An Enzymatic 2-Step Cofactor and Co-Product Recycling Cascade towards a Chiral 1,2-Diol. Part I: Cascade Design

Article abstract of DOI:10.1002/adsc.201900187

Alcohol dehydrogenases are of high interest for stereoselective syntheses of chiral building blocks such as 1,2-diols. As this class of enzymes requires nicotinamide cofactors, their application in biotechnological synthesis reactions is economically only feasible with appropriate cofactor regeneration. Therefore, a co-substrate is oxidized to the respective co-product that accumulates in equal concentration to the desired target product. Co-product removal during the course of the reaction shifts the reaction towards formation of the target product and minimizes undesired side effects. Here we describe an atom efficient enzymatic cofactor regeneration system where the co-product of the ADH is recycled as a substrate in another reaction set. A 2-step enzymatic cascade consisting of a thiamine diphosphate (ThDP)-dependent carboligase and an alcohol dehydrogenase is presented here as a model reaction. In the first step benzaldehyde and acetaldehyde react to a chiral 2-hydroxy ketone, which is subsequently reduced by to a 1,2-diol. By choice of an appropriate co-substrate (here: benzyl alcohol) for the cofactor regeneration in the alcohol dehydrogenases (ADH)-catalyzed step, the co-product (here: benzaldehyde) can be used as a substrate for the carboligation step. Even without any addition of benzaldehyde in the first reaction step, this cascade design yielded 1,2-diol concentrations of >100 mM with optical purities (ee, de) of up to 99%. Moreover, this approach overcomes the low benzaldehyde solubility in aqueous systems and optimizes the atom economy of the reaction by reduced waste production. The example presented here for the 2-step recycling cascade of (1R,2R)-1-phenylpropane-1,2-diol can be applied for any set of enzymes, where the co-products of one process step serve as substrates for a coupled reaction. (Figure presented.).

Full text of DOI:10.1002/adsc.201900187

Title: An Enzymatic 2-Step Cofactor and Co-Product Recycling
Cascade towards a Chiral 1,2-Diol. Part I: Cascade Design
Authors: Justyna Kulig, Torsten Sehl, Ursula Mackfeld, Wolfgang
Wiechert, Martina Pohl, and Dörte Rother
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201900187
Link to VoR: http://dx.doi.org/10.1002/adsc.201900187

Advanced Synthesis & Catalysis
10.1002/adsc.201900187
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
An Enzymatic 2-Step Cofactor and Co-Product Recycling
Cascade towards a Chiral 1,2-Diol. Part I: Cascade Design
a
a
a
a
a
Justyna Kulig, Torsten Sehl Ursula Mackfeld Wolfgang Wiechert Martina Pohl and
,
,
,
a,b *
Dörte Rother
a
Forschungszentrum Jülich GmbH, IBG-1: Biotechnology
Wilhelm-Johnen-Straße, 52428 Jülich (Germany)
b
RWTH Aachen University, ABBt
Aachen Biology and Biotechnology, 52074 Aachen (Germany)
*
Corresponding Author: Prof. Dörte Rother
Forschungszentrum Jülich GmbH, IBG-1: Biotechnology
Wilhelm-Johnen-Straße, 52428 Jülich (Germany)
Fax:
(+) 49 2461 61 3870
E-mail:
do.rother@fz-juelich.de
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.
Abstract. Alcohol dehydrogenases are of high interest for By choice of an appropriate co-substrate (here: benzyl
stereoselective syntheses of chiral building blocks such as alcohol) for the cofactor regeneration in the alcohol
1
,2-diols. As this class of enzymes requires nicotinamide dehydrogenases (ADH)-catalyzed step, the co-product
cofactors, their application in biotechnological synthesis (here: benzaldehyde) can be used as a substrate for the
reactions is economically only feasible with appropriate carboligation step. Even without any addition of
cofactor regeneration. Therefore, a co-substrate is oxidized benzaldehyde in the first reaction step, this cascade design
to the respective co-product that accumulates in equal yielded 1,2-diol concentrations of >100 mM with optical
concentration to the desired target product. Coproduct purities (ee, de) of up to 99 %. Moreover, this approach
removal during the course of the reaction shifts the reaction overcomes the low benzaldehyde solubility in aqueous
towards formation of the target product and minimizes systems and optimizes the atom economy of the reaction by
undesired side effects. Here we describe an atom efficient reduced waste production. The example presented here for
enzymatic cofactor regeneration system where the co- the 2-step recycling cascade of (1R,2R)-1-phenylpropane-
product of the ADH is recycled as a substrate in another 1,2-diol can be applied for any set of enzymes, where the
reaction set. A 2-step enzymatic cascade consisting of a co-products of one process step serve as substrates for a
thiamine diphosphate (ThDP)-dependent carboligase and an coupled reaction.
alcohol dehydrogenase is presented here as a model reaction.
In the first step benzaldehyde and acetaldehyde react to a Keywords: Enzymatic cascade reaction; vicinal 1,2-diols;
chiral 2-hydroxy ketone, which is subsequently reduced by cofactor and co-product recycling cascade; alcohol
to a 1,2-diol.
dehydrogenase; carboligase; chiral 2-hydroxy ketone
by an enzymatic 2-step cascade starting with the
carboligation of aldehydes catalyzed by thiamine
diphosphate (ThDP)-dependent enzymes towards
Introduction
[3c]
Chiral 1,2-diols find broad application as building
blocks for pharmaceuticals, agrochemicals, and
chemical catalysts.[ NAD(P)H-dependent alcohol
dehydrogenases (ADH) can catalyze synthesis of 1,2-
diols from prochiral 2-hydroxy ketones in high
different chiral 2-hydroxy ketones. In the second
step these 2-hydroxy ketones are reduced by alcohol
dehydrogenases (ADH) yielding vicinal chiral 1,2-
1]
[1f, 1g, 3a]
diols.
