1] Chemical production
turns away from generating hazardous substances and targets
the utilisation of renewable resources.[2],[3] Within the last
decades, the developments achieved in biological techniques
and tools have enabled the engineering of microorganisms for
catalytic conversions.[4] Through inexpensive, renewable
substrates and high cell density fermentation processes,
biotechnology is more widely introduced in industry.[5] Carboxylic
acid reductases (CARs, E.C. 1.2.1.30) are the enzymes of
choice to reduce carboxylic acids to aldehydes. Their 3-domain-
architecture as well as the reaction scheme are displayed in
Figure 1. Their ATP and NADPH dependent mechanism was
affirmed by the determination of protein crystal structures of
separate adenylation and reductase domains of bacterial CARs
and modelling.[6] CARs are frequently introduced into cascade
reactions in vivo. In many cases, endogenous enzymes such as
To broaden our understanding of CARs within one phylogenetic
category, one goal is to improve expression of TvCAR and to
explore its characteristics and limitations in more depth. The
second goal is to uncover further fungal CARs.
[a]
[b]
M. Horvat, S. Fritsche, Priv.-Doz. M. Winkler
acib - Austrian Center of Industrial Biotechnology
Petersgasse 14, 8010 Graz, Austria
E-mail: margit.winkler@tugraz.at
Prof. R. Kourist, Priv.-Doz. M. Winkler
Institute of Molecular Biotechnology
Graz University of Technology
Petersgasse 14, 8010 Graz, Austria
Supporting information for this article is given via a link at the end of the
document.
aldehyde
reductases,
decarbonylases
and
alcohol
dehydrogenases within the expression hosts are being utilized
1
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Figure 1. Schematic reaction mechanism of CAR enzymes (E.C.1.2.1.30). Left: ATP and substrate enter the active site of the adenylation domain. The structure
of the adenylation domain of Nocardia iowensis CAR (NiCAR) is shown in complex with AMP (PDB ID: 5MSC).[6] The substrate is then activated as an AMP
anhydride and pyrophosphate is released.[3] Middle: A thioester is formed at the phosphopanthetheine binding site (grey), and AMP is released. Right: the
thioester is translocated to the reductase domain (PDB ID: 5MSO).[6] The reductase domain incorporated in the scheme is from Mycobacterium marinum in
complex with NADP+. The last step is thioester reduction and release of the aldehyde and NADP+. Microsoft Power Point and ChemDraw were used to generate
the figure.
In this study, we explored the hidden potential of fungal CARs to
increase the number of biocatalytic tools for reducing carboxylic
acids and broaden the operational space for potential industrial
applications. We identified two new type IV fungal CARs and
described their phylogenetic relations to the family of CARs. Two
CARs from Trametes versicolor and one from Dichomitus
squalens were heterologously expressed in E. coli. Low
expression levels increased by co-expressing chaperone
systems GroE-GroES with either the trigger factor or the DnaJ-
DnaK-GrpE system to enhance the affinity for proteins to
promote their folding. To determine substrate specificities of type
IV CARs, specific activities towards a range of aliphatic and
aromatic substrates have been determined. We investigated the
effects of pH and temperature and finally the reduction of the
model substrate 3,4-dihydroxyphenylacetic acid (DOPAC) to 3-
hydroxytyrosol (3-HT). We improved the reduction of DOPAC to
3-HT, a highly potent antioxidant with various applications in
cosmetics and food supplements. The preparation of 3-HT from
DOPAC has the potential to attain a 45-fold value increase by
pursuing the biosynthetic route. We achieved a production of 0.4
g L-1 h-1 for 3- HT (2.4 mM h-1) that after further optimization may
lead to an alternative method for producing 3-HT.
representatives of fungal CARs are not only needed to diversify
the basis of phylogenetic analyses and corroborate conclusions
from such analyses, but also to eventually assemble a repertoire
of new enzymes with different characteristics. The protein
sequences of NiCAR,[21],[22] MmCAR,[8] SroCAR[23] and
AtCAR[17],[20] were taken as templates for a BLASTp search with
the restriction to organisms with experimentally confirmed ability
to reduce carboxylic acids. Hit sequences were further analyzed
for the presence of essential key residues and motifs.[16],[24]
Putative CAR sequences from Dichomitus squalens (DsCAR)
and Trametes versicolor (Tv2CAR) were top rated. Both CAR
candidates were chosen for further characterization, as
phylogenetic analysis revealed that DsCAR and Tv2CAR were
both type IV CARs, for which TvCAR is the only experimentally
confirmed CAR so far[19] (Figure 2). Sequence analyses
(Figure S1) of the CARs displayed in the phylogenetic tree
(Figure 2) revealed that type IV CAR sequences of Trametes
versicolor were 65.1% identical, whereas the DsCAR showed
39.6% sequence identity to the TvCAR and 39.8% to Tv2CAR,
respectively.
Expression and purification
New CARs were cloned and expressed from the pETDuet-1
plasmid in E.coli MG1655 RARE (DE3) as previously
established in our lab.[12],[14] This strain harbors specific knock
outs that reduce its ability to reduce aromatic aldehydes further
to their corresponding alcohols.[25] DsCAR was well expressed,
whereby approximately equal amounts were detected in soluble
and insoluble fraction (Table 1 and SI Figure S4A). This
Results and Discussion
Alignment and phylogenetic analysis
Up to date, 30 functional CARs have been described (list of
CARs with references see SI Table S1). Among these, only five
CARs do not originate from bacteria. These few CARs constitute
three of four phylogenetic types of CARs.[14],[18]–[20] Further
indicated
a
steady translation and protein folding of
DsCAR.[26],[27]
2
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Figure 2. A phylogenetic tree of fungal and bacterial CAR enzymes, categorized in four subtypes. Newly discovered sequences and previously described
CARs[8],[14],[15],[18],[19],[23] were incorporated in the phylogenetic analysis. Green branches represent CARs investigated in this study. *TtCAR was identified in
Thermothelomyces thermophilus, which is also known as Myceliophthora thermophila, Thielavia heterothallica ATCC 42464, or Sporiotrichum thermophile.
pETDuet-1:EcPPTase:Tv2CAR (Table 2). This construct
Determination of the total protein concentration of DsCAR
resulted in ~14 mg per 400 mL of cultivation [~4.2 g cell wet
weight (CWW)]. TvCAR showed an expression of ~10 mg
protein in the soluble fraction per 400 mL cultivation, (~4.1 g
CWW). Tv2CAR was expressed the weakest. Most protein was
found in the insoluble fraction. The amount of CAR was less
than ~5 mg per 400 mL of cultivation (~4.1 g CWW). According
to Sorensen and Morensen, the following strategies for
improving the solubility of proteins without engineering the target
was suggested: to investigate various temperatures, strains,
media, molecular chaperones or include tRNA complementation
plasmids.[26] An optimized expression medium and autoinduction
protocol at 20°C (SI Figure S4) as described by Studier showed
the best results for CARs in our previous studies and was used
as our expression standard.[28]
encodes the CAR with the weakest expression level Tv2CAR
(SI Figure S4).
