In Vivo Reduction of Medium- to Long-Chain Fatty Acids by Carboxylic Acid Reductase (CAR) Enzymes: Limitations and Solutions

Article abstract of DOI:10.1002/cctc.202000895

Fatty aldehyde production by chemical synthesis causes an immense burden to the environment. Within this study, we explored a sustainable, aldehyde-selective and mild alternative approach by utilizing carboxylic acid reductases (CARs). CARs from Neurospora crassa (NcCAR), Thermothelomyces thermophila (TtCAR), Nocardia iowensis (NiCAR), Mycobacterium marinum (MmCAR) and Trametes versicolor (TvCAR) were overexpressed in E. coli K-12 MG1655 RARE (DE3) and screened for medium- to long-chain fatty acid (C6–C18) reduction. MmCAR showed the broadest tolerance towards all carbon-chain lengths and was selected for further investigations of fatty aldehyde synthesis in whole cells. To yield relevant product concentrations, different limitations of CAR whole-cell conversions were elucidated and compensated. We coupled an in vitro cofactor recycling system to a whole-cell biocatalyst to support cofactor supply and achieved 12.36 g L^?1 of octanal (STY 0.458 g L^?1 h^?1) with less than 1.5 % of 1-octanol.

Full text of DOI:10.1002/cctc.202000895

The European Society Journal for Catalysis
Supported by
Accepted Article
Title: In vivo reduction of medium- to long-chain fatty acids by
carboxylic acid reductase (CAR) enzymes: limitations and
solutions
Authors: Melissa Horvat and Margit Winkler
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ChemCatChem
10.1002/cctc.202000895
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In vivo reduction of medium- to long-chain fatty acids by carboxylic
acid reductase (CAR) enzymes: limitations and solutions
[a]
[a,b]
Melissa Horvat and Margit Winkler
*
[
[
a]
b]
M. Horvat, Priv.-Doz. Dr. M. Winkler
acib - Austrian Center of Industrial Biotechnology
Krenngasse 37, 8010 Graz, Austria
E-mail: margit.winkler@tugraz.at
Priv.-Doz. Dr. 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.
Abstract: Fatty aldehyde production by chemical synthesis causes
an immense burden to the environment. Within in this study, we
explored a sustainable, aldehyde-selective and mild alternative
approach by utilizing carboxylic acid reductases (CARs). CARs from
Neurospora crassa (NcCAR), Thermothelomyces thermophila
adenylation domain (A- domain). Pyrophosphate is released. The
AMP-anhydride is being targeted by the 4´phosphopantetheine
moiety of the linker (peptidyl carrier protein (PCP) -domain) and
the substrate is shuttled to the reductase domain (R-domain) as
an enzyme bound thioester, while AMP is being released. In the
presence of NADPH, the thioester is reduced to the respective
aldehyde and released together with NADP+.[14] For activation of
(TtCAR), Nocardia iowensis (NiCAR), Mycobacterium marinum
MmCAR) and Trametes versicolor (TvCAR) were overexpressed in
(
E. coli K-12 MG1655 RARE (DE3) and screened for medium- to long-
chain fatty acid (C6-C18) reduction. MmCAR showed the broadest
tolerance towards all carbon-chain lengths and was selected for
further investigations of fatty aldehyde synthesis in whole cells. To
yield relevant product concentrations, different limitations of CAR
whole cell conversions were elucidated and compensated. We
coupled an in vitro cofactor recycling system to a whole cell
the
CAR
enzyme,
its
apo-form
needs
to
be
phosphopantetheinylated by a phosphopantetheinyl transferase
(PPTase) under coenzyme A (CoA) consumption to form active
holo-CAR.
The necessity of PPTases to activate PCPs has been studied
extensively. In nature, 4´-phosphopantethenylation is required for
pathways forming toxins or primary and secondary metabolites.[
Co-expression of PPTase with CAR enzyme was previously
shown to enhance CAR activity and is seen as the gold
standard.[14] Literature predominantly reports on the co-
expression of SfpPPTase with CAR enzymes. It is the best
15]
-
1
biocatalyst to support cofactor supply and achieved 12.36 g L of
-
1
-1
octanal (STY 0.458 g L h ) with less than 1.5 % of 1-octanol.
characterized PPTase with a wide substrate spectrum, which
should interact with PCP-domains of diverse CARs,[16] but the
Introduction
activation of apo- to holo-CAR has not yet been addressed
systematically.
Due to the dependency of the enzyme on cofactors, an in vivo
approach as seen in the production of vanillin[17] seemed most
feasible and cost-efficient in view of synthetic applications. To
explore the suitability of CAR enzymes for the production of
aliphatic aldehydes, the ideal process criteria would be the
following: at least gram per liter production, a broad substrate
In the early 20th century, aldehydes were identified as crucial
constituents of flavors and fragrances.[1] In particular, medium-
and long-chain aliphatic aldehydes have ever since transformed
the world of smells. In addition, aldehydes are seen as highly
valuable intermediates in pharmaceutical industry[2] as well as
precursors for products such as biofuels, biopolymers, etc.[3,4] The
reactive nature of aldehydes is a challenge for their production:
under oxidative conditions from alcohols, toxic catalysts are being
employed,[5] and reductive routes from carboxylic acids suffer
from poor atom economy and are prone to overreduction.
Additional activation of the carboxylic acid group and protection of
other reactive groups to minimize off-target reductions are in most
cases required.[6] All these factors are cost contributors and cause
an immense burden onto the environment.
acceptance by the enzyme, high biocatalyst yield/low biocatalyst
- 1
requirement (< 0.005 g L
cell wet weight (CWW)), no by-
products, no toxicity of substrates/products to the host organism,
sufficient supply of cofactors, high intracellular concentrations of
active CAR and a continuous process.[9] Several constraints were
observed with the in vivo CAR approach for vanillin production by
Hansen et al.[18] First, the metabolic flux of the carbon-source to
In the biocatalysis community, a specific two-electron reduction of
carboxylic acids to aldehydes by carboxylic acid reductases (CAR,
EC 1.2.1.30) was discovered.[7,8] Bioreductions facilitated by CAR
enzymes constitute a sustainable and environmentally-friendly
alternative to chemical approaches.[9] Due to their broad substrate
scope, including aromatic, aliphatic and heterocyclic carboxylic
acids, CARs are gaining interest.[10–12] Structural analysis recently
confirmed the catalytic steps in these enzymes that are composed
of 3-domains.[ First, the enzyme converts the substrate to an
AMP-anhydride in the presence of ATP and Mg2 in the
vanillin in de novo synthesis was difficult to control. Similar
difficulties in controlling the carbon flux were seen in the de novo
synthesis of fatty acids, which have been described
extensively.[19–21] The first and major challenge is targeting
[3]
specific carbon-chain lengths of fatty acids. A mixture of fatty
acids will result in a mixture of the respective aldehydes in a
reaction sequence with CARs due to the enzyme´s broad
substrate scope. In case of individual aldehydes as desired
products, the use of living cells in biotransformation mode
13]
+
(
feeding carboxylic acids) is typically more efficient compared to
1
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ChemCatChem
10.1002/cctc.202000895
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the de novo approach, due to the limited carbon flux to the desired
fatty acid as CAR substrate.[19] Herein we focused on this strategy.
investigated to pull the aldehyde intermediate from the reaction
towards follow-up products. Because of impurities in enzyme
preparations alcohol by-products were observed.[
32,33]
Fatty
The second challenge was that the host organisms showed high
levels of aldehyde overreduction by alcohol dehydrogenases
(
amines were targeted by Citoler et al. in a one-pot tandem
cascade performed by a CAR and a transaminase, while recycling
NADPH and ATP. In order to achieve high fatty amine
concentrations, an in situ product removal system (ISPR) was
applied and an end product yield of 0.927 g L - 1 dodecyl amine
was achieved.[33] ISPR is used to trap the compounds in the
ADHs) and/or aldo-keto reductases (AKR) (Figure 1).[18]
Overreduction of aldehydes to alcohols (Figure 1) is the
intracellular defense-mechanism of the expression host for coping
with high concentrations of these toxic compounds.[22] Of the
respective acid, aldehyde and alcohol, the fatty acid is the most
tolerated compound.[23] However, relatively low concentrations of
aliphatic acids and derivatives cause growth inhibition, cell
membrane perforation and leakage, thus disrupting the electron
transport chain, uncoupling oxidative phosphorylation and
organic layer and to improve product yields by reducing the
[34,35]
exposure of the cells to the cytotoxic compounds.