Such alcohol dehydrogenases require
expensive nicotinamide cofactors (NAD(P)H) to
deliver the necessary reduction equivalents.
Therefore in situ regeneration of such cofactors is
decisive for economic feasibility of these biocatalytic
stereoselectivities, mild reaction conditions, and
without toxic reagents[
1f, 3]
compared to classical
chemical methods such as Sharpless asymmetric
dihydroxylation, epoxide hydrolysis and asymmetric
aldol condensation[ Various 1,2-diols are accessible
[
4]
processes.
According to the classification of
2]
[5]
Chenault and Whitesides there are five general
1
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Advanced Synthesis & Catalysis
10.1002/adsc.201900187
methods for redox cofactor regeneration including
biological, chemical, electrochemical, photochemical,
and enzymatic methods. Enzymatic cofactor
phenylpropane-1,2-diol (PPD) (Scheme 1). Starting
from benzaldehyde and acetaldehyde, the thiamine
diphosphate (ThDP)-dependent benzaldehyde lyase
from Pseudomonas fluorescens (PfBAL) gives access
to the 2-hydroxy ketone (R)-2-hydroxy-1-phenyl-
propane-1-one, short (R)-2-HPP, with high regio- and
[4b, 6]
regeneration methods are most frequently used
and can be further subdivided into substrate-coupled
and enzyme-coupled approaches. In enzyme-coupled
approaches two different enzymes are involved, one
in the main reaction and the other catalyzes the
cofactor regeneration reaction. In the substrate-
coupled approach, the main reaction and the cofactor
regeneration is catalyzed by the same enzyme, but
requires a cosubstrate and generates coproducts. This
concept has the advantage of avoiding an additional
enzyme therefore keeping production costs in
[12]
enantioselectivity (ee > 99 %). For the subsequent
reduction to the respective 1,2-diol (1R,2R)-PPD the
alcohol dehydrogenase from Ralstonia sp. (RADH) is
well suited to reduce (R)-2-HPP with excellent
diastereoselectivities and high activities (up to
–
1 [3b]
~ 360 U mg ).
[7]
industrial scale applications lower.
Some
biocatalytic processes implemented via substrate-
coupled cofactor regeneration require removal of the
co-product to shift the equilibrium or to avoid
[4, 8]
negative effects on enzyme activity and stability.
No matter if enzyme- or substrate-coupled cofactor
regeneration is chosen, the co-substrate is usually
used in excess to shift the equilibrium of the main
reaction appropriately. In contrast to formate
dehydrogenases, which generate CO
2
as a co-product
leaves the
by oxidation of formate and that CO
2
Scheme 1. 1-Pot 2-step co-product recycling cascade
for the synthesis of (1R,2R)-1-phenylpropane-1,2-diol.
liquid phase, the resulting co-products produced by
glucose dehydrogenases (D-gluconate), and ADHs
(
1R,2R)-1-phenylpropane-1,2-diol (PPD) is formed by
(
e.g. acetone, acetaldehyde) accumulate in the
combining a carboligation step (1) and an oxidoreduction
step (2a). By using benzyl alcohol as a co-substrate for
NADPH cofactor regeneration, the co-product benzyl
alcohol (step 2b) can be recycled as a substrate in the
carboligation step (1). PfBAL = benzaldehyde lyase from
reaction mixture and must be separated from the
product either during the course of the reaction to
shift the reaction equilibrium or during downstream
processing. The use of “smart co-substrates”, like
1,4-butandiol or 1,6-hexandiol enables the use of only
Pseudomonas
fluorescens,
RADH
=
alcohol
a 0.5-fold molar concentration relative to the main
substrate.[ However, here also co-products are
produced, which increase the waste-stream and must
be separated during downstream processing. In so-
called recycling cascades, also known as “closed-loop”
or “self-sufficient” cascades,[10] oxidizing and
reducing steps are directly coupled and neither co-
substrate nor further regenerating enzymes are
required. This concept has the potential to
significantly improve the atom economy and reduce
the waste stream. However, this beneficial concept
9]
dehydrogenase from Ralstonia sp.
Results and Discussion
Based on previously optimized reaction conditions
[6a, 13]
[3b, 14]
for both enzymes, PfBAL
and RADH
, we
here focused on a feasibility study of the co-product
recycling concept towards chiral 1,2-diols in buffered
systems. Although in principle, benzyl alcohol (via
benzaldehyde) and ethanol (via acetaldehyde) could
theoretically be used as co-substrates in this recycling
cascade concept, we focused on benzyl alcohol due to
the higher activity of RADH towards large cyclic and
aromatic alcohols compared to small aliphatic
only
functions
if
NAD(P)H-based
oxidation/reduction can be coupled red/ox-neutral in
a desired cascade setup.
To broaden the operational window of smart
recycling cascades processes, we present a cascade
where the cofactor NADPH is regenerated in a way
that the co-product is a substrate for a coupled
reaction step. This recycling cascade was recently
applied by our group in micro-aqueous reaction
[3b, 14]
substrates.
A) Benzyl alcohol for cofactor regeneration
[11]
systems using whole lyophilized cells. In this study
here, we show its general application in buffered
systems using cell free catalyst formulations. Further,
we studied more deeply the reaction parameters
necessary in such buffered reaction environment and
identified limiting factors (such as kinetics and
thermodynamic equilibrium) in order to get optimal
conversion. We demonstrate this concept using a 2-
step cascade for the synthesis of (1R,2R)-1-
Since the PfBAL-catalyzed reaction was already
[12b, 13]
well characterized in buffered reaction systems,
the RADH reaction was initially optimized separately
with the special focus on the application of benzyl
alcohol as a co-substrate. As demonstrated earlier,
RADH exhibits different non-overlapping pH-optima
for reduction (pH 6.0-9.5) and oxidation (pH 10.0-
[14]
11.5).