E. coli strains were transformed with pETDuet-1:EcPPTase or
the empty pETDuet-1 vector, respectively. Both served as
negative controls. After cultivation and induction at standard
conditions, 50 OD units of each strain were analyzed by
conducting whole cell-mediated reduction of piperonylic acid 1a
to show the impact of the expression level on catalyst efficiency.
In parallel, another 50 OD units of each cultivation were used to
determine the levels of soluble and insoluble expression. All E.
coli BL21 (DE3) TaKaRa [pG-KEJ8]/ [pG-Tf2]/ [pGro7]/ [pTF16]
strains showed improved soluble expression levels compared to
the expression level of Tv2CAR without co-expression of
chaperones (see Table 2 and SI Figure S4).
Due to the highest conversions and best soluble expression
levels of Tv2CAR, the plasmids [pG-KEJ8] and [pG-Tf2] were
chosen for further experiments with TvCAR in E. coli MG1655
RARE (DE3). This specific E. coli strain was engineered to
maintain aromatic aldehydes and minimize their reduction to
alcohols by knocking-out genes encoding for aldo-
ketoreductases and alcohol dehydrogenases.[25] Chaperones
GroEL-GroES with additional helper proteins such as the
triggering factor (TF) or the DnaK-DnaJ-GrpE system positively
influenced the folding behavior of Tv2CAR and TvCAR (
Figure 3B). The increased level of soluble proteins found in the
cell translated to increased activity (
Table 1. CARs in this study.
Ratio
NCBI
Reference
Calculated
size [kDa]
soluble/
insoluble
fraction
Abbreviation
Organism
Trametes
TvCAR
Tv2CAR
DsCAR
XP_008043822.1
XP_008044321.1
XP_007365851.1
118.2
118.6
121.2
0.341
0.178
1.059
versicolor
Trametes
versicolor
Dichomitus
squalens
[a] The size of the CAR enzymes was calculated via ExPASy. [b] Protein bands
were quantified via G:Box HR16 by Syngene.
Figure 3A). An increase of up to ~40% in conversion was
achieved when Tv2CAR was co-expressed with GroEL-GroES
and DnaK-DnaJ-GrpE system (
Improvement of CAR expression levels
Figure 3A). Similar improvements of soluble expression were
achieved with the co-expression of GroEL-GroES with TF. TF
was described to bind to GroEL and increase its affinity for
proteins to promote their folding.[33] The overexpression of TF
showed similar effects to the overexpression of the DnaK-DnaJ-
GrpE chaperone team in preventing aggregation of Tv2/TvCAR,
which is due to the highly similar function of DnaK and TF in
folding nascent polypeptides.[31]
Whole cell-mediated conversions are dependent on the amount
of functional enzyme, cell wall transport and substrate
acceptance, among other factors (SI Table S4).[29],[30] In vitro
bioreductions catalyzed by TvCAR showed that only the soluble
protein fraction was functional and responsible for reductase
activity (SI Figure S5). Hence, an increase in soluble protein
should increase the specific catalyst activity. Therefore, E. coli
strains which were known to be beneficial for protein expression,
translation and folding[31],[32] were transformed with plasmid
3
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Table 2. Ratios of soluble to insoluble protein fractions of Tv2CAR with co-expressed chaperones or expressed in optimized expression hosts and catalyst
efficiency determined by whole cell biotransformations [%].
Expression system of Tv2CAR [a]
E. coli strain/plasmid feature
Ratio soluble/
insoluble protein
fraction [b]
Absolute amounts of
soluble protein [mg] [c]
Conversion [d]
[%]
Tv2CAR in E. coli K-12 MG1655 RARE
(DE3)
Engineered for reduced reduction of aromatic
aldehydes
0.178
4.7
25.8 ± 7.0
Tv2CAR [pGro7] in E. coli BL21 (DE3)
Tv2CAR [pKJE7] in E. coli BL21 (DE3)
Tv2CAR [pG-KJE8] in E. coli BL21
(DE3)
Co-expression GroES-GroEL
0.327
0.221
8.7
5.8
38.9 ± 3.9
43.2 ± 3.9
Co-expression DnaK-DnaJ-GrpE
Co-expression GroES-GroEL-DnaK-DnaJ-GrpE
0.385
10.2
63.3 ± 4.7
Tv2CAR [pTF16] in E. coli BL21 (DE3)
Tv2CAR [pG-TF2] in E. coli BL21 (DE3)
Tv2CAR in E. coli BL21 CodonPlus
(DE3)-RP
Co-expression of trigger factor (Tf)
Co-expression of GroES-GroEL-Tf
Contain extra copies of the argU and proL genes
for increasing tRNA pool
0.297
0.393
7.9
19.4 ± 4.7
59.0 ± 3.3
10.4
N/A
N/A
N/A
N/A
N/A
N/A
N/A
19.6 ± 4.7
14.9 ± 0.7
11.8 ± 0.4
25.9 ± 4.7
Tv2CAR in E. coli Origami B (DE3)
Mutations in lacZY, trxB and gor for enhancing
disulfide bonds
Tv2CAR in E. coli Rosetta-gami (DE3)
Tv2CAR in E. coli Tuner (DE3)
Combine codon bias and enhance disulfide bond
formation
lac permease (lacY) mutation for controlled
induction
N/A
[a]
[b]
Chaperone plasmid set was commercially available at TaKaRa BIO Inc (catalogue number 3340).
Protein bands were quantified via G:Box HR16 by
Syngene. N/A: not applicable, bands too faint for evaluation. [c] Absolute amounts of soluble protein was estimated based on 400 mL shake flask cultivation of E.
coli K12 MG1655 RARE (DE3) [pETDuet-1:EcPPtase:Tv2CAR] followed by a Ni-affinity purification of Tv2CAR. [d] Whole cell-mediated reductions of piperonylic
acid were performed in triplicates.