Due to
decrease in cell viability when organic solvents are used for ISPR,
biocompatible solvents can be beneficial to achieve high product
titers. Cell viability ensures intracellular cofactor regeneration,
and biocompatible solvents could be highly advantageous in this
respect.[36]
[
24]
ultimately diminish ATP production. 30 mM octanoic acid, e.g.,
caused 46% of increased leakage of ions in E. coli K-12 MG1655
cell membranes. Perforation of the cell membranes is the result
of a missing defense strategy in E.coli.[25,26]
An optimized whole cell system for acid reduction to fatty
aldehydes as end products is of high demand, but only few
studies actually describe targeting fatty aldehydes. With the
exception of Hansen et al.,[ no study aimed for a comprehensive
picture of advantages and limitations of CAR mediated whole cell
applications aiming for aldehyde products. In this study, CAR
18]
enzymes
from
Neurospora
crassa
(NcCAR),[34]
[
37]
Thermothelomyces thermophila (TtCAR),
Nocardia iowensis
Figure 1. Reduction of carboxylic acids to aldehydes and overreduction to
(NiCAR),[38] Mycobacterium marinum (MmCAR)[27] and Trametes
versicolor (TvCAR)[39] were overexpressed in E. coli K-12
MG1655 RARE (DE3) and screened for medium- to long-chain
fatty acid (C6-C18) reduction. MmCAR showed the broadest
tolerance towards all carbon-chain lengths and was selected for
further investigations of fatty aldehyde synthesis in whole cells.
To yield relevant product concentrations, a systematic evaluation
of various parameters of both the cultivation phase and the
biotransformation phase were carried out.
alcohol.
The majority of literature reports on alcohols as the desired
products of CAR mediated acid reductions. For example, Akthar
et al. were the first to describe the medium- to long-chain fatty
acid reduction by Mycobacterium marinum CAR to a mixture of
fatty alcohols and alkanes for de novo biofuel production in
milligram-scale.[27] Ahsan et al. targeted fatty diols by utilizing an
enzyme cascade incorporating endogenous aldehyde reductase
activity of E. coli. By doing so, 4.6 g L - 1 of 1,12-dodecanediol, a
very important monomer for polyester production, were produced,
demonstrating the feasibility of gram-scale production with a
whole cell catalyst. However, 30 OD units (~30 mg mL - 1 CWW)
of E. coli were applied and production of 1,8-octanediol resulted
Results and Discussion
Substrate scope of CARs in single phase reactions in vitro
A toolbox of CARs is available today for enzymatic reduction
of carboxylic acids to aldehydes. For production of fatty
aldehydes, the most suitable CAR needed to be identified.
Therefore, fungal and bacterial CARs of three phylogenetic
groups[40] were screened for fatty acid reductase activities.
-
1
in only 0.56 g L
product concentration. The explanation for
decreased product titer might be increased toxicity of the shorter
carbon-chain compound compared to 1,12-dodecanediol.[28]
Further, a study targeting C10-C12 aldehydes could not limit
overreduction to less than 80% in E. coli MG00 of E. coli K-12
MG1655 origin.[29]
The substrate specificity of CARs was described to be
determined by the adenylation core domain of CARs.[
Because sequence similarities between different subtypes
and between fungal and bacterial origins are below 25%,
substrate spectra vary.[40,42]
41]
Targeting aldehydes, Hansen et al. established an enzyme
cascade to produce vanillin glucoside which is the naturally
occurring compound in vanilla pods, via de novo biosynthesis.[18]
Additionally, yeast strains used for vanillin glucoside production
were engineered to minimize overreduction to vanillyl alcohol.
ADH and AHR genes responsible for overreduction of vanillin
were identified and deleted. In a similar study, the E. coli K-12
MG1655 (DE3) strain was modified for aromatic aldehyde
accumulation by using the same approach.[30]
Substrate solubility limited the spectrophotometric assay to
medium chain aliphatic acids. Moreover, NADPH depletion
is only an indirect method. Chromatography based methods
directly detect the desired aldehyde
as a result of over-reduction. An in vitro activity assay method
was developed for quantitation of unreacted acid , desired
aldehyde and undesired alcohol using gas
b and possible alcohol c
a
b
c
As an alternative to cellular systems, CAR mediated synthesis of
aldehydes was recently shown. ATP is recycled in vitro with two
polyphosphate kinases, and NADPH by exploiting a commercially
available GDH. For eliminating the CAR inhibitor pyrophosphate
from the reaction, a pyrophosphatase from E. coli was also
implemented.[31] In similar approaches, cascade reactions were
chromatography (GC). Due to the lack of commercially
available product standards, substrates without product
standards were measured via gas chromatography-mass
spectrometry detection (GC-MS). All other fatty acids,
aldehydes and alcohols were quantified with gas
2
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chromatography-flame ionization detection (GC-FID) using
calibration curves.
Single-phase bioreduction of E. coli whole cells expressing
MmCAR and EcPPTase
Recently, biosynthesis of fatty acids and derivatives is
gaining attention due to their potential of replacing
petroleum-based process.[3,43] We aimed for an MmCAR
producing E. coli biocatalyst in in vivo to take advantage of
cellular metabolism for ATP and NADPH supply.
Figure 2 summarizes the substrate scope of CARs. First,
almost all CARs converted all listed compounds in Figure 2
at least in trace amounts. This highlights the broad substrate
tolerance of CARs. Substrate preferences agreed well with
literature. Type III NcCAR was outstanding for hexanoic acid
E. coli K-12 MG1655 RARE (DE3) had been engineered towards
reduced overreduction of aromatic aldehydes, such as
benzaldehyde and vanillin, to the respective alcohol by knocking-
out six alcohol dehydrogenase and aldo-keto reductase genes
and one activator gene by Kunjapur et al. A 55-fold improvement
in vanillin titers compared to the wild type strain was achieved.[
This strain seemed promising for aliphatic aldehyde production as
it also showed less overreduction of octanal.[44]
1
a (Figure 2). Type IV TvCAR had shown a tendency
towards aliphatic substrates in comparison to other type IV
CARs such as DsCAR and Tv2CAR.[37] Second, as indicated
by color, short chain aliphatic acids (hexanoic acid 1a and
heptanoic acid 2a) showed high conversions compared to
low conversions of long-chain fatty acids (oleic acid 14a and
linoleic acid 15a). Especially NcCAR showed outstanding
activity with 1a. When it comes to medium-chain substrates
30]
(
octanoic acid 3a
acid 5a, 8-methylnonanoic acid 6a, decanoic acid 7a, 9-
decenoic acid 8a undecanoic acid 9a 10-undecenoic
, nonanoic acid 4a, 4-methylnonanoic
,
,
acid 10a, and 5Z-dodecanoic acid 11a), type I (MmCAR and
NiCAR) and IV (TvCAR) CARs outperform type III CARs
Figure 3. Reduction of the model substrate octanoic acid (3a) to octanal (3b)
and overreduction to 1-octanol (3c).
(
NcCAR and TtCAR). Comparing branched and unbranched
substrates 4-6a, the presence of a terminal methyl-group
6a) increased conversion, whereas a branch in the middle
of the carbon chain (5a) reduced product formation with all
CARs. The best in vitro conversions were observed for 6a
Long-chain aliphatic substrates (myristoleic acid 12a,
palmitoleic acid 13a and oleic acid 14a were mostly
First bioreductions were conducted with this strain, harboring a
single plasmid for co-expression of MmCAR and EcPPTase.
Certain OD units of the E. coli strain co-expressing PPTase and
MmCAR were harvested and resuspended in reaction buffer.
Substrate and additives were added. After incubation of the
bioconversions at 28°C, the aqueous solution was extracted twice
with ethyl acetate and internal standard and measured via GC-
FID. Figure 4 shows full conversion of 6 mM 3a after 4.5 h at 28°C
with 50 OD units (corresponding to approximately 50 mg of cell
wet weight (CWW)). Independent of cell density, the product was
almost exclusively 3c, indicating that the engineered E. coli strain
still harbors alcohol dehydrogenases and aldo-keto reductases
with the ability for 3b reduction (Figure 3). Bioreductions with
(
.
)
accepted by bacterial and type IV fungal CARs. As a general
trend, CARs performed better with unsaturated substrates
compared to saturated compounds. This was also observed
in Citoler et al. in cascade reactions to fatty amines.[33]
Further, short-chain aliphatic substrates were more accepted
by type III CARs, whereas long-chain aliphatic substrates
were best accepted by type I bacterial CARs .