Furthermore, stability tests indicated that
RADH is rather stable between pH 5.5 and pH 8.0
2
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Advanced Synthesis & Catalysis
10.1002/adsc.201900187
(
half-life: ~60-70 h), whereas higher pH-values
[14]
induced progressive inactivation,
making the
application of RADH for the simultaneous oxidation
of benzy alcohol and the reduction of 2-HPP
specifically challenging.
Based on these previous results, the RADH-
catalyzed reduction rac-2-HPP[3b,
14]
was tested
between pH 8.0 to 9.0 (Figure 1-A) for determining
optimal reaction conditions for the cascade setup. The
results indicate 1.7-fold higher conversions of rac-2-
HPP to the respective 1,2-diols (1R,2R)- and (1R,2S)-
PPD at pH 9.0 compared to pH 8.0 (35 % for pH 8
and 59 % for pH 9). The applied buffer species
(
TEA-HCl or TRIS-HCl) had no influence at pH 8.0,
resulting in 35 % conversion in both cases (Figure 1-
A). Thus, a concentration range of 10-250 mM
benzyl alcohol was tested to convert 10 mM of rac 2-
HPP (Figure 1-B). Higher concentrations were not
investigated in order to avoid the formation of a two-
phase system due to the limited solubility of benzyl
alcohol in aqueous media. Although RADH accepts
[3b, 14]
both enantiomers of rac-2-HPP
experiments conversion of rac-2-HPP exceeded 59 %
Figure 1-B). This could be partly due to the fact that
the (R)-enantiomer is 24-fold faster converted
in none of these
(
compared to the (S)-enantiomer.[
3b]
Additionally,
deactivation of RADH under reaction conditions
could occur. To test whether the RADH concentration
was limiting the conversion, it was increased from
0
.05 mg/mL to 0.30 mg/mL, which indeed resulted in
almost full conversion (98.5 %; Figure 1-C).
These results prove that benzyl alcohol is a suitable
co-substrate for cofactor regeneration in RADH-
catalyzed reductions for single step reactions of 2-
HPP to the 1,2-diol.
B) 2-Step cofactor and co-product recycling
cascade
After identification of optimal reaction conditions
for the RADH-catalyzed reduction of HPP, the 1-pot
2
-step cascade was investigated by combining the
carboligation and the reduction step with in-situ
benzaldehyde removal (see Scheme 1). The cascade
reaction was evaluated using two starting conditions
(
see Figure 2 and SI Fig. S1), which differed in the
initial concentrations of benzaldehyde. In route A the
one-pot recycling cascade was started with
benzaldehyde (10 mM), acetaldehyde (150 mM),
+
benzyl alcohol (120 mM) and NADP (in different
concentrations).
Small
concentrations
of
Figure 1. Reaction optimization for RADH-catalyzed
reduction of 2-HPP reactions with benzyl alcohol as co-
substrate for cofactor regeneration.
benzaldehyde were added in this route to detect
weather starting material of benzaldehyde is
important to start the cascade reaction. In route B no
benzaldehyde was added at all, so that the
benzaldehyde (required for the first carboligation
step) had to be completely generated by recycling
through oxidation of benzyl alcohol. Both cascades
were optimized with respect to pH and the
A) Identification of optimal pH for the oxidoreduction. B)
Optimization of benzyl alcohol concentration. C)
Optimization of the RADH concentration. All reactions
were carried out in duplicates at 20 °C as endpoint-
determination for 24 h under constant shaking (500 rpm) in
aliquots (1 mL each) in closed glass vials.
+
concentrations of NADP and RADH, respectively
(
Figure 2).
3
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Advanced Synthesis & Catalysis
10.1002/adsc.201900187
Conversions were calculated based on the total
Proof-of-principle successfully demonstrated
In all shown cases of the optimization using route
A (Figure 2; entry A-1 and B-1), higher 1,2-diol
concentrations than the initially added benzaldehyde
concentration (10 mM benzaldehyde) were found.
This clearly demonstrates that the recycling of the
benzaldehyde formed as a co-product via the RADH-
catalyzed oxidation of benzyl alcohol was successful
in the first reaction step catalyzed by PfBAL (Figure
3). The highest value of 93.5 mM indicates that at
least 83.5 mM 1,2-diol was formed via co-product
recycling. Here an overall conversion (relatative to
the benzaldehyde and benzyl alcohol) of 72 % was
reached. Moreover, even if no benzaldehyde was
added initially (route B) the 1,2-diol is formed to
concentrations of 82.0 mM. This corresponds to 68 %
conversion of the benzyl alcohol.
amount of aromatic substrate (benzaldehyde) and co-
substrate (benzyl alcohol), which finally gave the 1,2-
diol. Thus, 100 % conversion refers to 130 mM 1,2-
diol for route A (10 mM benzaldehyde + 120 mM
benzyl alcohol) and 120 mM for route B (120 mM
benzyl alcohol), respectively. As a consequence, 1,2-
diol concentrations greater than the initial
benzaldehyde concentration (route A: 10 mM
benzaldehyde; route B: 0 mM) are indicative for the
successful co-product (benzaldehyde) recycling in the
first reaction step (catalyzed by PfBAL).
