3D-Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4-Hydroxystilbene

Article abstract of DOI:10.1002/chem.201904206

Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regio- and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agarose-based modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactor-free phenacrylate decarboxylase enzymes suitable for the production of 4-vinylphenol and test their applicability for the encapsulation and direct 3D printing of disk-shaped agarose-based modules that can be used for compartmentalized flow microreactors. Using the most active and stable phenacrylate decarboxylase from Enterobacter spec. in a setup with four parallel reactors and a subsequent palladium(II) acetate-catalysed Heck reaction, 4-hydroxystilbene was synthesized from p-coumaric acid with a total yield of 14.7 % on a milligram scale. We believe that, due to the convenient direct immobilization of any thermostable enzyme and straightforward tuning of the reaction sequence by stacking of modules with different catalytic activities, this simple process will facilitate the establishment and use of cascade reactions and will therefore be of great advantage for many research approaches.

Full text of DOI:10.1002/chem.201904206

A Journal of
Accepted Article
Title: 3D-Printed phenacrylate decarboxylase flow reactors for the
chemoenzymatic synthesis of 4‑hydroxystilbene
Authors: Martin Peng, Esther Mittmann, Lukas Wenger, Jürgen
Hubbuch, Martin K. M. Engqvist, Christof M. Niemeyer, and
Kersten S. Rabe
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201904206
Link to VoR: http://dx.doi.org/10.1002/chem.201904206
Supported by