1
00 OD units showed poor mass balance already after 4.5 h,
which could be due to its utilization as a C-source. Up to 5.82 mM
of 1- octanol (97.0%) was detected under these conditions via
-1
GC-FID. This correlates to a productivity of 0.745 g L or a space
time yield of 0.165 g L- 1 h .
- 1
Figure 4. Single-phase bioreduction of E. coli K-12 MG1655 RARE (DE3)
expressing MmCAR and EcPPTase with different cell densities converting
6
mM 3a. 50 OD units correspond to approximately 50 mg cell wet weight
(
CWW). Bioconversions were incubated at 28°C for 4.5 h on a tissue culture
Figure 2. Substrate scope of CARs towards saturated, unsaturated and
branched fatty acids: hexanoic acid 1a, heptanoic acid 2a, octanoic acid 3a,
nonanoic acid 4a, 4-methylnonanoic acid 5a, 8-methylnonanoic acid 6a,
decanoic acid 7a, 9-decenoic acid 8a, undecanoic acid 9a, 10-undecenoic acid
rotator. White: 3a. Grey: 3b. Black striped: 3c (see Figure 3 for reaction scheme).
Error bars are shown for biological triplicates.
10a, 5Z-dodecenoic acid 11a, myristoleic acid 12a, palmitoleic acid 13a, oleic
acid 14a and linoleic acid 15a. Substrates 5a, 6a, 8a, 11a, 12a and 14a and 15a
were detected with GC-MS all others via GC-FID. (Retention times and chemical
structures are summarized in Table S6 and S7). X not determined.
3
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Time dependent reactions with 50 OD units of biocatalyst
This first experiment revealed the challenge to obtain
aldehyde instead of alcohol despite the CARs product
selectivity. We previously showed that in situ product
removal (ISPR) with n-hexane suppressed cinnamyl alcohol
formation to the benefit of cinnamaldehyde titers.[34] Using a
second organic layer puts additional constraints on the
biotransformation itself as well as on the industrial
applicability, such as process robustness, safety aspects
improve the reaction system, we investigated these
hypotheses.
Overreduction, a mechanism to minimize cytotoxicity of certain
compounds by the cell, constantly interfered with aldehyde titers.
For this reason, not many studies focused on fatty aldehydes as
[46]
the final product. On the contrary, they exploited this effect to
optimize fatty alcohol [
28,47]
or alkane
[27,48]
titers. To overcome
overreduction of fatty aldehydes, we screened 30 single deletion
E. coli BW25113 strains from the Keio Collection[49] to find a strain,
which might have a decreased defense mechanism for fatty
aldehydes (data not shown). Especially genes coding for alcohol
dehydrogenases and aldo-keto reductases were chosen as
targets. By incubation of the strains with medium- to long-chain
fatty aldehydes and determining alcohol concentrations, strains
with low alcohol concentrations should give indications of genes
responsible for overreduction of fatty aldehydes. Next to aldehyde
overreduction, also aldehyde oxidation to its respective fatty acid
was observed, which was most likely due to aldehyde
(
e.g. flammability of solvents) and the market of the
product.[ Furthermore, organic solvents such as n-hexane
and n-heptane, are cytotoxic.[33,34,45] This includes growth
inhibition, due to interaction of the solvent with cell
membranes. Effects on cell integrity and cell vitality must,
therefore, be expected in an ISPR approach with living (i.e.
ATP regenerating) cells. However, trapping the aldehyde in
an organic layer to reduce its harming effects increased cell
vitality and integrity compared to a single-phase system and
therefore increased conversion levels and aldehyde titers
35]
[
50]
dehydrogenases. In the best-case scenario, only one or two
genes would be identified as responsible for overreduction of fatty
aldehydes and would need to be engineered in the E. coli genome
for aliphatic aldehyde production. In Table S1 the four gene
deletion strains with the lowest alcohol concentrations for five
substrates (3a, 7a, 9a, 10a and 13a) were compared to E. coli K-
12 MG1655 RARE (DE3). None of the non-essential genes of the
Keio collection seems to exhibit the exclusive reduction capacity
for any of the tested substrates. A single gene deletion is certainly
not enough for any of the investigated substrates to fully suppress
alcohol formation.
(
Figure 5). A number of non-water miscible solvents were
tested for ISPR. A positive trend towards 3b production was
observed. A second organic layer resulted in at least 50% of
3b formation. After 3 h, full conversion was observed for
short-chain alkanes as second layer (n-hexane and
n- heptane), whereas only 80% of 3a was converted using
n- dodecane as trapping layer. The mass balance indicated
that 3a, 3b and 3c were fully extracted from the aqueous
phase. Decreased formation of 3c indicates that 3b is
efficiently exported from the cells and detoxification by over-
reduction is the secondary mechanism after 3b expulsion.
To our delight, E. coli K-12 MG1655 RARE (DE3) was the best
strain in this comparison. It was interesting to observe that gene
deletions of yahK, yjgB, yqhC and yqhD in the E. coli K-12
MG1655 RARE strain was found beneficial for minimizing
overreduction of fatty aldehydes, specifically 3a, in this study.
Deletions of yeaE, dkgB and dkgA might be specific to aromatic
substrates, because these genes did not show a positive effect
for aliphatic aldehyde reduction.
Reduced biocatalyst amount resulted in less than 1%
overreduction
Next, we aimed to determine the optimal biocatalyst
concentration. High biocatalyst amounts introduce more CAR, but
less biomass in the biotransformation should decrease
endogenous co-factor pools and enzymes with aldehyde
reductase activity. Indeed, by reducing biomass to 1-5 OD units
of E. coli K-12 MG1655 RARE (DE3) expressing EcPPTase and
MmCAR, only ~ 1% of 3c was formed within 5 h (Table 1,
Figure S3), at reduced 3b formation. Bioconversions with 50 OD
units of E. coli cells expressing CAR and PPTase showed nearly
Figure 5. Biphasic bioreduction converting 6 mM of 3a for aldehyde trapping
and comparison to single phase reaction. 50 OD units of E. coli K-12 MG1655
RARE expressing EcPPTase and MmCAR were incubated at 28°C for 3 h on a
tissue culture rotator. White: 3a. Grey: 3b. Black striped: 3c (see Figure 3 for
reaction scheme). Quantification by GC-FID.
6
0% of 3c. For industrial applications, biomass is a major cost-
factor. Hence, a reduction of biocatalyst to keep alcohol formation
low would be beneficial for multiple reasons.
Building on the positive effect of ISPR on aldehyde formation,
we next increased substrate concentrations. After 18 h, 30.8
mM 3b and 3c were produced from 50 mM of 3a (Figure S2).
Table 1. Product formation of biphasic bioreduction of 10 mM 3a with increasing
amount of E. coli MG1655 RARE cells, co-expressing EcPPTase and MmCAR.
This correlates to
a
productivity of 3.0 g L - 1 or
0
.168 g L – 1 h - 1. Evident from Figure S2, in vivo CAR
reactions discontinued after few hours. When aldehyde
formation stops, surplus NADPH leads to depletion of the
desired product and alcohol formation. Possible reasons for
preliminary stop of acid reduction may be poor stability of the
CAR or limited ATP and NADPH supply due to the effects of
solvent, products and/or substrate on cell viability. To
1 OD
0.10
1
2 OD
0.23
1
5 OD
0.48
1
10 OD
0.93
8
20 OD
1.75
26
50 OD
3.30
59
3
b+3c [mM][a]
3c in product [%][a]
[
a]
All standard deviations were <0.1%. Technical triplicates were measured via
GC-FID.
4
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Table 2. Comparison of MmCAR expression at different temperatures.
Expression parameters of MmPPTase and
MmCAR
CWW per 450 ml
cultivation [g]
In vivo product formation
of 3b+3c [mM][
Partially purified enzyme
yield [mg]
specific activity for 3a
[U/mg]
a]
auto induction at 20°C
auto induction at 25°C
auto induction at 28°C
5.91
6.15
6.53
6.02 ± 0.20
5.50 ± 0.23
3.21 ± 0.17
42.1
64.9
55.8
0.32± 0.02
0.18± 0.01
0.10± 0.03
[a]Conditions for biotransformation: 24 h post induction, 50 OD units, biphasic setup, 28°C, tissue culture rotator, 3 h, 6 mM 3a, quantification via GC-FID, standard
deviations are shown for three biotransformation reactions.