+
NADP concentration
In order to identify the optimal cofactor
+
concentration, NADP concentration was varied
within a range of 0.2-0.8 mM while the other
parameter for route A and route B were kept constant
(
see Figure 2). Whereas the conversions were about
three-fold higher following route A (see Figure 2;
A-1) compared to route B (see Figure 2; B-1), the
Moreover, in all reactions with RADH
concentrations <0.33 mg/mL, an optical purity of
>97 % (in some cases even >99 %; see SI Chapter 3,
Fig. S3-S5) was reached for both route A and B. The
optical impurities of <3% were caused by the isomers
(1S,2R)- and (1R,2S)-PPD. While the formation of
the (1S,2R)-isomer can be explained by the imperfect
stereoselectivity of RADH, the formation of the
(1R,2S)-diol is most likely caused by keto-enol
tautomerism of the 1,2-diol. With higher RADH
concentrations (>0.65 mg/mL) the optical purity
dropped to 95-96 %. In both routes (A, B) small
amounts of benzoin (<1 mM) were formed as a side
product of the PfBAL-catalyzed carboligation step.
+
influence of the NADP concentration was almost
negligible. In both cases, NADP+ concentrations
>
0.2 mM had no influence on amount of formed 1,2-
diol product (Figure 2; A-1 and B-1) or even on the
reaction velocity (see SI Chapter 3 Fig. S3-S5).
Optimization of pH and buffer
As previously demonstrated, RADH shows different
pH optima for the reduction and oxidation reaction,
with pH 9.0 being a compromise for the simultaneous
oxidation and reduction reactions.[ The challenging
task for the co-product recycling cascade was to
combine these requirements with conditions that are
14]
also optimal for the PfBAL[
12b, 13]
and the RADH
[14]
enzymes in the 1-pot 2-step cascade. Thus, we
investigated the cascade within a pH range of 8.0, 8.5
and 9.0 using TEA-buffer. At pH 8.5-9.0, slightly
higher 1,2-diol values were observed in both routes A
and B. Although the trend was not clear, higher pH
values were seen as beneficial within the cascade due
Limitations of the 1-pot 2-step cascade
The maximal conversions obtained under
optimized conditions did not exceed 72 % (93.5 mM;
route A), suggesting that full conversion might be
hampered by an unfavourable thermodynamic
equilibrium. Therefore, a rough estimation on the
theoretical equilibrium constant was calculated (see
SI, Chapter 4). According to these rough calculations,
conversions up to 99 % should be possible via route
B starting from 120 mM benzyl alcohol and 150 mM
acetaldehyde, whereas a significant lower theoretical
total conversion of ~90% was calculated for route A
(SI Tab. S2). The difference in theoretical total
conversion for route A (~99%) and route B (~90%)
are based on the ratio of the aromatic substrates
(benzyl alcohol + benzaldehyde) and acetaldehyde
(150mM). For route A the ratio is 130mM/150mM
and for route B 120mM/150mM.
to the activity/stability profiles of both enzymes
PfBAL and RADH.[
12b, 13-14]
Additionally, the TEA-
buffer (buffer range pH 7.0-8.3) was compared to
TRIS-HCl (buffer range pH 7.5-9.0) at pH 9.0 for
route A. As slightly better results were obtained using
TRIS buffer, which additionally has a higher buffer
capacity at pH 9.0, this buffer was used for the
subsequent studies.
RADH concentration
To investigate a limitation caused by reaction
velocity and/or enzyme stability, the RADH enzyme
concentration was varied form 0.10 mg/mL to
Further increasing the RADH concentration to
–1
+
2
.00 mg/mL. Here, we found a strong influence of the
RADH concentration on the amount of the formed
,2-diol product in both routes A and B (Figure 2;
A-3 and B-3). The highest tested RADH
concentration resulted in final product
3.75 mg mL and NADP concentration to 0.8 mM
did not result in full theoretical conversion. Here
demonstrated for route A, 93.2 mM (Figure 3)
1,2-diol was observed, corresponding to a conversion
of 72 %.
1
a
concentration of 82.8 mM (Figure 2, A-3) via route A
and 75.7 mM via route B (Figure 2, B-3).
4
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Advanced Synthesis & Catalysis
10.1002/adsc.201900187
Figure 2. Optimization of the 1-pot 2-step cofactor and co-product recycling cascade based on route A and route B
Constant parameter: 50 mM buffer, 0.8 mM CaCl , 2.5 mM MgSO , 0.15 mM ThDP, 0.05 mg/mL PfBAL
Varied parameter: pH: 8.0-9.0, NADP : 0.2-0.8 mM, TEA or TRIS-HCl and RADH: 0.1-2.0 mg/mL
2
4
+
Route A:
Route B:
benzaldehyde (10 mM), acetaldehyde (150 mM), benzyl alcohol (120 mM)
acetaldehyde (150 mM), benzyl alcohol (120 mM)
All reactions were carried out in a 1 mL at 20 °C with 150 rpm shaking and measured within a maximum of 240 h.
5
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Advanced Synthesis & Catalysis
10.1002/adsc.201900187
for both PfBAL and RADH increases the conversion.
Under similar conditions, the thermodynamically
maximal conversion of ~90% (for route A) was
reached by maintaining high optical purities of >99 %
ee and >99 % de.