O
O
O
O
O
O
O
O
E. coli RARE EcPPTase CAR
OH
E. coli endogenous ADH/ADRs
OH
H
citrate, glucose, air
citrate, glucose, air
1c
1b
1a
Figure 3A) Expression analysis of Tv2CAR and TvCAR, co-expressed with chaperones. Lane 1p-s: insoluble (p) and soluble (s) fraction of Tv2CAR. Lane 2 p-s:
insoluble (p) and soluble (s) fraction of TvCAR. Lane 3p-s: insoluble (p) and soluble (s) fraction of Tv2CAR with [pG-TF2]. Lane 4p-s: insoluble (p) and soluble (s)
fraction of Tv2CAR with [pG-KEJ8]. Lane 5p-s: insoluble (p) and soluble (s) fraction of TvCAR with [pG-TF2]. Lane 6p-s: insoluble (p) and soluble (s) fraction of
TvCAR with [pG-KEJ8]. SDS 4-12% Bis-Tris-gel of soluble and insoluble fractions of Tv2CAR and TvCAR with or without co-expression of chaperones. 60 min at
200 V in MOPS-buffer; staining: Simply Blue™ solution, ladder: PageRuler™ Prestained Protein Ladder (10-140 kDa). B) Whole cell-mediated reduction of 1a by
Tv2CAR (~120 kDa) or TvCAR (~120 kDa) expressed in E. coli K-12 MG1655 RARE (DE3) [pETDuet-1:EcPPTase:XyCAR] with or without chosen co-expression
of chaperones GroEL (~60 kDa) -GroES (~22 kDa) and trigger factor (~56 kDa) [pG-TF2] or GroEL-GroES with DnaK (~70 kDa) -DnaJ (~40 kDa) -GrpE (~22
kDa) [pG-KEJ8]. Beige: 1a; orange: 1b; red striped: 1c. Error bars show the standard deviation of three sets of technical triplicates. C) Reaction scheme of whole
cell-mediated reduction of 1a by CAR.
4
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Determination of substrate scope
bicine- NaOH buffer and showed a preference for neutral to
basic aqueous environment (Figure 4B-C).
TvCAR and Tv2CAR showed highly similar specific activities for
fatty acids ranging from C5-C10, which is most likely due to high
sequence similarity (~65.3%). In addition to medium-chain
aliphatic substrates tested previously,[19] TvCAR reduces short
chain aliphatic acids. The comparison of the initial rate activities
for the reduction of acids over a period of ten minutes indicated
benzoic acid 2a to be the preferred substrate of Tv2CAR and
DsCAR (Table 3). Tv2CAR and DsCAR showed higher activity
for aromatic acids than for the aliphatic carboxylic acids. DsCAR
seemed to be significantly less efficient for fatty acid reduction
than the CARs from T. versicolor. All CARs were tolerant to all
tested substrates, with a clear preference towards aromatic and
shorter aliphatic acids. Interestingly, TvCAR showed lower
specific activity for benzoic acid as compared to aliphatic
substrates. This supports the hypothesis that each organism
might have multiple CARs for reducing different substrates.[19]
[a]
Table 3. Specific activities
of CARs for various aromatic and aliphatic
substrates determined by monitoring NADPH depletion. Chemical structures of
substrates are displayed in the supporting information Figure S3.
TvCAR
Tv2CAR
DsCAR
[b]
Substrate
[U mg-1]
[U mg-1]
[U mg-1]
Aromatic substrates
Piperonylic acid (1a)
Benzoic acid (2a)
DOPAC (3a)
0.79 ± 0.05
0.35 ± 0.02
0.43 ± 0.00
0.87 ± 0.08
0.78 ± 0.03
0.30 ± 0.01
0.46 ± 0.01
0.82 ± 0.06
0.50 ± 0.02
Aliphatic substrates
Pentanoic acid (4a)
Hexanoic acid (5a)
Heptanoic acid (6a)
Octanoic acid (7a)
Nonanoic acid (8a)
Decanoic acid (9a)
0.65 ± 0.03
0.40 ± 0.10
0.41 ± 0.03
0.24 ± 0.00
0.31 ± 0.07
0.31 ± 0.02
0.22 ± 0.02
0.47 ± 0.02
0.24 ± 0.01
0.26 ± 0.02
0.21 ± 0.01
0.05 ± 0.01
0.03 ± 0.03
0.45 ± 0.03
0.34 ± 0.02
0.14 ± 0.02
0.30 ± 0.03
0.30 ± 0.09
[a]
One activity unit is defined as the amount of enzyme preparation catalyzing
[b]
the oxidation of 1 µM of NADPH per minute.
Standard deviations were
Figure 4. Relative activities of type IV fungal CARs in response to different pH
values. Overlapping buffers containing 10 mM MgCl2 were used to cover the
pH range of 5.5-9.0. 7a (TvCAR) and 2a (Tv2CAR and DsCAR) was used as
substrates and the activity relative to the highest activity at 100% is presented.
calculated on basis of four measurements. Blank reactions were conducted
with no ATP.
Biochemical characterization of CARs
A) TvCAR. B) Tv2CAR. C) DsCAR. : sodium citrate buffer, : potassium
phosphate buffer, : MES-NaOH buffer, : bicine-NaOH buffer, x : Tris-HCl
buffer. Error bars show the standard deviation of three technical triplicates.
Enzymes evolved to perform their function in specific cellular
environments. Therefore, they should be adapted to the
biophysical characteristics, e.g. pH and temperature, of their
respective cellular compartment. Many enzyme properties, such
as activity and stability, are pH dependent. [34]
Commonly used buffer constituents are acetate or citrate. Being
carboxylic acids, they may partly be reduced by CARs.
However, CAR activity for citric acid was below detection limit
under these conditions. The inhibition effect of potassium
phosphate buffer is most likely due to binding of phosphate at
the ATP-binding site in the adenylation domain.
Effect of reaction pH
For determination of the optimal pH environment for the fungal
type IV CARs their activity at different pH and buffer systems
was assessed. Correlating with their preferred substrate, an
aliphatic substrate (7a) was used for the pH assessment of
TvCAR and an aromatic substrate (2a) for Tv2CAR and DsCAR.
The relative activity analysis of TvCAR revealed a preferred
neutral to acidic environment. The optimum pH for TvCAR was
identified as pH 7.0 in 100 mM MES-NaOH. Overall, TvCAR
showed activity in a rather broad range from pH 6.0-8.0 without
losing activity considerably (Figure 4A). This observation was
consistent with the biophysical characterizations of other
CARs.[10],[24] The pH optimum determined for Tv2CAR and
DsCAR showed the highest relative activity at pH 7.5 in 100 mM
Effect of temperature
Activity and stability are important characteristics when it comes
to temperature dependent properties of enzymes.[34] From a
biocatalytic point of view, high activity is important while stability
can be enhanced in most cases by additives e.g. DTT, NaCl,
etc. and protein engineering. To investigate the stability of type
IV fungal CARs, the midpoint temperatures of protein unfolding
transitions (Tm) and the temperature optimum of TvCAR,
Tv2CAR and DsCAR were determined.