CAR-expression and activation
A further increase in biocatalyst efficiency was expected by
increasing the levels of functional MmCAR in the cells. The co-
EcPPTase is efficient, but not exhaustive. By increasing the
expression temperature to 25°C more soluble CAR molecules
were produced due to higher efficiency of the expression
machinery of E. coli at this temperature. However,
posttranslational modification of MmCAR seemed to become
insufficient. Correctly folded CAR proteins are necessary for Sfp-
like PPTases such as EcPPTase. PPTases recognize PCP
protein structure and not only the primary amino acid sequence.
This was the conclusion by studying the interactions of PCP mimic
polypeptides of the conserved sequence (19AA) or whole PCP
proteins.[15] Several studies of carrier proteins and their 4´-
phosphopantetheinylation described that apo- and holo-carrier
polypeptides were both found in cell lysates. The ratio of apo- to
holo- carrier protein was directly linked to the presence or activity
of PPTases. Additionally, it was speculated that the amount of
apo- to holo- carrier proteins regulates metabolic processes.[
This supports the assumption that different ratios of correctly
folded apo- and holo-CARs could be present in the soluble
fraction dependent on the amount and the efficiency of the
PPTase.
expression of a PPTase is vital to obtain functional CAR
14]
(
Figure S4).[ Without co-expression of a PPTase only 4.6% of
activity was observed compared to 100% activity with co-
expressed PPTase (Figure 7). In our hands, higher soluble
expression levels of MmCAR did not correlate to higher activity
levels (Table 2). Partially purified CARs that were produced at
either 20°C, 25°C or 28°C showed that the highest yield of soluble
MmCAR was obtained at 25°C (64.9 mg). However, highest
specific activity was clearly obtained with MmCAR produced at
2
0°C.
[
51]
Incorrectly folded proteins should be found as inclusion bodies,
however, folding was not a limitation for MmCAR, since soluble
protein content increased. Consequently, we hypothesized that
insufficient posttranslational modification would lead to
inhomogeneous CAR, in other words, a mixture of soluble non-
activated apo-CAR and activated holo-CAR. Hence, the soluble
MmCAR expressed at 25°C would have a higher content of apo-
CAR compared to holo-CAR, whereas CARs expressed at 20°C
have a higher portion of holo-CARs. To examine this assumption,
in vitro studies of partially purified MmCAR were conducted. If the
MmCAR preparation undergoes 4´-phosphopantetheinylation in
vitro, the specific activity of the preparation should increase and
deliver a first evidence of the presence of apo-CAR despite co-
expression of PPTase.
52]
For activating CAR molecules not only the presence of a PPTase
plays an important role, but also intracellular CoA is required.[
Because equimolar concentrations of apo-CAR and CoA
15]
[14]
molecules are necessary for full modification by PPTases, high
soluble CAR concentrations will lead to CoA depletion. It was
described that decreasing CoA levels activated the acyl carrier
protein hydrolase (AcpH, also called ACP phosphodiesterase) in
E. coli and AcpH removed the 4´- phosphopantetheine arm from
In vitro activation of MmCAR with GkPPTase
[53]
its carrier protein.
This regulation mechanism may be one
Partially purified MmCAR, was additionally activated in vitro with
a novel, thermostable PPTase from Geobacillus kaustophilus and
CoA for 1 h before MmCAR activity was determined (Figure 7).
Partially purified MmCAR had been co-expressed with EcPPTase
prior purification and in vitro activation with GkPPTase. Cloning
details of GkPPTase can be found in supplementary information,
section Materials and Methods. GkPPTase was purified by heat-
precipitation of background proteins before use.
reason for high apo-CAR content under conditions of strong
expression. Two possible investigations for improved modification
of apo-CARs would be attractive: increased CoA levels for higher
holo-CAR concentration and the identification of a PPTase with
improved apo-CAR activation potential.
The optimal PPTase for improved MmCAR activation
Up to date, the PPTase Sfp from Bacillus subtilis, the name giving
PPTase of the subclass comprising PPTases for the activation of
CARs, has been studied and applied most extensively. Sfp was
The in vitro post-translational modification of purified MmCAR by
GkPPTase in the presence of CoA clearly increased activity
(
Figure 7). MmCAR expressed at 20°C showed 70% conversion,
shown to be able to modify all types of carrier proteins and has a
which was increased by 10% by in vitro activation. Fifty percent
conversion was achieved by enzyme preparations expressed at
2
Consequently, the holo-CAR fraction must have been doubled.
The increased activity confirms that CAR expression at both
temperatures resulted in a mixed population of apo-CAR and
holo-CAR. At low expression temperature (20°C), 4´-
phosphopantetheinlation by endogenous and overexpressed
- 1
K
m
of 0.7 μM and a kcat of 102 min for the co-substrate CoA,
[16]
which is the 4´-phosphopantetheinyl group donor (Figure S4).
It showed a clear pH optimum at pH 6.0 with activity loss of more
than 70% at pH 5.0 or 7.0.
5°C, which could be increased to full conversion of 6 mM 16a.
5
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ChemCatChem
10.1002/cctc.202000895
Figure 6. Biphasic bioreductions with 50 OD units of E. coli K-12 MG1655 RARE expressing MmCAR and 5 different PPTases at 20°C or 25°C. Bioconversions were
conducted with 10 mM 3a, at 28°C for 3 h on a tissue culture rotator. White: 3a. Grey: 3b. Black striped: 3c (see Figure 3 for reaction scheme). Error bars are shown
for biological triplicates.
expressed at 20°C (Figure S5). When activities were compared, it
was apparent that more soluble CAR again (Figure 6)
corresponded to significantly less activity, either due to incorrect
folding or lack of essential decoration. Comparing the different
PPTases, activity levels varied. The best conversions were
observed by MmCAR modified by either SfpPPTase (45.6%) or
MmPPTase (57.6% conversion), both clearly outperforming
EcPPTase (35.5%). A 1.6-fold improvement was achieved. These
results indicate that in case of MmCAR, its native PPTase from
Mycobacterium marinum seems to be a better choice for activating
apo-MmCAR to holo-MmCAR.
Interestingly, high expression levels of (soluble) PPTases did not
increase activation of apo-MmCAR. The expression level of
MmPPTase at 20°C, for example, showed a small PPTase protein
band compared to NcPPTase expressed at 20°C (Figure S6).
However, higher activity levels were detected with MmPPTase,
suggesting that interaction of the PPTase with the CAR was more
important than its mere amount.
Despite this improvement, insufficient 4´- phosphopante-
theinylation at higher expression temperatures was – by far – not
fully compensated, suggesting that the intracellular CoA pool might
Figure 7. In vitro conversions of 6 mM 16a. MmCAR and EcPPTase, were co-
expressed at 20°C or 25°C. MmCARs were purified and pretreated with heat-
be limiting.
purified GkPPTase and CoA. Activation time: 1 h. Reaction time: 3 h at 28°C.
Triplicates were measured via HPLC-UV at 254 nm. White: 16a. Grey: 16b. Black
Precursor supplementation to increase CoA supply
CoA is, at least, needed equimolar to apo-CARs.[
striped: 16c.
14]
The
biosynthesis pathway of CoA in E. coli (Figure S7)[58] is highly
energy-consuming: to form one molecule of CoA, one NADPH and
four ATPs are required. According to literature, one of the major
rate-controlling steps is the conversion of pantothenate to
In E. coli, the homologous gene for transcribing the Sfp-type
PPTase was identified as entD. This EcPPTase was used as the
co-expressed PPTase for activation of apo-CARs in this and other
studies.[
18,34,37,54,55]
To co-express a PPTase that binds and
4
´- phosphopantothenate by the pantothenate kinase PanK (also
activates the PCP-domain from MmCAR perhaps more efficiently
than EcPPTase, the endogenous PPTases from known CAR
origins (Nocardia iowensis, Neurospora crassa, Mycobacterium
marinum) as well as SfpPPTase from Bacillus subtilis, which was
widely used in CAR research,[12,27,56,57] were cloned and co-
expressed with MmCAR. Biphasic whole cell bioconversions of 3a
were carried out to study the effect. Specifically, co-expression at
[59]
known as CoaA). CoA concentrations regulate PanK activity by
competing for the ATP binding site. Therefore, the energy state
[60]
of the cells plays an important role. High intracellular ATP levels will
increase concentrations of CoA levels available for
4
´- phosphopantetheinylation and override the competitive
[61]
behavior of CoA. This interaction between ATP and CoA levels
lead to the hypothesis that an increase in CoA level during
expression would most likely be beneficial for the formation of holo-
CAR.
2
0°C and 25°C was performed in E. coli K-12 MG1655 RARE to
compare activities of cells with high amounts of soluble CAR
produced at 25°C) to rather low amounts (produced at 20°C).