Conclusion
We developed a step-efficient recycling cascade for
the highly stereo selective production of (1R,2R)-1-
phenylpropane-1,2-diol starting from inexpensive
benzaldehyde and acetaldehyde as substrates for the
carboligation step and benzyl alcohol as co-substrates
for the cofactor regeneration step. To increase the
atom-efficiency, the co-substrate (here: benzyl
alcohol) was chosen such that the resulting co-
product (here: benzaldehyde) is used as a substrate in
the carboligation step of the synthetic enzyme
cascade. After optimization of the one-pot 2-step
cascade, 93.5 mM (1R,2R)-1-phenylpropane-1,2-diol
was produced starting with 10 mM benzaldehyde and
150 mM acetaldehyde by successful recycling of the
co-product benzyl alcohol (added initially: 120 mM)
according to route A. Moreover, even without any
addition of benzaldehyde 82 mM of the final product
was formed with high stereoselectivity (>96-99 %
isomeric purity) in route B. By addition of 50 mM
fresh benzyl alcohol, a product concentration of
102 mM was reached in this monophase batch
reaction system. We could further demonstrate that
the batch systems are limited by the activity and
stability specifically of the RADH. An increase of the
RADH concentration from 0.1 mg/mL to 2 mg/mL
and keeping the concentration of PfBAL constant
(0.05 mg/mL) resulted in an increase in conversion
from about 15 % to 72 % (route A). At these highest
conversion values, the benzyl alcohol concentration
might cause kinetic limitations since the RADH is not
Figure 3. Conversion curve of the 2-step recycling
cascade with 3.75 mg mL RADH concentration.
–1
Reaction conditions: TRIS-HCl buffer (50 mM)
supplemented with CaCl
ThDP (0.15 mM), pH 9.0, NADP
2
(0.8 mM), MgSO
4
(2.5 mM),
(0.8 mM),
+
benzaldehyde (10 mM), acetaldehyde (150 mM), benzyl
alcohol (120 mM), PfBAL (0.05 mg/mL), RADH
(
●
3.75 mg/mL). Symbols: ◆benzyl alcohol, ○benzaldehyde,
(R)-2-HPP, ◇1,2-diol.
Reactions were carried out at 20 °C with constant shaking
150 rpm). Samples were taken in defined time intervals.
After 96 h the reaction solution was split and to one aliquot
0 mM fresh benzyl alcohol was added ( 1,2-diol) to
(
5
another not (◇1,2-diol) and the reaction further incubated
at 20 °C with constant shaking (150 rpm).
Considering kinetic limitations, we found that at
this maximum conversion, the residual concentration
of benzyl alcohol was 17 mM (Figure 3), which is in
the range of the K
M
-value of RADH for benzyl
[14]
alcohol under the conditions tested (12.3±2.7 mM),
Thus, the enzyme is not operating under Vmax
conditions anymore when 72 % conversion (route A)
was reached. Indeed, the product concentration could
be further increased to 102 mM by addition of further
benzyl alcohol (50 mM) after 96 h (Figure 3; blue
diamonds). Since thermodynamic parameter seems
not to be the limiting factor, enzyme activity might
hamper the reaction to go to theoretical maximal
conversion of ~90 % (for route A).
vmax. Here, on the one hand a fed-batch strategy might
be useful to further increase the 1,2-diol product
concentration and to reach the theoretical conversion
of ~90% (route A) and >99 % (route B). On the other
hand, the activity and stability issue was solved in
part II of the paper where ~90% (for route A) was
reached by maintaining high optical purities of >99 %
[17]
ee and >99 % de.
To overcome such limitations in future, one option
might be to continuously fed benzyl alcohol to the
process to ensure maximal RADH activity in fed-
batch reactions. Another possibility would be to
increase the operation window of both enzymes
Our method describes a process strategy for more
sustainable and eco-friendly synthetic enzyme
cascades employing nicotine amide red-ox cofactor
recycling without any waste production. This
approach optimizes the atom economy of a reaction,
reduces waste production and thus increases process
economy can be applied for any set of enzymes,
where the co-products of one process step serve as
substrates for another reaction step.
(
PfBAL and RADH) with respect to their activity
profile. In this part I of the paper we did not address
the inactivation of the enzymes over time under
process conditions, however this was considered in
part II. Moreover, the addition of even higher enzyme
loads is not beneficial for industrial application since
the reaction process costs might be significantly
[17]
increased. The results of part II of the publication
indicate that an improvement of the enzyme stability
6
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Advanced Synthesis & Catalysis
10.1002/adsc.201900187
with CaCl
2
(0.8 mM). The optimal RADH concentration
Experimental Section
was tested by varying the enzyme concentration between
0
.05–0.30 mg mL–1 under standard biotransformation
Materials and Methods
conditions, using benzyl alcohol (100 mM) as co-substrate.
Chemicals
All chemicals were of high analytical grade. Tris-2-
B) 2-Step cofactor and co-product recycling cascade
(
(
hydroxyethyl)amine (TEA) was purchased from Sigma
Steinheim, Germany), 2-amino-2-hydroxymethylpropane-
Two different routes were studied differing in substrate
supply strategy (Figure 2 and SI Fig. S1). Depending on
the mode, initial amounts of substrates and co-substrates
1
2 2
,3-diol (TRIS) and calcium chloride (CaCl ∙ 2 H O) were
from Merck (Darmstadt, Germany), benzyl alcohol and
benzaldehyde from Sigma-Aldrich (Steinheim, Germany),
were varied. Route A:
acetaldehyde (150 mM),
benzaldehyde (10 mM), and benzyl alcohol (120 mM).
Route B: acetaldehyde (150 mM) and benzyl alcohol
(120 mM). All reaction were performed in 50 mM buffer,
4
and acetaldehyde and magnesium sulphate (MgSO ) from
Fluka (Steinheim, Germany). Cofactors: thiamine
+
diphosphate (ThDP) and NADP were from AppliChem
0
0
.8 mM CaCl
.05 mg/mL PfBAL. For NADP optimization, the NADP
2
,
2.5 mM MgSO
4
,
0.15 mM ThDP,
(
Darmstadt, Germany) and Biomol (Hamburg, Germany),
+
+
respectively. (R)-2-Hydroxy-1-phenylpropan-1-one ((R)-2-
HPP)[
2
3a, 12a]
concentration was varied in the range of 0.2 mM to
0.8 mM. For pH optimization TEA-HCl (pH 8.0-9.0) and
TRIS-HCl buffer systems (50 mM, pH 9.0) were tested. To
and rac-2-hydroxy-1-phenylpropan-1-one (rac-
-HPP)[ were synthesized as described in the literature.