5
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To determine thermostabilities of type IV fungal CARs, thermal
shift assays were performed in different buffer systems and a pH
range from 4.6-9.0.[35] TvCAR showed a Tm of 55°C in potassium
phosphate buffer at pH 6.5 (Table 4). The addition of 0.1 M
NaCl increased the unfolding temperature by 1°C (SI Figure
S6). Using the thermal shift assay for type III CAR from
Neurospora crassa (NcCAR), the Tm was 51°C in potassium
phosphate buffer with 0.1 M NaCl, pH 7.0 (Table 4) whereas
Tv2CAR and DsCAR showed a Tm of 52°C in potassium
phosphate buffer at pH 7.5. Notably, the reported Tm of NcCAR
at 45°C was determined by NanoDSF.[14] For storage of TvCAR,
and CARs in general, phosphate buffer showed stabilizing
effects, which is most likely explained by the binding of
phosphate in the adenylation domain.
typically performed at 28°C.[14]–[16],[24] Our results (Figure 5)
confirmed the reported temperature optima, as type IV fungal
CARs showed 100% activity from 25°C to 35°C. For resting cells
conversions utilizing E. coli cells, 37°C may be of interest to
facilitate co-factor recycling. However, CARs seemed to become
slightly less active at 35°C and above.
Figure 5. Relative activity of partially purified type IV fungal CARs in response
to temperatures from 25-50°C. Samples were incubated for 1 h and activity
was indirectly determined through NADPH-consumption. 2a was used as
substrate and the activity relative to the highest activity at 100% is presented.
Error bars show the standard deviation of three technical triplicates. Blue
diamonds: TvCAR. Red squares: Tv2CAR. Green triangles: DsCAR.
higher temperatures.[14] This observation confirmed also the
results of the thermal shift assays when compared with NcCAR.
Consequently, it can be concluded that type IV fungal CARs
showed no significantly reduced activity up to 35°C and TvCAR
even remained active up to 50°C when incubated for 1 h at the
specific temperatures. Up to date only the bacterial MavCAR
was described to be more thermostable and active at
temperatures higher than 40°C.[10] Hence, TvCAR would be a
good fungal candidate when increased thermostability is
needed. The broad substrate scope of TvCAR, its improved
heterologous expression, the good specific activity of DOPAC 3a
(~0.43 U mg- 1) and the acceptance of acidic pH conditions made
TvCAR a promising candidate for the overreduction of DOPAC
to 3-HT.
Table 4. Thermostability assessment of TvCAR, Tv2CAR, DsCAR and
NcCAR.
[a]
Reaction
buffer
NcCAR
[Tm]
TvCAR
[Tm]
Tv2CAR
[Tm]
DsCAR
[Tm]
[b]
PP, pH 6.5
PP, pH 6.5,
with 0.1 M
NaCl
PP, pH 7.0,
PP, pH 7.0,
with 0.1 M
NaCl
PP, pH 7.5
PP, pH 7.5,
with 0.1 M
NaCl
50.7 ± 0.7
51.0 ± 0.6
51.5 ± 0.6
55.3 ± 0.6
55.0 ± 0.0
55.0 ± 0.0
55.0 ± 0.0
54.0 ± 0.0
54.0 ± 0.0
50.6 ± 0.5
50.3 ± 0.5
52.0 ± 0.0
51.0 ± 0.0
52.0 ± 0.0
52.0 ± 0.0
49.0 ± 0.0
51.0 ± 0.0
52.0 ± 0.0
52.3 ± 0.5
52.0 ± 0.0
52.3 ± 0.5
51.0 ±
0.00
51.0 ± 0.6
50.3 ± 0.6
Isoelectric point
[a]
Midpoint temperatures of protein unfolding transitions (Tm) determined by
The isoelectric point (pI) of TvCAR was both calculated and
determined to minimize pH induced protein aggregation during
e.g. purification procedures. The calculated pI of TvCAR was
5.84, determined with ExPASy: SIB bioinformatics tool
(https://web.expasy.org/compute_pi/), and was compared with
an experimentally determined pI (Supporting information Figure
S7). A range of 5.0 -5.4 for the pI of TvCAR was determined by
isoelectric focusing.
[b]
thermal shift assay. Each reaction was performed in technical triplicates. All
reaction buffers contained 10 mM MgCl2. Potassium phosphate buffer is
abbreviated PP.
Comparison of the most thermostable type IV fungal TvCAR with
the type III NcCAR (SI Figure S6A and B) suggests that TvCAR
is slightly more thermostable under a wider range of pH than
NcCAR, which only showed a peak above 50°C, when incubated
in phosphate buffer.
Synthetic application of TvCAR for bioreduction of DOPAC
to 3-HT
To determine the optimal operational temperature for in vitro
reactions, purified CAR preparations were used for NADPH-
depletion analysis at temperatures varying from 25°C-45°C.
Triplicate reactions proceeded for 1 h and relative activities were
determined after workup. Previous studies of CAR enzymes
were
TvCAR readily reduced 3,4-dihydroxyphenylacetic acid
(DOPAC, 3a) (Table 3). We previously investigated the
reduction of 3a to the antioxidant 3-hydroxytyrosol (3-HT, 3c)
using a whole cell biocatalyst, because the antioxidant 3-HT was
in the spotlight of numerous studies to uncover its beneficial
effects on human health.[36] 3-HT is abundant in olives (Olea
europaea) and olive oil, and the health-promoting Mediterranean
diet is partly ascribed to this compound. Due to its
Therefore, whole cell-mediated conversions such as performed
with piperonylic acid 1a are preferably performed at 28°C. 35%
activity was observed at 50°C for TvCAR, which correlates to the
results of the stability assays. However, Tv2CAR and DsCAR
lost almost all activity at 45°C. In comparison to characterized
fungal CARs, TvCAR seemed to be slightly more active at
anticarcinogenic,
anti-inflammatory,
antiapoptotic
and
neuroprotective activities, 3-HT is already used in foods and
cosmetics.[29],[37],[38] The high cost of purified 3c, however, limits
its availability on the market and is the bottleneck for its use.[36]
6
This article is protected by copyright. All rights reserved.

10.1002/cctc.201900333
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FULL PAPER
The first generation catalyst for 3c preparation harbored
bacterial type I CAR from Nocardia iowensis (NiCAR) as the key
not satisfying due to the evaporation of the aqueous phase
overnight in open reaction tubes. Summarizing, 3-HT formation
was significantly more efficient using the second-generation
biocatalyst with TvCAR as the key enzyme for acid reduction.
enzyme. The PPTase was co-expressed from
a second
plasmid.[39] Due to the whole cell approach, necessary cofactors
were recycled intracellularly. After extensive optimization of the
whole cell biotransformation, finally 27.9 mM of 3c could be
converted in 21 h using 30 mM of 3a at a relatively high cell
density of OD120.[40]
Conclusions
HO
HO
OH
HO
HO
OH
HO
HO
H
New methods for the reduction of underivatized carboxylic acids
are of high interest in view of sustainable chemistry that is
needed for the valorization of renewable feedstock. Carboxylic
acid reductases hold high potential in this respect. Uncovering
more diversity in CARs from different phylogenetic subtypes is
essential to build a useful toolbox for a broader substrate scope
and extended operational space. We contributed to this field by
extending the phylogenetic type IV fungal CARs with Tv2CAR
and DsCAR. Low expression levels of Tv2CAR and TvCAR
were improved by co-expressing chaperone systems GroEL-
GroES and DnaK-DnaJ-GrpE or the TF. The increase of soluble
protein correlated to the increase in activity when whole cell-
mediated biotransformations were conducted. Investigation of
specific activities from the same species (i.e. Trametes
versicolor) suggested that species are using multiple CARs for
different substrates. Hence, Tv2CAR demonstrated a tendency
towards aromatic and TvCAR towards aliphatic substrates.