(
In accordance with earlier results, high amounts of soluble MmCAR
were produced at 25°C and lower amounts of soluble protein when
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FULL PAPER
Table 3 Comparison of MmCAR expression with supplementation of D-pantothenate at different temperatures.
CWW per
Expression parameters of MmPPTase
and MmCAR
5 mM of
supplementation
In vivo product formation of
3b+3c [mM]*
IMAC purified
enzyme yield [mg]
specific activity for
1
450 ml
3a [U mg -
]
cultivation [g]
autoinduction at 20°C
autoinduction at 20°C
autoinduction at 20°C
autoinduction at 20°C
autoinduction at 25°C
auto induction at 28°C
auto induction at 20°C
L-valine
β-alanine
L-aspartate
D-pantothenate
D-pantothenate
D-pantothenate
No precursor
5.73
5.82
5.79
5.89
6.39
6.65
5.91
3.91± 0.74
1.95± 0.94
4.65± 0.15
16.7± 1.17
21.4± 0.40
14.6± 0.13
6.02 ± 0.20
52.9
43.6
46.7
48.9
67.0
49.7
44.1
0.20± 0.02
0.28± 0.03
0.29± 0.05
0.85± 0.08
0.76± 0.05
0.70± 0.06
0.27± 0.04
*
Conditions for biotransformation: 24 h induction, 5 OD units, 28°C, tissue culture rotator, 3 h, 30 mM of 3a, determined via GC-FID, standard deviations are shown
for triplicates.
Figure 8. Effect of CoA precursor supplementation during induction on biphasic bioreductions. 5 OD units of E. coli K-12 MG1655 RARE expressing MmCAR and
MmPPTase were incubated with 30 mM 3a at 28°C for 16 h. White: 3a. Grey: 3b. Black striped: 3c (see Figure 3 for reaction scheme). Error bars are shown for
biological triplicates.
To increase CoA levels, inexpensive precursors of CoA were
supplemented into growth and/or CAR expression media and the
effect on carboxylate reduction capacity of the whole cell
biocatalyst was examined. Because most bacteria, including
Escherichia coli synthesize pantothenate, the key intermediate in
CoA biosynthesis, from the amino acid L- aspartate and an
intermediate in L-valine biosynthesis,[62] L- aspartic acid and
L- valine were tested. L- Aspartate is decarboxylated to β- alanine
in intact cells and further coupled to pantoate to form
pantothenate. Therefore, L- aspartate, L- valine, β- alanine and
D- pantothenate were supplemented. Five mM of each and
selected combinations were added either directly into the growth
media or supplemented before expression was initiated. To study
the effect, 5 OD units of E. coli cells were used for biphasic
bioreductions to assess activity and indirectly the amount of holo-
CAR. Supplementation of CoA precursors at the begin of E. coli
cultivation only resulted in slight increase of CAR activity
3b (and minor amounts of 3c), suggesting that the increase in
activity by 35% was due to higher holo-CAR content. All
supplementations of D- pantothenate with either L- valine,
β- alanine or L- aspartate showed increased activities of about
24% to 29%. Therefore, supplementation of solely
D- pantothenate improved CAR activation the most.
It was reported that E. coli is not growth inhibited when precursors
such as pantothenate were supplemented.[64] This observation
was confirmed herein (Table 3). Equal amounts of CWW (~5.8 g)
were harvested from 450 mL cultures, incubated at 20°C during
the expression phase. Further, protein yields of CARs in E. coli
with or without supplementation were compared (Table 3).
CAR expression at 20°C yielded ~45 mg of Ni-affinity
chromatography purified CAR, independent of the supplemented
precursor. Purity levels and protein yields after Ni-affinity
chromatography were also the same (Figure S9). Specific activity
of MmCAR expressed with D- pantothenate increased from 0.25
to 0.85 U mg - 1, a 3.4-fold improvement. To confirm this result,
E. coli K-12 MG1655 RARE (DE3) expressing MmCAR in the
presence of MmPPTase were supplemented with 5 mM
D- pantothenate and expressed at either 25°C or 28°C. Specific
activities determined for purified enzyme preparations also
resulted in increased specific activities (0.76 and 0.70 U mg - 1,
Figure S10). The minor decrease in specific activity compared to
CARs expressed at 20°C hints towards a new bottleneck.
[
63]
(
Figure S8). However, supplementation of precursors and
combinations thereof at the begin of the expression phase
showed improved activities compared to E. coli K-12 MG1655
RARE (DE3) expressing MmPPTase and MmCAR without
supplementation (Figure 8, no precursor). E. coli cells without
addition of precursors showed conversions of 14%, similarly to
cells supplemented with either L- valine, β- alanine and/or
L- aspartate. Surprisingly, cells expressed with additional
D- pantothenate showed conversions of 49% forming 14.6 mM of
7
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Figure 9. Bioreductions with or without addition of 10 mM cofactor. Biphasic bioconversions were conducted with 5 OD units of E. coli K-12 MG1655 RARE
expressing MmCAR and MmPPTases and 50 mM 3a, incubated at 28°C for 5 h on a tissue culture rotator. White: 3a. Grey: 3b. Black-striped: 3c (see Figure 3 for
reaction scheme). Error bars are shown for biological triplicates.
reaction mix. MrPPK and SmPPK were enriched by Ni-affinity
chromatography (Figure S11). The first attempt of combining the
in vitro recycling system with the biphasic bioreduction showed
promising results (Figure S12). Cell-mediated bioreductions
showed high product formation even with substrate
concentrations above 30 mM by utilizing only 5 OD units of cells.
ATP and NADPH supply in whole cell biotransformations
Cofactor regeneration is essential for CAR reactions, rendering in
vivo applications the most economic option. As discussed above,
the decrease of CAR activity with time could be the result of
various factors: deactivation of the CAR, depletion of cofactors,
product inhibition, disintegration of membranes, cell lysis etc.
Empiric cofactor feed experience showed that feed or
extracellular recycling of NADPH had a positive effect on yields.[8]
In order to investigate whether CAR deactivation or cofactor
depletion was the major factor for stagnating conversion
In vitro recycling meets in vivo bioconversion
The contribution of cellular metabolism for ATP/NADPH recycling
was investigated by comparison of whole cells with the respective
CFE. Therefore, bioreductions with 5 OD units or CFE samples
equivalent to 5 OD units were incubated with the in vitro recycling
system and compared. Notably, CFE samples of CAR incubated
showed lower product yields (~65-72%, (Figure S12)) compared
to the cell-based bioreductions. This could be due to higher initial
reaction rates within the viable cell before the recycling system
becomes effective or adds to the in vivo recycling of ATP and
NADPH. To optimize ratios of regeneration enzymes for cell-
based application, 2 equivalents of regeneration enzymes were
compared to the standard set-up.[31] Product formation for 60 mM
of 3a, using the standard set-up, converted 24.4 mM of 3a.
Bioconversions with additional PPKs reached up to 40.9 mM
conversion using twice the amount of PPKs for the regeneration
of ATP (Nr.9 and 15 in Figure S12). 3.2% of 3c was detected,
which was most likely due to the high concentrations of recycling
enzymes used and consequently more introduction of more
background activity. In order to increase product formation, higher
amounts of PPKs as previously described seemed beneficial.
The potential of cell-driven co-factor recycling combined with
external ATP/NADPH recycling, was explored by monitoring
reactions with various substrate concentrations of 3a
(10- 200 mM) (Figure 11). A direct comparison of bioreduction
with and without recycling shows the impact of the recycling
system.
(
Figure S2), external cofactors were added to bioreductions.
Either 10 mM of ATP and/or NAD(P)H were added at the
beginning of the bioreductions. This experiment should
investigate whether the cells’ metabolism for generating cofactors
was sufficient to fuel the CAR reaction up to its full potential. In
Figure 9, a trend of increased product yield by cofactor addition
could be observed. Feed of both ATP and NAD(P)H increased
product yield the most. Addition of ATP alone increased
production more than feed of NAD(P)H alone (Figure 9). 14.9 mM
of 3b were produced by 5 OD units of E. coli K-12 MG1655 RARE
(
3
DE3) by adding 10 mM of ATP and NADPH and less than 1% of
c was detected. Supplementation of the reaction mix with ATP
alone increased the product yield by 2.3-fold, whereas NAD(P)H
addition did only slightly improve conversion as compared to the
reaction without additional cofactors. Also extra NADH, the less
costly alternative to NADPH, promotes CAR activity in whole cell
bioreductions.[8] Based on these results, it can be concluded that
the CAR reaction was limited in cofactor and could not unfold its
full potential.