15]
find optimal RADH concentrations, catalyst loads from
.10 mg mL–1 to 2.00 mg mL
–1
were examined. For
0
Expression and purification of the recombinant enzymes
reaction details of the varied parameter see description
Figure 2. All reactions were carried out at a 1 mL scale in
screwed glass vessels equipped with a Teflon membrane at
0 °C under constant shaking (500 rpm). Samples (100 µL)
were taken in defined time intervals for 240 h.
Expression and purification of His-tagged benzaldehyde
lyase from Pseudomonas fluorescens (PfBAL) employing
metal chelate affinity chromatography (Ni-NTA column)
2
was carried out as described elsewhere.[
12b]
Alcohol
dehydrogenase from Ralstonia sp. (RADH) was expressed
and purified by ion exchange chromatography (anion
exchanger) as recently published.[
8]
Determination of the thermodynamic equilibrium constant
The equilibrium constant (Keq) for the carboligation
reaction was calculated according to Formula 1:
Determination of protein concentration
[
2–퐻푃푃]ꢀꢁ
Protein concentration was determined by the Bradford
푒푞
퐵퐴퐿
퐾
=
(1)
method[
16]
using bovine serum albumin as a standard
[푎푐푒푡푎푙푑푒ℎ푦푑푒]ꢀꢁ∙[푏푒푛푧푎푙푑푒ℎ푦푑푒]ꢀꢁ
(
Sigma-Aldrich, Steinheim, Germany).
The equilibrium constant for the oxidoreduction step was
calculated according to Formula 2 and Formula 3:
A) Benzyl alcohol for cofactor regeneration
Optimization of cofactor regeneration was carried out at a
1,2–푑ꢂ표푙]ꢀꢁ ∙ [푁퐴퐷푃+]ꢀꢁ
[
푒푞
퐾
= [2–퐻푃푃]ꢀꢁ
(2)
(3)
1
mL scale in screwed glass vials equipped with a Teflon-
푅퐴퐷퐻–푟푒푑.
∙ [푁퐴퐷푃퐻]ꢀꢁ
membrane in order to avoid evaporation of aldehydes.
Reactions were conducted at 20 °C for 24 h with constant
shaking of 500 rpm (Eppendorf thermomixer, Hamburg,
Germany) and stopped by extraction with ethyl acetate
[
푏푒푛푧푎푙푑푒ℎ푦푑푒]ꢀꢁ ∙ [푁퐴퐷푃퐻]ꢀꢁ
푒푞
퐾
=
[푏푒푛푧푦푙 푎푙푐표ℎ표푙]ꢀꢁ ∙ [푁퐴퐷푃+]ꢀꢁ
푅퐴퐷퐻–표푥.
–1
Equilibrium constants were calculated using initial and
final concentrations of all available compounds in the
reaction vessel measured for the respective route. The
theoretical overall conversions of the 2-step recycling
cascade were calculated with MATLAB (The MathWorks,
Germany). For details concerning calculations and scripts
see SI, Chapter 4.
(
50 % v v ). All reactions were performed in duplicate.
Conversions were determined using GC-analysis based on
a calibration with the formed product 1-phenylpropane-
1
,2-diol. For instrumental analysis and origins of reference
compounds see section Chiral analysis.
All reactions were conducted in TRIS-HCl buffer (50 mM)
supplemented with CaCl (0.8 mM), pH 9.0, if not stated
2
otherwise. For biotransformations the following standard
Synthetic cascade reaction with 3.75 mg mL–1 RADH
concentration using route A
conditions were applied (if not stated otherwise): rac-2-
+
HPP (10 mM), NADP
(
(0.2 mM) and RADH
–1
0.05 mg mL ). To determine the optimal co-substrate
Reactions were conducted under the following conditions:
TRIS-HCl buffer system (50 mM), pH 9.0, supplemented
concentration benzyl alcohol (10 mM to 250 mM) was
tested under standard conditions. To identify the optimal
pH and buffer conditions, batch experiments were
performed in TRIS-HCl (50 mM, pH 8.0–9.0) and in TEA-
HCl (50 mM, pH 8.0) buffer systems, both supplemented
2 4
with CaCl (0.8 mM), MgSO (2.5 mM), ThDP (0.15 mM),
+
benzyl alcohol (120 mM) and NADP (0.8 mM). Reactions
were carried out at 20 °C with constant shaking (500 rpm)
in closed glass vials. After 96 h additional fresh 50 mM
7
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Advanced Synthesis & Catalysis
10.1002/adsc.201900187
benzyl alcohol was added to the reaction vessel. Samples
were taken in defined time intervals and reaction progress
was measured by GC and HPLC (see paragraph Chiral
analysis).
References
[
1] a) M. Kurina-Sanz, F. R. Bisogno, I. Lavandera, A. A.
Orden, V. Gotor, Advanced Synthesis & Catalysis
2
009, 351, 1842-1848; b) R. Agudo, G.-D. Roiban, R.
Lonsdale, A. Ilie, M. T. Reetz, The Journal of
Organic Chemistry 2015, 80, 950-956; c) D. Gala, D.
J. DiBenedetto, J. E. Clark, B. L. Murphy, D. P.
Schumacher, M. Steinman, Tetrahedron Letters 1996,
Chiral
Chromatography (HPLC)
analysis
-
High
Performance
Liquid
Concentrations of substrates (benzaldehyde and benzyl
alcohol) and the intermediately formed (R)-2-HPP were
determined by HPLC-analysis. Therefore 10 µL sample
3
7, 611-614; d) J. Wachtmeister, A. Jakoblinnert, J.