TvCAR showed a pH optimum at pH 6.5-7.5 whereas Tv2CAR
and DsCAR seemed to prefer a pH of pH 7.0-8.0. Type IV fungal
CARs optimal activity in vitro was observed at 25-30°C. These
biophysical parameters correlated with previously described
E. coli BL21 GroEL/GroES EcPPTase TvCAR
citrate, glucose, air
E. coli endogenous ADH/ADRs
citrate, glucose, air
O
O
3b
3c
3a
Figure 6. A-B) Scheme and whole cell-mediated biotransformation of 3a
to 3c with 50 OD units of E. coli BL21 (DE3) [pG-KEJ8] [pETDuet-
1:EcPPTase:TvCAR]. The 3-HT formation over different time points at
28°C is depicted. Error bars show the standard deviation of three technical
triplicates. The red line symbolizes the benchmark of 27.9 mM 3-HT previously
CARs. TvCAR´s Tm of 55°C indicated
a higher stability
compared to type III fungal CAR, NcCAR, as well as other type
IV CARs. Due to the tolerance of acidic pH environments and
the higher stability of TvCAR, it was finally applied for the
reduction of DOPAC to 3-HT. 58.2 mM of 3-HT were converted
in whole cell biotransformations when 60 mM of DOPAC was
used within 24 h without any optimizations, which is nearly a
duplication compared to the previous benchmark.
achieved with 120 OD units over 21 h..[40]
: 10 mM 3a used as substrate.
:
25 mM 3a used as substrate. : 40 mM 3a used as substrate. X: 60 mM 3a
used as substrate.
We opted for E. coli BL21 (DE3) as the expression host for
TvCAR and EcPPTase expression via the pETDuet-1 vector in
the presence of [pG-KEJ8], to create a second-generation
catalyst for 3c preparation. 3c formation was monitored, using
50 OD units of E. coli BL21 (DE3) [pG-KEJ8] [pETDuet-
1:EcPPTase:TvCAR]. This corresponds to 48 mg mL- 1 of wet
cells. With 3a concentrations varying from 10 to 60 mM, overall
up to 58.2 mM of 3c were formed within 24 h. This corresponds
to 0.37 g L-1 h-1 (Figure 6), in comparison to 0.21 g L- 1 h- 1 using
the first generation strain.[40] In 21 h, 49.4 mM of 3c were
detected when 60 mM of 3a was used. By co-expressing
chaperones to increase the amount of soluble CAR protein
within the E. coli cells, using the validated pETDuet-
1:EcPPtase:TvCAR expression set-up with the TvCAR as well
as using the E. coli BL21 (DE3) strain to facilitate high
Experimental Section
General
All chemicals were obtained from Sigma-Aldrich/Merck or Roth, except
otherwise noted. No further purification steps were performed. HPLC-MS
grade acetonitrile was purchased from J.T.Baker/Avantor Performance
Materials, Deventer, The Netherlands. Helium gas for GC-analysis was
purchased from Linde Gas, Austria. For combining vector and insert
fragments, the Gibson Assembly® was performed with the Gibson
Assembly® HiFi 1-Step Kit by Synthetic Genomics® (Cat. Nr. GA1100-10).
expression
levels
and
the
overreduction
of
3,4-
Phylogenetic analysis
dihydroxypheylacetaldehyde 3b to 3c,
a
significant
improvement of the synthesis of 3-HT (3c) was demonstrated. At
the same time, the amount of cells was reduced from OD120 to
OD50 which lead to a four-fold increase of biocatalyst yield
(0.11 g/g versus 0.027 g/g based on cell wet weight,
respectively) and a 1.7-fold improvement of the space-time-
yield. A closed vessel set-up by using 15 mL glass tubes
improved the mass balance. Previously, the mass balance was
30 functional CARs were analyzed (SI Table S1). The platform of
phylogeny.fr provided the ready-to-use pipeline of MUSCLE for multiple
alignment, PhyML for tree building, and TreeDyn for tree rendering.[41]
Expression and purification
Gene assembly, cultivation and expression
7
This article is protected by copyright. All rights reserved.

10.1002/cctc.201900333
ChemCatChem
FULL PAPER
Gene sequences were equipped with an N-terminal His6-tag and codon-
optimized for E. coli K12 via the IDT optimizing tool. The gene fragments
were ordered from IDT (SI Table S3). To combine the gene fragments,
overlap extension PCR was conducted. PCR was carried out under the
following conditions: 50 μL total volume, 200 μM of each dNTP, 0.2 μM of
each primer (SI Table S2), and 2.5 U of Q5 High-Fidelity DNA
polymerase. Cycling conditions were as follows: initial denaturation at
98°C for 30 s, followed by 25 cycles of denaturation at 98°C for 30 s,
annealing at 56°C for 30 s, extension at 72°C for 4 min, and a final
extension at 72°C for 4 min. The plasmid pETDuet-1 with
phosphopantetheinyltransferase from E. coli (Genbank Accession Nr.
CAQ31055.1) was used for vector and gene assembly via the method by
Gibson et al.[42] The correct construct was confirmed by automated
sequencing (Microsynth) and a control digestion.
Frozen aliquots of Ni-affinity chromatography-purified CARs were thawed,
re-buffered to 50 mM MES, pH 7.5 and loaded onto a QFF HiTrap 5 mL
FF column. The anion-exchange chromatography was conducted with
Äkta Pure 100 with a fraction collector F950 (Unicorn 6.3 software, GE
Healthcare) by applying a concentration gradient (0-100%) of 0.5 M NaCl.
CARs eluted at approximately 27% of NaCl, which correlates to 0.135 M
of NaCl. Protein concentrations were determined with a Nanodrop 2000c
spectrophotometer (Thermo Scientific).
Tv2CAR co-expressed with various chaperone and modified E. coli
strains
E. coli strains listed in Table 2. were transfected with pETDuet-
1:EcPPTase:HTTv2CAR (SI Figure S2). For the co-induction of the
chaperone proteins, 0.5 mg mL-1 L-arabinose (pG-KEJ8) and/or 5 ng mL-1
tetracycline ([pG-KJE8], [pG-TF2]) was added before the temperature
was adjusted to 20°C.