Addition of ATP and NADPH positively influenced 3b yield,
indicating that decreased metabolic activity was diminishing the
reaction rate with time rather than CAR deactivation. To support
whole cell-based cofactor recycling, an in vitro recycling system
was coupled to standard biphasic bioconversions with 5 OD units
of cells (Figure 10). The in vitro multi-enzyme system used was
described to fully recycle ATP and NADPH and prevent undesired
by-products (pyrophosphate, PP) in cell-free synthesis of
aldehydes from acids.[31] Regeneration of ATP was achieved
through the simultaneous action of polyphosphate kinases (PPK)
from Meiothermus ruber and Sinorhizobium meliloti and NADPH
was regenerated by a commercial glucose dehydrogenase
After 41 h, bioreductions with 5 OD units without in vitro
regeneration system produced 32.4 mM of 3b and 3c (4.2 g L 1),
including only 1.2% of 3c. Hence, even after long incubation times,
aldehyde overreduction was not predominant. Additionally, low
biomass input was beneficial for mass balance. Testing higher
substrate concentrations without the in vitro recycling system
(data not shown) fully inhibited the reaction and showed only
traces of 3b and 3c (4.05 mM of at 50 mM 3a load). By contrast,
full conversion was reached in the presence of the ATP/NADPH
recycling system. Figure 11 shows that 89.2 mM of 100 mM 3a
were reduced with only 5 OD units of cells by applying in vitro
-
(
GDH- 105).[31] Enzymes used for the in vitro recycling of ATP and
NADPH are listed in Table S4. All enzymes were expressed in
E. coli MG1655 RARE (DE3) to keep background activities at a
minimum. EcPPase was added as cell free extract (CFE) to the
8
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ATP/NADPH recycling in addition to cell mediated co-factor
supply. Less than 1% of 3c (0.78 mM) was detected even after
of 0.458 g L - 1 h - 1 was achieved. Temperature, pH, and agitation
control (Figure S13B) as well as a reduction of the volume of the
organic layer resulted in a faster conversion of 3a (Figure S13A)
as compared to the 1 mL scale reactions. The crude product in
650 mL of n-heptane, including 7.67 g of 3b, was washed with
4
1
2
1 h of reduction. 89.2 mM product formation correspond to
1.42 g L - 1 of 3b and 3c or a STY of 0.28 g L – 1 h - 1. Up to the
4 h time point of incubation, the reaction proceeded at a constant
reaction rate. The STY of the first 24 h was 0.418 g L – 1 h – 1
,
3
NaHCO and distilled. 10.4 g partially purified product was
emphasizing a high potential to produce fatty aldehydes. Even
though the reaction rate decreased after 24 h of incubation,
optimizations of the in vitro system prolonged cofactor supply and
increased the reaction rate compared to the bioreductions without
extracellular ATP/NADPH regeneration. Consequently, it pushed
the reaction further and increased product formation in the
presence of high substrate concentrations. This is the first time
that significant product amounts were formed at 200 mM
substrate concentration with only 5 OD units of cells.
obtained. Lab-scale distillation resulted in a loss of 42% of 3b.
NMR and GC analysis (Figure S14) revealed that the product
contained predominantly n-heptane, as well as very minor
amounts of octanoic acid, 1-octanol and cell membrane-based
long-chain fatty acids and/or derivatives. GC-FID analysis using
calibration curves for quantification revealed that 4.67 g thereof
was 3b.
Figure 11. Biphasic bioreductions of 3a with 5 OD units of E. coli K-12 MG1655
RARE expressing MmCAR and MmPPTase with external regeneration of ATP
and NADPH. Bioconversions were incubated at 28°C for up to 41 h on a tissue
culture rotator. Filled circle: 200 mM 3a with regeneration system. Filled
diamond: 100 mM 3a, with regeneration system. Filled triangles: 50 mM 3a with
regeneration. Filled rectangles: 30 mM 3a with regeneration system; Triangles:
50 mM 3a, no regeneration system; Rectangles: 30 mM 3a, no regeneration
system. See Figure 3 for reaction scheme.
Improved cell viability by biocompatible organic layer
Due to ISPR, higher aldehyde concentrations were achieved and
less alcohol as by-product was formed. However, not every
solvent is suitable for a biphasic conversion. n-Heptane, the
standard solvent used in this study, was reported to show
hazardous attributes towards bacterial life, causing membrane
perforation and leakage.[66] To avoid leakage and maintain the
ability of cofactor regeneration, biocompatible solvents could be a
solution. Therefore, a variety of non-water miscible biocompatible
solvents were evaluated as second phase for ISPR. Product
formation and cell viability were determined.
Figure 10. Biphasic bioreduction set-up with in vitro ATP/NADPH regeneration
system. Adapted from Strohmeier et al.[31]
Summarizing,
a
major limiting factor in whole cell
biotransformation is cellular metabolism. The impact of the
stability of the carboxylate reductase requires further systematic
investigations.
After biphasic bioreductions (Figure 12), selection pressure was
applied on the used E. coli cells to screen for viable cells. Dilutions
6
of 10 of cell suspension were plated, grown overnight at 37°C
and colony forming units (CFUs) were counted (Figure 13). Only
Preparative Gram scale production of aldehyde
2
5% of cells resuspended in reaction buffer containing 30 mM of
Fluctuations of small-scale reactions in, e.g. pH, may impact the
results. Feedback control for essential parameters can increase
productivities significantly.[65] Our next aim was to upscale the
reaction based on the extensive optimization of CAR mediated
reduction of fatty acids. Hence, preparative biphasic
bioreductions with cofactor regeneration under controlled
conditions were performed in a bioreactor (450 mL reaction
volume) with a total of 9.37 g of 3a as the substrate (Figure S13A).
After 27 h of incubation, 92.1% of the substrate was converted.
The product comprised of only 1.6% of 3c and 98.4% 3b. A STY
3
a and incubated for 5 h without a second phase (-solvent)
survived compared to non- incubated cells without a carboxylic
acid substrate.
n-Hexadecane showed no loss of CFUs for substrate 3a
compared to cells which were incubated in the tissue culture
rotator for 5 h without any substrate and only a 25% of loss
compared to cells with no substrate nor incubation. Also,
farnesene showed promising results. In Brennan et al. also
farnesene and dioctyl phthalate as well as n-hexadecane showed
beneficial results regarding cell viability when S. cerevisiae
9
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ChemCatChem
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FULL PAPER
cultures were incubated with toxic substances.[67] Because Conclusion
n- hexadecane and farnesene delivered highest product titers of
3
b and 3c (Figure 12) and showed improved viability after
Hardly any studies with the use of carboxylate reductases
(CARs) in vivo were aiming for high aldehyde titres, and
these were focused on aromatic aldehydes such as vanillin
or piperonal.[18,34] Within this study, the main focus lay on the
production of fatty aldehydes by CAR enzymes. We
screened 15 fatty acid substrates with 5 CARs to find the
most suited CAR for the desired application. MmCAR
showed the most promising and broadest substrate scope.
Hence, E. coli expressing MmCAR was used as the
biocatalyst for a systematic optimization of various cultivation
and reaction parameters, using octanoic acid (3a) as the
substrate. Various bottlenecks were identified and strategies
to circumvent these bottlenecks were evaluated in order to
gain industrially relevant aldehyde yields. Schwendenwein et
bioreductions with 3a (Figure 13), these would be the most
promising ISPR solvents. Recently, Yunus and Jones applied
isopropyl myristate as a second layer for fatty alcohol production
by reducing 3a with MmCAR and endogenous AHRs in
Synechocystis sp. PCC 6803 strain as host organism.[19]
According to our results and experiments reported,[67] the
application of hexadecane would most likely also increase
product titers in algae by maintaining viability and ability for
essential cofactor regeneration for the CAR reaction.
al. had shown that within 3 h, 30 mM piperonylic acid (16a
)
can be reduced by 50 g L -1 E. coli cells expressing NcCAR
-1
-1
and EcPPTase, which corresponds to 1.5 g L h . Here we
achieved similar productivities with 10 times less biocatalyst
input and a non-tailored microbial host. In Table 4, relevant
parameters are summarized. 92.1 mM of octanal (3b) and
1
.54 mM of 1-octanol (3c) were produced within 27 h under
controlled conditions. Overreduction was kept at only 1.6%
without additional gene deletions. After isolation of the target
compound from the organic layer, 4.67 g of octanal (3b) were
obtained in a mixture with residual solvent. All together, this
systematic investigation of the whole cell approach for
medium- to long-chain fatty aldehyde production led to an
approximate 16.6 times increase in productivity (from
-
1
- 1
0
.745 g L
to 12.36 g L ), while using 10 times less
Figure 12. Biphasic bioreductions of 30 mM 3a with 5 OD units of E. coli K-12
MG1655 RARE expressing MmCAR and MmPPTase using biocompatible
solvents. Bioconversions were incubated at 28°C for 5 h on a tissue culture
rotator. White: 3a. Grey: 3b. Black striped: 3c (see Figure 3 for reaction scheme).