Kulig, H. Offermann, D. Rother, ChemCatChem
014, 6, 1051-1058; e) J. Wachtmeister, A.
2
(
10-times
diluted
in
acetonitrile
with
2-
Jakoblinnert, D. Rother, Organic Process Research &
Development 2016, 20, 1744-1753; f) S. M. Husain,
T. Stillger, P. Dünkelmann, M. Lödige, L. Walter, E.
Breitling, M. Pohl, M. Bürchner, I. Krossing, M.
Müller, D. Romano, F. Molinari, Advanced Synthesis
methylbenzaldehyde as internal standard) was analyzed by
chiral HPLC using a HiBar 250-4 LiChrosphere 100 RP-8
–1
(
5 µm) column (Merck KGaA, Germany; flow: 1 mL min ,
temperature: 25 °C). As mobile phase, a gradient of 25-
–
1
6
0 % (v v ) acetonitrile was used, mixed with ultrapure
–
1
&
Catalysis 2011, 353, 2359-2362; g) L. Muschallik,
water (0-12 min: 25 % (v v ) acetonitrile, 12-13 min:
gradient 25-60 % (v v ) acetonitrile, 13-20 min: 60 %
v v ) acetonitrile, 20-23 min: 60-25 % (v v ) acetonitrile,
3-25 min: 25 % (v v ) acetonitrile). Absorption was
–1
D. Molinnus, J. Bongaerts, M. Pohl, T. Wagner, M. J.
Schöning, P. Siegert, T. Selmer, Journal of
Biotechnology 2017, 258, 41-50; h) B. M. Nestl, S. C.
Hammer, B. A. Nebel, B. Hauer, Angewandte Chemie
International Edition 2014, 53, 3070-3095.
–
1
–1
(
2
–
1
determined at 200 nm (benzyl alcohol) and 250 nm (2-HPP
and benzaldehyde) with the following retention times:
R
R
t,(benzyl alcohol) = 7.6 min, Rt,(2-HPP) = 10.5 min and
t,(benzaldehyde) = 16.2 min.
[
2] a) J. C. Griffith, K. M. Jones, S. Picon, M. J. Rawling,
B. M. Kariuki, M. Campbell, N. C. O. Tomkinson,
Journal of the American Chemical Society 2010, 132,
1
4409-14411; b) R. S. Mohan, K. Gavardinas, S.
Chiral analysis - Gas Chromatography (GC)
Kyere, D. L. Whalen, The Journal of Organic
Chemistry 2000, 65, 1407-1413; c) Y. Ashikari, T.
Nokami, J.-i. Yoshida, Organic Letters 2012, 14,
938-941; d) W. Notz, B. List, Journal of the
American Chemical Society 2000, 122, 7386-7387; e)
C. C. Torres, C. H. Campos, J. L. G. Fierro, P. Reyes,
D. Ruiz, Journal of Molecular Catalysis A: Chemical
2014, 392, 321-328; f) K. B. Sharpless, W. Amberg,
Y. L. Bennani, G. A. Crispino, J. Hartung, K. S.
Jeong, H. L. Kwong, K. Morikawa, Z. M. Wang, The
Journal of Organic Chemistry 1992, 57, 2768-2771.
Final concentration and diastereomeric excess (de) of the
cascade product (non-derivatized stereoisomers of 1-
phenyl-propane-1,2-diol) were determined via GC-analysis.
Therefore samples were extracted with ethyl acetate
including 1-dodecanol as internal standard. 5 µL of the
obtained organic phase were analyzed by gas
chromatography using a chiral CP-Chirasil-DEX CB
(
Varian, Germany) column (25 m x 0.25 mm x 0.25 µm)
with a flame ionisation detector (FID) and hydrogen as
carrier gas. The following GC-program was applied:
1
40 °C injection temperature, isothermic run for 30 min.
[
3] a) D. Kihumbu, T. Stillger, W. Hummel, A. Liese,
Tetrahedron: Asymmetry 2002, 13, 1069-1072; b) J.
Kulig, R. C. Simon, C. A. Rose, S. M. Husain, M.
Häckh, S. Lüdeke, K. Zeitler, W. Kroutil, M. Pohl, D.
Rother, Catalysis Science & Technology 2012, 2,
Retention times: Rt,(benzaldehyde) = 3.1 min and Rt,(benzyl alcohol)
=
phenylpropan-1,2-diol: Rt,(1S,2S) = 24.1 min, Rt,(1R,2R)
2
chemical syntheses of reference compounds and the
assignment of absolute configuration are described
elsewhere.[
4.9 min. Retention times of the four stereoisomers of
=
5.8 min, Rt,(1S,2R) = 27.4 min, Rt,(1R,2S) = 28.5 min. Detailed
1
580-1589; c) M. Pohl, C. Dresen, M. Beigi, M.
Müller, Acyloin and Benzoin Condensations, in
Enzyme Catalysis in Organic Synthesis, Wiley ‐
3b]
VCH Verlag GmbH
&
Co. KGaA, 2012,
doi:10.1002/9783527639861.ch22.
Acknowledgements
[4] a) S. Kara, J. H. Schrittwieser, F. Hollmann, M. B.
Ansorge-Schumacher, Applied Microbiology and
Biotechnology 2014, 98, 1517-1529; b) A.
Weckbecker, H. Gröger, W. Hummel, in Biosystems
Engineering I: Creating Superior Biocatalysts (Eds.:
C. Wittmann, R. Krull), Springer Berlin Heidelberg,
Berlin, Heidelberg, 2010, pp. 195-242. c) L. Josa-
Culleré, A.S.K. Lahdenperä, A. Ribaucourt, G.T.