E. coli K12 MG1655 RARE (DE3) or BL21 (DE3), respectively, was
transformed with the plasmid [pETDuet-1:EcPPTase:HTXyCAR] and
colonies selected via LB/Amp. Protein expression was performed via the
autoinduction protocol described by Studier.[28] After 24 h of expression
at 20°C, the cells were harvested by centrifugation and used for
biotransformations or the pellet was stored at -20°C until further use.
Co-expression of beneficial chaperones with weakly expressed TvCAR
and Tv2CAR
E. coli K-12 MG1655 RARE (DE3) pETDuet-1:EcPPTase:HTTvCAR and
E. coli K-12 MG1655 RARE (DE3) pETDuet- 1:EcPPTase:HTTv2CAR
were transfected with either pG-KJE8 or gG-TF2 to exploit the diminished
aldehyde reduction of E. coli K-12 MG1655 RARE (DE3). For the co-
induction of the chaperone proteins, 0.5 mg mL-1 L-arabinose (pG-KEJ8)
and/or 5 ng mL-1 tetracycline (pG-KJE8, pG-TF2) was added before the
temperature was downregulated to 20°C.
Expression analysis
The culture supernatant was discarded, and the pellet was resuspended
in 50 mM MES buffer, pH 7.5, containing 10 mM MgCl2, 1 mM EDTA,
and 1 mM DTT. The cell suspensions were transferred to pulping bottles
and sonicated for 2x3 min with 80% duty cycle and output control level 8
with extensive cooling. Whole cell lysates were immediately cooled on
ice and centrifuged in 50 mL tubes for 45 min at 41,533 rcf and 4°C to
pellet cell debris. The supernatant was further used for Ni-affinity
chromatography. The pellet was resuspended in 2 mL of 6 M urea and
incubated for 40 min at 60°C and 700 rpm. After centrifugation for 15 min
at 16,168 rcf, the concentration of the supernatant was determined via
the BCA-Assay. Successful protein expression was visualized by SDS-
PAGE analysis under reducing conditions. Protein samples were
denatured in NuPAGE LDS Sample Buffer (4X) for 10 min at 80°C. In
general, 15 µL sample solution containing 10 µg protein for cell lysate
and 5 µg for purified protein in 1X NuPAGE™ LDS Sample Buffer were
loaded onto a NuPAGE Bis-Tris gels 4–12% polyacrylamide gels and run
at 25 mA per gel using the XCell SureLock™ Mini-Cell Electrophoresis
System with a PowerEase® 500 Power Supply. Staining was performed
with SimplyBlueTM SafeStain solution (LC6065, Novex®) according to the
manufacturers protocol. After staining, the gel was washed with dH2O
and destained overnight in dH2O. Bands corresponding to soluble and
insoluble protein were analyzed via G:Box HR16 by Syngene (SnapGene,
software by Syngene). The software Gene Tools by Syngene was used
for quantification of the protein bands.
Enzyme characterization
Resting cell bioconversions of 1a determined by HPLC-UV
Resting cell bioconversions: OD600 values were determined and culture
aliquots corresponding to 50 OD600 units were transferred into the wells
of a 24-deep well plate. Specifically, 48.4 mg mL-1 of cells were used for
each reaction. Cells were harvested by centrifugation for 20 min at
3,220 rcf and 4°C. Afterwards, the supernatants were removed and
pellets were carefully resuspended in 200 mM potassium phosphate
buffer, pH 7.4, containing 10 mM piperonylic acid 1a in KOH 100 mM,
48 mM glucose, 24 mM sodium citrate and 4 mM MgSO4. Bioconversions
were carried out in 24-deep well plates covered with oxygen permeable
membranes for 2 h at 28°C and 320 rpm. The reactions were then
stopped by transferring 500 µL of cell suspension into an Eppendorf tube
and adding 1.5 mL of MeOH. Further, the suspensions were centrifuged
for 5 min at 16,168 rcf and 22°C. 200 µL of the supernatant were
transferred into 96-well plates (V-bottom, polypropylene). At least three
individual experiments were conducted. Conversions with 1a were
determined by HPLC. 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 thermostated column compartment and a G1315B Diode Array
Detector was used. The analysis of 1a, 1b and 1c was carried out with a
Kinetex 2.6μ Biphenyl 100A HPLC column (Phenomenex). The mobile
phases were ammonium acetate (5 mM) and 0.5% v/v acetic acid in
water and acetonitrile (ACN) at a flow‐rate of 0.26 mLꢀmin−1. 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). The compounds were detected at 254 nm
(DAD) (SI Table S7).
Ni-affinity chromatography
Cell free extracts (CFEs) were prepared by resuspending harvested cell
pellets in 20 mM potassium phosphate buffer, 500 mM NaCl, pH 7.4,
supplied with 10 mM imidazole for purification of recombinant protein.
The prepared CFE was filtered through a 0.22 μm syringe filter prior to
purification. Protein purification was conducted with prepacked, ready-to-
use column or with Äkta Pure 100 with a fraction collector F950 (Unicorn
6.3 software, GE Healthcare) by applying a concentration gradient of
imidazole. For buffer exchange and removal of imidazole after
purification, proteins were desalted using PD-10 columns, previously
equilibrated with desalting buffer (50 mM MES, pH 7.5, containing 10 mM
MgCl2, 1 mM EDTA, and 1 mM DTT). Aliquots of the protein solution
were shock frozen in liquid nitrogen and stored at −80ꢀ°C. Protein
Determination of substrate scope by NADPH-depletion monitoring
The NADPH depletion assay was used to determine initial rate activities
for the reduction of short to middle-long aliphatic acids and aromatic
acids. Therefore, carboxylic acids were dissolved in DMSO or KOH (each
100 mM). The assay composition was as follows: the substrate (100 mM
stock solution, 5 mM end concentration) was added to MES buffer
concentrations
spectrophotometer (Thermo Scientific).
were
determined
with
a
Nanodrop
2000c
Anion-exchange chromatography
8
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10.1002/cctc.201900333
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FULL PAPER
(50 mM, pH 7.5, containing 10 mM MgCl2, 1 mM EDTA, and 1 mM DTT).
Subsequently, 0.5 mM NADPH,1.0 mM ATP and 10 μL of CAR enzyme
preparation from Ni‐affinity chromatography (1.0 mg ꢀmL−1) were added
to a final volume of 200 µL. The depletion of NADPH was followed on a
Synergy Mx Plate reader (BioTek) at 340 nm and 28ꢀ°C for 10 min. Blank
reactions (ATP omitted) were carried out in parallel and each reaction
was carried out in triplicates.
The temperature stability of CARs was investigated after two step
purification by Ni-affinity chromatography and anion exchange
chromatography by monitoring the fluorescence of a solvatochromic dye
(ThermoFluor®)[35],[43] 20 mg -1 of each CAR was added to SYPRO
Orange (1:200 dilution) and 100 mM buffer pH 4.6-9.0. Sodium acetate
pH 4.6-5.5, sodium citrate pH 5.5-6.5, MES-NaOH pH 5.5-6.5, potassium
phosphate pH 6.0-8.0, HEPES-NaOH 7.5-8.0, bicine-NaOH pH 7.0-9.0
and Tris-HCl pH 8.0-9.0 were used. The samples were measured in
triplicates with CFX96 touch detection system (Bio-Rad). The
temperature profile started from 20°C to 95°C in a gradient of 1°C min-1.