Error bars are shown for biological triplicates.
biocatalyst.
The gradual improvement of whole cell bioreduction of
octanoic acid (3a) is summarized in Table 4. By aiming for
fatty aldehydes as the main product, toxicity effects in E. coli
were addressed and circumvented by ISPR. We identified
limitations in enzyme expression and post-translational
modification, and improved CAR activation: Increased supply
of 4´-phosphopantetheine, via increased CoA levels and a
more suited PPTase improved activation of apo-CAR. The
optimal PPTase for modifying the PCP- domain of MmCAR
originated also from Mycobacterium marinum.
During CAR expression in E. coli, limitations in CoA levels
and possibly ATP levels constrained holo-CAR
concentrations. During bioreduction, cofactor supply was
also insufficient. Especially ATP supplementation improved
whole cell conversion. Hence, a coupling of the whole cell
biocatalyst with an in vitro regeneration system for ATP and
NADPH produced more than 7 g L-1 of octanal (3b). To
enhance productivity without applying an in vitro cofactor
regeneration strategy, biocompatible solvents, e.g. n-
hexadecane or farnesene, increased productivity by an
increase in cell viability of E. coli. Most-likely they enable
longer and more proficient cofactor regeneration and
decreased membrane perforation. Due to overwhelming
endogenous activity of alcohol dehydrogenases and aldo-
keto reductases reducing the target aldehyde further to
alcohol, a decreased biomass amount and a second organic
layer were key to limit 1-octanol (3c) formation to below 1%.
Figure 13. CFU count of plated 106 colonies of E. coli K-12 MG1655 RARE
expressing MmCAR and MmPPTase after biphasic bioreduction of 30 mM 3a
with biocompatible solvents. Plates were incubated at 37°C for 5 h. Error bars
are shown for triplicates.
1
0
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ChemCatChem
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Table 4. Summary of fatty aldehyde production[a] by whole cell biocatalyst.
controlled
reaction
bioreactor)
single
n-
reduced
biomass
D-
ATP/NADPH
supplementation
5 OD
+recycling
parameter
MmPPTase[e]
phase[
b]
heptane
[c]
[d]
pantothenate
[f]
[g]
[h]
(
substrate [mM]
6.0
50.0
10.0
10.0
5.63
30.0
50.0
100.0
100.0
Aldehyde and
alcohol [mM]
alcohol
5
.82[f]
23.60
0.48
14.43
14.89
89.10
96.40
97.0
22.9
18.00
3.03
1.0
6.9
1.3
16.00
1.85
0.8
1.2
1.6
[
% in product]
time [h]
4.5
5.00
0.06
3.00
0.72
5.00
1.91
41.00
11.42
27.00
12.36
productivity
0
.745
-
1
[g L
]
STY [g L – 1 h – 1
]
0.165
0.050
0.168
0.050
0.012
0.005
0.241
0.050
0.116
0.005
0.382
0.005
0.279
0.005
0.458
1.000
CWW [g]
biocatalyst yield
14.90
60.52
12.31
14.44
370.01
381.81
2284.70
12.36
–
1
[g g CWW]
[a]
[b]
[c]
[d]
[e]
[f]
Bioreductions were carried out at 28°C on a tissue culture rotator.
MmCAR and EcPPTase, 50 OD units.
MmCAR and EcPPTase, 50 OD units, aqueous/n-heptane 1:1.
MmCAR and MmPPTase, 5 OD units, aqueous/n-heptane1:1.
MmCAR and MmPPTase expressed in the presence of 5 mM
D-pantothenate, 5 OD units, aqueous/n-heptane 1:1.
MmCAR and MmPPTase expressed in the presence of 5 mM
D
-pantothenate, 5 OD units, aqueous/n-heptane 1:1, addition of 10 mM ATP and/or
NADPH.
[
g]
+
MmCAR and MmPPTase expressed in the presence of 5 mM
PPKs, GDH and PPase.
D
-pantothenate, 5 OD units, aqueous/n-heptane 1:1, addition of NADP , ATP, polyP,
[
h]
+
MmCAR and MmPPTase expressed in the presence of 5 mM
PPKs, GDH and PPase in bioreactor.
D
-pantothenate, 5 OD units, aqueous/n-heptane 2:1, addition of NADP , ATP, polyP,
[f]
3c was used for calculating parameters. Less than 0.1% of 3b was found due to overreduction.
incubated at 75°C for 10 min to purify GkPPTase from non-
thermostable E. coli background proteins. The CFE was then
centrifuged for 30 min at 16,100 rcf and 4°C. The supernatant
was used for in vitro 4´phosphopante-theinylation of apo-CAR to
give active holo-CAR.
Experimental Section
General: HPLC‐MS grade acetonitrile was purchased from VWR.
All other chemicals were obtained from Sigma–Aldrich or Roth
and used without further purification. Exceptions are described
below. Gasses for GC-analysis were obtained from Linde Gas,
Austria
Additional activation of MmCAR with GkPPTase: Pre-activation:
Partially purified MmCAR (90 µg) was incubated with 1 mM CoA
and 100 µg heat purified GkPPTase at 28°C for 1 h and 500 rpm
in an Eppendorf Thermomixer. For the assay, 6 mM 16a in KOH
were added to 100 mM HEPES, pH 7.5, containing 10 mM
MgCl . 30 µg of preactivated MmCAR was added and the
2
reduction started with 10 mM ATP and NADPH. This in vitro
activity assay with preactivated CAR was incubated at 28°C for
Strains, primers and enzymes: See Table S3-S5.
Cultivation and expression of E. coli strains harboring CAR
enzymes (auto-induction protocol): Cultivation and
expression procedures were performed as described
[
68]
previously.
The auto-induction medium LB 5052 with the
3
h. The reaction was stopped by addition of 120 µL of MeOH
appropriate antibiotic was inoculated with either 0.2% or 2% (v/v)
inoculation volume and incubated at 37°C and 130 rpm for 4 h
and the expression phase was conducted at 20°C, 25°C or 28°C
for 12-14 h or 24 h.
and proteins removed by centrifugation at 16,100 rcf for 40 min.
The supernatant was transferred into a microtiter plate and
analyzed by HPLC-UV. As control, pre-activation reactions with
GkPPTase were incubated without CoA. Also, a buffer control
was incubated at 28°C for 1 h.
For increasing the intracellular CoA pool, 5 mM of either
L
- valine, β- alanine,
L
- aspartic acid or
D
- pantothenic acid
Substrate scope of CARs - in vitro: Aliphatic acid substrate,
hemicalcium salt dissolved in ddH
induction.
2
O was added before
5
mM dissolved in DMSO, were added to 50 mM MES, pH 7.5,
containing 10 mM MgCl . 90 µg of partially purified enzyme
2
-
1
preparation (1 mg mL of IMAC purified CARs) were added to
the reaction mix. The reaction was started by addition of 1.2 mM
ATP and NADPH. The in vitro activity assay was performed in a
total volume of 600 µL in Eppendorf reaction tubes at 28°C and
Cultivation and expression of in vitro cofactor recycling
enzymes: E. coli K-12 MG1655 RARE cells were transfected
with the plasmids listed in Table S4. Cultivation and expression
[
31]
was performed as described in Strohmeier et al.
7
00 rpm for 16-18 h in a Thermomixer. To stop the reaction, the
solution was acidified to pH 2.0 with 3 M HCl. Extraction of fatty
acid , aldehyde and alcohol were achieved by two repeated
Expression analysis of CAR enzymes, cell disruption and
Ni-affinity chromatography purification of enzymes:
Methods were conducted as described in Horvat et al.
a
b
c
extractions with equal amount of ethyl acetate, including 0.01%
of n- tetradecane as internal standard (IS). Afterwards, the
mixture was vortexed for 10 min and centrifuged for 15 min at
[
68]
1
6,100 rcf and 4°C. The organic phase containing the extracted
fatty , and IS was transferred to a fresh Eppendorf tube
and dried with Na SO (approximately 10% (w/v)). 190 µL of the
Heat purification of GkPPTase: Cell free extract (CFE) was
prepared by resuspending the cell pellet of E. coli K-12 MG1655
RARE pMS470:GkPPTase in 200 mM MES, pH 7.5. Cells were
disrupted by ultrasonication and CFE prepared by centrifugation
for 35 min at 65,563 rcf and 4°C. 1.5 mL of the CFE was
a, b, c
2
4
mixture were transferred into glass vials for GC analysis. The
biotransformation reactions were carried out in technical
1
1
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ChemCatChem
10.1002/cctc.202000895
FULL PAPER
triplicates. Quantitation was performed based on linear
interpolation of calibration curves with concentrations of
standards from 0.156 mM to 10 mM.
and HMBC) were recorded for identification and confirmation of the
structure. Deuterated solvents were purchased from euriso-top .