Höfler, S. Gargiulo, Y.-Y. Liu, J.-He Xu, J. Cassidy,
F. Paradisi, D. J. Opperman, F. Hollmann, C. E. Paul,
Catalysts 2019, 9, 207-218.
We thank Prof. Wolfgang Kroutil (University of Graz, Austria)
for providing the radh gene and Dr. Christiane Claaßen for her
held with the NMR data processing.
This work was financed by the Marie Curie Initial Training
Network “BIOTRAINS – a European biotechnology training
network for the support of chemical manufacturing”, grant
agreement no. 238531 and by the Helmholtz Association in frame
of the W2/W3-professor program (grant no. W2/W3-110).
8
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201900187
[
[
5] H. K. Chenault, G. M. Whitesides, Applied
Biochemistry and Biotechnology 1987, 14, 147-197.
[14] J. Kulig, A. Frese, W. Kroutil, M. Pohl, D. Rother,
Biotechnology and Bioengineering 2013, 110, 1838-
1
848.
6] a) R. Wichmann, D. Vasic-Racki, in Technology
Transfer in Biotechnology: From lab to Industry to
Production (Ed.: U. Kragl), Springer Berlin
Heidelberg, Berlin, Heidelberg, 2005, pp. 225-260; b)
F. Hollmann, I. W. C. E. Arends, K. Buehler,
ChemCatChem 2010, 2, 762-782.
[15] C. Chen, X. Feng, G. Zhang, Q. Zhao, G. Huang,
ChemInform 2009, 40.
[
[
16] M. M. Bradford, Analytical Biochemistry 1976, 72,
48-254.
2
17] V. D. Jäger, M. Piqueray, S. Seide, M. Pohl, W.
[
7] a) F. d. M. Bastos, A. G. dos Santos, J. Jones, E. G.
Oestreicher, G. F. Pinto, L. M. C. Paiva,
Biotechnology Techniques 1999, 13, 661-664; b) F.
M. Bastos, T. K. França, G. D. C. Machado, G. F.
Pinto, E. G. Oestreicher, L. M. C. Paiva, Journal of
Molecular Catalysis B: Enzymatic 2002, 19-20, 459-
Wiechert, K.-E. Jaeger, U. Krauss, Adv. Synth. Catal.
2
019, 361, DOI: 10.1002/adsc.201900189.
4
65; c) T. R. Röthig, K. D. Kulbe, F. Bückmann, G.
Carrea, Biotechnology Letters 1990, 12, 353-356; d) J.
Peters, T. Zelinski, T. Minuth, M.-R. Kula,
Tetrahedron: Asymmetry 1993, 4, 1683-1692; e) T.
Daußmann, T. C. Rosen, P. Dünkelmann,
Engineering in Life Sciences 2006, 6, 125-129.
[
[
8] a) M. Julliard, J. Le Petit, P. Ritz, Biotechnology and
Bioengineering 1986, 28, 1774-1779; b) R. Abu, J. M.
Woodley, ChemCatChem 2015, 7, 3094-3105.
9] a) S. Kara, D. Spickermann, J. H. Schrittwieser, C.
Leggewie, W. J. H. van Berkel, I. W. C. E. Arends, F.
Hollmann, Green Chemistry 2013, 15, 330-335; b) A.
Bornadel, R. Hatti-Kaul, F. Hollmann, S. Kara,
ChemCatChem 2015, 7, 2442-2445.
[
10] a) T. Sehl, H. C. Hailes, J. M. Ward, R. Wardenga, E.
von Lieres, H. Offermann, R. Westphal, M. Pohl, D.
Rother, Angewandte Chemie International Edition
2
013, 52, 6772-6775; b) R. C. Simon, N. Richter, E.
Busto, W. Kroutil, ACS Catalysis 2014, 4, 129-143;
c) J. Wachtmeister, D. Rother, Current Opinion in
Biotechnology 2016, 42, 169-177; d) J. H.
Schrittwieser, S. Velikogne, M. Hall, W. Kroutil,
Chemical Reviews 2018, 118, 270-348; e) P. Gupta,
N. Mahajan, New Journal of Chemistry 2018, 42,
1
2296-12327. e) P. Gupta, N. Mahajan, New Journal
of Chemistry 2018, 42, 12296-12327. f) S. Liu, X.
Zhang, F. Liu, M. Xu, T. Yang, M. Long, J. Zhou, T.
Osire, S. Yang, Z. Rao, ACS Synthetic Biology 2019,
DOI: 10.1021/acssynbio.8b00364
[
[
11] R. Oeggl, T. Maßmann, A. Jupke, D. Rother, ACS
Sustainable Chemistry & Engineering 2018, 6,
1
1819-11826.
12] a) A. S. Demir, M. Pohl, E. Janzen, M. Müller,
Journal of the Chemical Society, Perkin Transactions
1
2001, 633-635; b) E. Janzen, M. Müller, D. Kolter-
Jung, M. M. Kneen, M. J. McLeish, M. Pohl,
Bioorganic Chemistry 2006, 34, 345-361.
[
13] a) P. D. de María, T. Stillger, M. Pohl, S. Wallert, K.
Drauz, H. Gröger, H. Trauthwein, A. Liese, Journal
of Molecular Catalysis B: Enzymatic 2006, 38, 43-47;
b) T. Stillger, M. Pohl, C. Wandrey, A. Liese,
Organic Process Research & Development 2006, 10,
1
172-1177.
9
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10.1002/adsc.201900187
FULL PAPER
An Enzymatic 2-Step Cofactor and Co-Product
Recycling Cascade towards a Chiral 1,2-Diol.
Part I: Cascade Design
Adv. Synth. Catal. Year, Volume, Page – Page
Justyna Kulig, Torsten Sehl, Ursula Mackfeld,
Wolfgang Wiechert, Martina Pohl, Dörte Rother
*
1
0
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