The fluorescence emission of the dye was determined with HEX
fluorescence emission filter to monitor unfolding of the protein. The Tm is
defined as the temperature at the inflection point of the corresponding
melting curve.
In vitro activity assay for determination of pH optima of TvCAR
2 mM octanoic acid (7a) in DMSO (0.1 M) were added to 100 mM MES
buffer, pH 7.5, containing 10 mM MgCl2, 1 mM EDTA, and 1 mM DTT.
90 µL of enzyme preparation from Ni‐affinity chromatography (1 mg mL-1)
were added to the reaction mix. 1.2 mM ATP and NADPH were added to
start the reaction. The in vitro activity assay was performed in a total
volume of 600 µL in Eppendorf reaction tubes at 28°C and 700 rpm for
3 h in a Thermomixer comfort (Eppendorf). To stop the reaction, the
solution was acidified to pH 2.0 with 3 M HCl. Extraction of fatty acid,
aldehyde and alcohol was achieved with two times 400 µL of ethyl
acetate, including 0.01% tetradecane as internal standard (IS).
Afterwards, the mixture was vortexed for 10 min and centrifuged for
15 min at 16,168 rcf and 4°C. The organic phase containing the extracted
fatty acid, aldehyde, alcohol and IS was transferred to a fresh Eppendorf
tube and dried with Na2SO4 (approximately 10% w/v). 190 µL of the
mixture were transferred into glass vials for GC analysis. For the analysis
of fatty acid concentration, at least technical triplicates were carried out.
Quantitation was performed based on linear interpolation of calibration
curves with concentrations of standards from 0.156 mM to 30 mM.
Improved bioconversion of DOPAC to 3-hydroxytyrosol by TvCAR
Resting cell bioconversions of DOPAC (3a) determined by HPLC-MS
Resting cell bioconversions: OD600 values were determined and culture
aliquots corresponding to 50 OD units were transferred into Pyrex™
tubes. Cell pellets were harvested by centrifugation for 20 min at
3,220 rcf and 4°C. Afterwards, the supernatants were removed and
pellets were carefully resuspended in 1.5 mL of 200 mM MES buffer, pH
6.0, containing 10-60 mM DOPAC (3a), 10 mM MgCl2, 20 mM glucose,
48 mM sodium citrate and 8 mM MgSO4. Bioconversions were carried
out in glass cultivating tubes up to 24 h at 28°C in a tissue rotator. The
suspensions were transferred to Eppendorf tubes and centrifuged for
40 min at 16,168 rcf. 200 µL of the supernatant were transferred into 96-
well plates (V-bottom, polypropylene). At least three individual
experiments, i.e. technical triplicates, were conducted.
Analysis of ,7a-c was carried out on an Optima
1 MS (100%
dimethylpolysiloxane; 30 m, 0.25 mm in diameter and 0.25 μm film
thickness) on a Shimadzu GCMS-QP2010 SE equipped with a mass
selective detector. Sample aliquots of 1 μL were injected in split mode
(split ratio 6.5:1) at 240°C injector temperature and 320°C detector
temperature with helium as carrier gas. The temperature program was as
follows: 70°C for 5 min and a ramp to 300°C at 15°C min-1. The total run
time was 24.26 min (SI Table S5). The mass selective detector was
operated in a mass range of 30-500 m/z. GC-MS results were evaluated
with the GC-MS Data Analysis software LabSolution by Shimadzu.
Conversions with 3a were determined by HPLC-MS. For HPLC-MS
measurements, 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 mass-selective detector (MSD) were used. Samples
were filtered through AcroPrep 96 Filter Plate (Pall Life Science) with
0.2 mm
polypropylene
membrane
prior
to
analysis.
3,4-
Determination of optimal pH of Tv2CAR and DsCAR
Dihydroxyphenylacetic acid (3a), 3,4-dihydroxyphenylacetaldehyde (3b),
and 3-HT (3c) were separated on a Chromolith Performance RP-18
endcapped 100–4.6 column (Merck) at 30°C by using a 95% aqueous
eluent (0.1% formic acid) and 5% ACN at a flow of 0.8 mL min-1
(SI Table S8).
CARs were investigated in the pH range of 5.5-9.0 in intervals of 0.5 pH
units. All buffers (100 mM, supplemented with 10 MgCl2), were adjusted
to the desired pH value with either NaOH or HCl at 25°C. Specifically,
MES-NaOH pH 6.0-7.5, potassium phosphate pH 6.0-7.5, bicine-NaOH
pH 7.0-8.0 and Tris-HCl pH 7.5-9.0 were applied. All other parameters
were applied as described in in vitro activity assay. Activity was
calculated relative to the maximum rate at 100%.
Acknowledgements
Giuseppe Fiume, Daniel Schwendenwein and Sandy Schmidt
are greatly acknowledged for technical assistance. The authors
thank Fredi Brühlmann and Andreas Taglieber for valuable
discussions as well as Kristala L. J. Prather for generously
providing Escherichia coli K-12 MG1655 RARE (DE3). The
Austrian Research Promotion Agency FFG (P 860262) and
Firmenich SA are kindly acknowledged for the financial support.
This research has also been supported by the Austrian BMWFW,
BMVIT, SFG, Standort Tirol, Government of Lower Austria and
ZIT through Austrian FFG-COMET-Funding Program.
Determination of optimal temperature
HEPES-NaOH buffer (100 mM), pH 7.5, containing 10 mM MgCl2, 1 mM
EDTA and 1 mM DTT was prepared at temperatures ranging from 25 to
50°C. The enzyme preparations were preincubated for 1 h at each
temperature. Residual activity was determined as described in in vitro
activity assay. Activity was calculated relative to the maximum rate at
100%.
Determination of pI of TvCAR
Ni-affinity chromatography purified samples, in 10 mM MES buffer, were
mixed with SERVALYT™ carrier ampholytes and separated on
a
Rotofor® Cell (Bio-Rad). The pI was determined through determination of
the pH of each protein fraction and loading each protein fraction onto
SDS-PAGE.
Keywords: carboxylate reductase (CAR) • in vivo
biotransformation • carboxylic acid • reduction • antioxidant
[1]
[2]
Y. H. P. Zhang, Biotechnol. Adv., 2015, 33, 1467–1483.
Thermostability determination via thermal shift assays
U. Hanefeld, R. A. Sheldon, Isabel Arends, Electrochemistry, 2007,
9
This article is protected by copyright. All rights reserved.