®
1
H NMR (300 MHz, CDCl
-), 1.70 – 1.51 (m, 2H, CO-CH
7.5 Hz, 21H, -CH - octanal and heptane), 0.87 (t, J = 6.5 Hz, 11H, -
, octanal and heptane) ppm.
3
) δ 9.75 (s, 1H, CHO), 2.50 – 2.32 (m, 2H,
Specific activity of CAR via NADPH-depletion monitoring: The
NADPH depletion assay was used to determine initial rate
activities for the reduction of aliphatic carboxylates as
CO-CH
2
2
-CH -), 1.40 – 1.13 (m, J =
2
2
CH
3
[
68]
described,
DMSO.
however, carboxylic acids were dissolved in
13
C NMR (75.5 MHz, CDCl
, heptane), 31.8 (CH ), 29.3 (CH
22.8 (2 CH , heptane), 22.7 (CH ), 22.3 (CH
14.2 (CH ) ppm.
3
) δ 203.0 (CHO), 44.1 (CO-CH
), 29.2 (2 CH , 3b and heptane),
), 14.2 (2 CH , heptane),
2
-), 32.0 (2
CH
2
2
2
2
In vivo bioreduction of octanoic acid (3a): Reactions with an
immiscible solvent phase (n-heptane) were carried out in 15-mL
Pyrex tubes and incubated at 28°C in a tissue culture tube
rotator. The reactions with a total volume of 2 mL contained
2
2
2
3
3
GC-FID analysis (method 1): GC-FID measurements were
performed for substances for which product standards were
available (Table S6). A ZP-5 column (crosslinked 5% Ph-Me
Siloxane; 30 m, 0.32 mm in diameter and 0.25 μm film thickness)
on a Shimadzu GC 2010 Plus equipped with an FID was used.
Sample aliquots of 1 μL were injected in split mode (split ratio
10:1) at 240°C injector temperature and 320°C detector
5
1
00 mM HEPES or MES buffer, pH 7.5, supplemented with
00 mM glucose, 10 mM MgSO and octanoic acid (3a), ranging
4
from 1- 200 mM.
In vivo bioreduction of 3a with external co-factor supply: The
standard reaction contained 3a (50 mM), 1 mM ATP, 0.5 mM
+
NADP , 5 OD units of cells resuspended in 100 mM HEPES,
temperature with N
quantification of 3a
followed by temperature gradients to 130°C at 5°C min and to
2
as carrier gas. The temperature program for
3b 3c was as follows: hold at 70°C for 4 min,
-
1
pH 7.5, GDH-105 (0.2 U mL ), 100 mM β-
MgCl , 140 mM polyP (related to ortho-phosphate units, medium
chain length: 25) (Merck), MrPPK (100 μg mL ), EcPPase
D
-glucose, 70 mM
/
/
-
1
2
-
1
- 1
300°C at 45°C per min and a hold at 300°C for 2 min. The total
-
1
- 1
(
25 μg mL ) and SmPPK (40 μg mL ). SmPPK and MrPPK
runtime for 3a/3b/3c was 21.45 min. This method was adapted
[
31]
were prepared and purified as described. EcPPase was used
as cell-free lysate. Also, AMP (1 mM) was tested as catalytic
cofactor. Additionally, the necessity of all components was
tested by omitting one enzyme at the time.
for C6, C7, C9, C10 C11, C11:1 and C16:1 fatty
acids/aldehydes/alcohols. The total run time ranged from 14.75
to 21.45 min. GC-FID results were evaluated with the GC-FID
Data Analysis software LabSolution by Shimadzu.
Controlled bioreduction of 3a with in vitro co-factor regeneration
in bioreactor: E. coli K-12 MG1655 RARE expressing MmPPTase
and MmCAR were cultivated in TB-media and the autoinduction
GC-FID analysis for biocompatible solvents: Biocompatible
solvents are characterized by relatively high boiling points and
their retention times interfered especially with long-chain
substrate and/or product peaks or internal standard. Individual
method adjustments were made to the main method (method 2).
A list of solvents and corresponding methods used is given in
Table S9.
protocol using 2% inoculation volume and addition of 5 mM
pantothenic acid hemicalcium salt prior induction.
D-
1
3
g CWW (~1000 OD) was harvested and resuspended in
00 mM HEPES, pH 7.5. The reaction mix in a 1 L Biostat B unit
+
(Sartorius) contained 3.13 g of 3a, 1 mM ATP, 0.5 mM NADP ,
GC-MS analysis: Gas chromatography-mass spectrometry (GC-
MS) measurements were performed for substances for which
only the substrate was available. A detailed list of retention times
and structures is summarized in Table S7. 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 was used. 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 oven temperature program was as
follows: 70°C for 5 min and a temperature gradient to 300°C at
-
1
GDH-105 (0.2 U mL ), 100 mM β-
2
D-glucose, 70 mM MgCl ,
1
40 mM polyP (related to ortho-phosphate units, medium chain
-
1
length: 25) (Merck), purified MrPPK (300 μg mL ), CFE of
EcPPase (25 μg mL ) and purified SmPPK (120 μg mL ) in an
-
1
- 1
overall aqueous volume of 300 mL. Air was supplied with
-
1
0
.3 L min for the first 5 h to support cellular metabolism. 2M
NaOH was used for pH regulation to pH 7.5. 150 mL of n-
heptane was used as organic ISPR layer. Reactions were stirred
at 300 rpm. Samples were analyzed at intervals. Bioreductions
under controlled conditions were performed three times.
-
1
1
5°C min followed by a hold for 5 min at 300°C. The total run
time was 24.26 min. The mass selective detector was operated
in a mass range of 30-500 m/z and an ionization voltage of 70 eV.
GC-MS results were evaluated with the GC-MS Shimadzu Data
Analysis software LabSolution.
Purification of aliphatic compounds of controlled bioreduction
and NMR analysis: After 27 h, the layers were separated by
centrifugation at 15,039 rcf for 35 min. The aqueous layer was
extracted once with 100 mL of n-heptane. The combined n-heptane
fractions, containing 3a, 3b and 3c, were washed three times with
saturated NaHCO
3
solution to remove unreacted octanoic acid. n-
HPLC-UV detection of 16a, b and c: Measurements were
Heptane was removed in a fractional distillation apparatus, equipped
with a water-cooled condenser. The overhead vapor temperature did
not exceed 98°C. 10.4 g of partially purified product was obtained and
analyzed via gas chromatography-flame ionization detection (GC-
FID) and NMR. The product was composed of 44% octanal (3b) in
residual n-heptane, as quantified by GC-FID. For recording NMR
spectra a Bruker Avance III 300 MHz FT NMR spectrometer with
autosampler was used ( H: 300.36 MHz; C: 75.5 MHz). The residual
protonated solvent signals serve as internal standard for interpretation
of the chemical shifts δ. To facilitate the interpretation, the C-spectra
were proton decoupled. In addition, 2D spectra (H,H-COSY, HSQC
performed by HPLC-UV. Equipment and method used are
[
68]
described in Horvat et al.
Retention times and chemical
structure are shown in Table S8.
Acknowledgements
1
13
The authors thank Anna Katharia Migglautsch for the workup
of the prep-scale reaction, Daniel Schwendenwein for
identifing limitations in the correlation of expression and
activities of CARs, Fredi Brühlmann and Andreas Taglieber
1
2
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.202000895
FULL PAPER
for valuable discussions and Kristala L.J. Prather for
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Entry for the Table of Contents
A mild, sustainable and selective biocatalytical approach to aldehydes is presented. Carboxylic acid reductases (CARs) were employed for fatty
acid (C6-C18) reduction. Cell viability, cofactor supply, functional expression, and in-situ product removal were systematically investigated and
-
1
1
2.36 g L of octanal was produced from octanoic acid
1
5
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