10.1002/cctc.201900333
ChemCatChem
FULL PAPER
2, 1–20.
Nordlund, Anal. Biochem., 2006, 357, 289–298.
[3]
[4]
[5]
[6]
R. Cernansky, Nature, 2015, 519, 379–380.
[36]
[37]
[38]
Y. Achmon and A. Fishman, Appl. Microbiol. Biotechnol., 2014, 99,
1119–1130.
J. D. Keasling, Science, 2003, 330, 1355–1358.
J. D. Keasling, Metab. Eng., 2012, 14, 189–195.
D. Gahloth, M. S. Dunstan, D. Quaglia, E. Klumbys, M. P. Lockhart-
Cairns, A. M. Hill, S. R. Derrington, N. S. Scrutton, N. J. Turner and
D. Leys, Nat. Chem. Biol., 2017, 13, 975–981.
M. Ahsan, S. Sung, H. Jeon, M. Patil, T. Chung and H. Yun,
Catalysts, 2017, 8, 4.
E. B. Bali, V. Ergin, L. Rackova, O. Bayraktar, N. Küçükboyaci and
Ç. Karasu, Planta Med., 2014, 80, 984–992.
S. Burattini, S. Salucci, V. Baldassarri, A. Accorsi, E. Piatti, A.
Madrona, J. L. Espartero, M. Candiracci, G. Zappia and E. Falcieri,
Food Chem. Toxicol., 2013, 55, 248–256.
[7]
[8]
[39]
[40]
[41]
K. Napora-Wijata, Ch. Rijksen, K. Robinson, A. V. Osorio Lozada
and M. Winkler, WO 2014/090923 Al, 2014, 1–65.
K. Napora-Wijata, K. Robins, A. Osorio-Lozada and M. Winkler,
ChemCatChem, 2014, 6, 1089–1095.
M. K. Akhtar, N. J. Turner and P. R. Jones, Proc. Natl. Acad. Sci. U.
S. A., 2013, 110, 87–92.
[9]
Z. Hu, US 2010/0105963 A1, 2010, 1-46.
[10]
L. Kramer, E. D. Hankore, Y. Liu, K. Liu, E. Jimenez, J. Guo and W.
Niu, ChemBioChem, 2018, 19, 1452–1460.
A. Dereeper, V. Guignon, G. Blanc, S. Audic, S. Buffet, F. Chevenet,
J.-F. Dufayard, S. Guindon, V. Lefort, M. Lescot, J.-M. Claverie and
O. Gascuel, Nucleic Acids Res., 2008, 36, 465–469.
D. G. Gibson, L. Young, R.-Y. Chuang, J. C. Venter, C. A.
Hutchison and H. O. Smith, Nat. Methods, 2009, 6, 343–5.
J. J. Lavinder, S. B. Hari, B. J. Sullivan and T. J. Magliery, J. Am.
Chem. Soc., 2009, 131, 3794–3795.
[11]
[12]
G. Qu, J. Guo, D. Yang and Z. Sun, Green Chem., 2018, 20, 777–
792.
[42]
[43]
H. Stolterfoht, D. Schwendenwein, C. W. Sensen, F. Rudroff and M.
Winkler, J. Biotechnol., 2017, 257, 222–232.
[13]
[14]
G. G. Gross, Eur. J. Biochem., 1972, 31, 585–592.
D. Schwendenwein, G. Fiume, H. Weber, F. Rudroff and M. Winkler,
Adv. Synth. Catal., 2016, 358, 3414–3421.
[15]
[16]
P. Venkitasubramanian, L. Daniels and J. P. N. Rosazza, J. Biol.
Chem., 2007, 282, 478–485.
H. Stolterfoht, G. Steinkellner, D. Schwendenwein, T. Pavkov-Keller,
K. Gruber and M. Winkler, Front. Microbiol., 2018, 9, 250.
M. Wang, M. Beissner and H. Zhao, Chem. Biol., 2014, 21, 257–63.
C. Li, Y. Matsuda, H. Gao, D. Hu, X. S. Yao and I. Abe,
ChemBioChem, 2016, 17, 904–907.
[17]
[18]
[19]
M. Winkler and C. K. Winkler, Monatshefte für Chemie - Chem.
Mon., 2016, 147, 575–578.
[20]
[21]
[22]
M. Wang and H. Zhao, ACS Catal., 2014, 4, 1219–1225.
T. Li and J. P. N. Rosazza, J. Bacteriol., 1997, 179, 3482–3487.
A. He, T. Li, L. Daniels, I. Fotheringham and J. P. N. Rosazza, Appl.
Environ. Microbiol., 2004, 70, 1874–1881.
[23]
[24]
Y. Duan, P. Yao, X. Chen, X. Liu, R. Zhang, J. Feng, Q. Wu and D.
Zhu, J. Mol. Catal. B Enzym., 2015, 115, 1–7.
W. Finnigan, A. Thomas, H. Cromar, B. Gough, R. Snajdrova, J. P.
Adams, J. A. Littlechild and N. J. Harmer, ChemCatChem, 2017, 9,
1005–1017.
[25]
[26]
[27]
A. M. Kunjapur, Y. Tarasova and K. L. J. Prather, J. Am. Chem.
Soc., 2014, 136, 11644–11654.
H. P. Sørensen and K. K. Mortensen, Microb. Cell Fact., 2005, 4, 1–
8.
A. K. Hess, P. Saffert, K. Liebeton and Z. Ignatova, PLoS One, 2015,
10, 1–14.
[28]
[29]
F. W. Studier, Protein Expr. Purif., 2005, 41, 207–234.
C. C. R. Carvalho and R. M. M. Fonseca, Dyn. Biochem. Process
Biotechnol. Mol. Biol., 2007, 1, 32–39.
[30]
[31]
B. Lin and Y. Tao, Microb. Cell Fact., 2017, 16, 1–12.
K. Nishihara, M. Kanemori, H. Yanagi and T. Yura, 2000, 66, 884–
889.
[32]
[33]
K. Nishihara, M. Kanemori, M. Kitagawa, H. Yanagi and T. Yura,
Appl. Environ. Microbiol., 1998, 64, 1694–1699.
O. Kandror, M. Sherman, R. Moerschell and A. L. Goldberg, J. Biol.
Chem., 1997, 272, 1730–1734.
[34]
[35]
K. Talley and E. Alexov, Proteins, 2010, 78, 2699–2706.
U. B. Ericsson, B. M. Hallberg, G. T. DeTitta, N. Dekker and P.
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10.1002/cctc.201900333
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Characterization
of
type
IV
carboxylate reductases (CARs) for
whole cell-mediated preparation of 3-
hydroxytyrosol
Expanding the toolbox: Fragrance and flavor industries could not imagine
business without aldehydes. Mild and selective reduction of carboxylic acids to
aldehydes is achieved with new carboxylic acid reductase enzymes (CARs).
11
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