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COMMUNICATION
3D-Printed phenacrylate decarboxylase flow reactors for the
chemoenzymatic synthesis of 4-hydroxystilbene
Martin Peng[a], Esther Mittmann[a], Lukas Wenger[b,c], Jürgen Hubbuch[b,c], Martin K. M. Engqvist[d],
Christof M. Niemeyer[a] and Kersten S. Rabe*[a]
Abstract: Continuous flow systems for chemical synthesis are
becoming a major focus in organic chemistry and there is a growing
interest in the integration of biocatalysts due to their high regio- and
stereoselectivity. Methods established for 3D bioprinting enable the
fast and simple production of agarose-based modules for biocatalytic
reactors if thermally stable enzymes are available. We report here on
the characterization of four different cofactor-free phenacrylate
Figure 1: Manufacturing process of agarose-based, compartmentalized
biocatalytic flow reactors. The enzyme is mixed with a liquid agarose solution
(at 42 °C) and immediately cooled down on ice and then stored at 8 °C. Using
a 3D printer, the bioink is melted at 60 °C and printed onto a cooled substrate
on which the bioink solidifies to form biocatalytic modules. These are integrated
into flow reactors that are transfused with a substrate solution to facilitate
continuous synthesis. The reactor productivity can be adjusted by variable
numbers of reaction modules.[4]
decarboxylase enzymes suitable for the production of 4-vinylphenol
and test their applicability for the encapsulation and direct 3D printing
of disk-shaped agarose-based modules that can be used for
compartmentalized flow micro reactors. Using the most active and
stable phenacrylate decarboxylases from Enterobacter spec. in a
setup with four parallel reactors and a subsequent palladium(II)
acetate-catalyzed Heck reaction, 4-hydroxystilbene was synthesized
from p-coumaric acid with a total yield of 14.7 % on the mg-scale. We
believe that, due to the convenient direct immobilization of any
thermostable enzyme and straightforward tuning of the reaction
sequence by stacking of modules with different catalytic activities, this
simple process will facilitate the establishment and use of cascade
reactions and will therefore be of great advantage for many research
approaches.
for chemoenzymatic routes by combining reactions that are
otherwise incompatible due to reaction conditions,product
inhibition or unwanted side reactions.[3a, 5] For the realization of
such compartmentalized biocatalytic processes in continuous
flow, the fast and simple immobilization of enzymes inside the
microstructured flow reactors is an essential step to ensure
efficient spatial separation of different reactions.[6] In order to
enable the simple rapid prototyping of biocatalytic reaction
modules for compartmentalized flow micro reactors, we have
recently established a fabrication process using direct 3D printing
of inexpensive biodegradable agarose-based hydrogel bioinks
containing thermostable enzymes (Figure 1).
3D printing (also known as additive manufacturing) is a fabrication
process employing layer-by-layer deposition of materials to
produce complex 3D structures with a high flexibility in design and
minimal waste of material[7] which has found applications ranging
from the aerospace and construction industry to smart materials,
medicine and biotechnology.[8] 3D printing offer a simple and
efficient route to rapid prototyping, the iterative process of
fabricating and evaluating prototypes, dramatically reducing
turnaround times in process development. Furthermore, 3D
printing facilitates the production of extremely complex
geometries[7] enabling the fabrication of sophisticated reactor
designs in chemical and biochemical engineering.[9]
Sustainable production processes based on renewable biomass
require biocatalysts with high conversion rates as well as regio-
and stereoselectivity.[1] Enzymes, as nature’s main catalysts, are
enabling the realization of such processes. Furthermore, they can
be precisely tailored to fit with the process requirements such as
substrate spectrum, regio- and stereoselectivity and
physicochemical stability by using protein engineering.[2] To
improve biotechnological production processes, individual
reaction steps can be compartmentalized and arranged into
reaction cascades.[3] This allows high process control even
[a]
M. Peng, E. Mittmann, Prof. Dr. C. M. Niemeyer, Dr. K. S. Rabe
Karlsruhe Institute of Technology (KIT)
Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-
Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany
E-mail: kersten.rabe@kit.edu
To enhance the compatibility with biological materials a variety of
methods and materials for extrusion-based 3D bioprinting can
now generate hydrogel structures customized for specific
applications.[10] Enzymes can be entrapped inside these
hydrogels without the need for enzyme-specific adaptations,
offering protection against organic solvents[4] but also modulating
the reaction rate through mass transfer limitations, since the 3D-
printed structures are relatively thick.[11] Such issues are less
prevalent when using for example packed-bed reactors
containing 0.1-1 mm Ca-alginate beads, packed-bed nanofiber
reactors or all-enzyme-hydrogel reactors, containing no carrier
material at all,[6a, 6e, 12] however the capability of the 3D bioprinting
for the on-demand production of biocatalytic modules, only
Web: https://www.ibg.kit.edu/ibg1/
L. Wenger, Prof. Dr. J. Hubbuch
Karlsruhe Institute of Technology (KIT)
Institute of Functional Interfaces, Hermann-von-Helmholtz-Platz 1,
D-76344 Eggenstein-Leopoldshafen, Germany
L. Wenger, Prof. Dr. J. Hubbuch
[b]
[c]
Karlsruhe Institute of Technology (KIT)
Institute of Engineering in Life Sciences, Section IV: Biomolecular
Separation Engineering, Fritz-Haber-Weg 2, D-76131 Karlsruhe,
Germany
M. K. M. Engqvist
Chalmers University of Technology
Department of Biology and Biological Engineering, Division of
Systems and Synthetic Biology, Kemivägen 10, SE-412 96
Gothenburg, Sweden
[d]
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201904206
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COMMUNICATION
requiring unmodified, thermostable enzymes, has great potential
for fast and simple immobilization approaches.
In order to extend the earlier proof-of-concept study to other
synthetically relevant enzymes, we here report on the 3D printing
of biocatalytic modules to be integrated in a chemoenzymatic
reaction cascade producing 4-hydroxystilbene 3 (Scheme 1).
4-Hydroxystilbene is an important precursor of the
pharmacologically relevant class of 2,3-dihydrobenzofuran-based
compounds.[13]
To realize the chemoenzymatic reaction cascade (Scheme 1), we
considered prokaryotic phenacrylate decarboxylases (PAD,
EC 4.1.1.102) that can catalyse the formation of 4-vinylphenols
from para-hydroxyphenacrylic acids without the supply of
additional redox equivalents.[14] Furthermore, unlike other
cofactor-dependent PADs, these enzymes do not require a
prenylated flavin cofactor, which typically requires production by
coexpression of the flavin prenyltransferase UbiX.[14-15] In a
second step of the cascade, the enzymatically produced
vinylphenols can then be further functionalized, for example, by
,
aid of other enzymes[16] oxidative dimerization[17] or by
organometallic coupling reactions[18] as demonstrated here.
Since the printing of biocatalytic active reactor models requires an
initial heating step at 60 °C for 15 min, thermostable enzymes are
required, which can be obtained by utilizing naturally occurring
Figure 2: A) EsPAD (20 kDa, lane 1), LpPAD (22 kDa, lane 2), BmPAD (20 kDa,
lane 3) and LbPAD (23 kDa, lane 4) were produced as pure enzymes, as
indicated by the coomassie stained 15 % SDS Tris-glycine polyacrylamide gel.
M: PageRuler Prestained Protein Ladder (Thermo Scientific). B) Enzymatic
activity of all four PAD enzymes at 28 °C with p-coumaric acid 1 (filled bars) and
caffeic acid 5 (empty bars) as substrate. C) Average thermal inactivation curves
with 95 % confidence interval of all four purified PAD enzymes after 10 min
incubation and their corresponding T50-values (for individual data sets see
Figure S2-S5). Analyses B and C were carried out in technical duplicates and
biological triplicates.
19]
thermostable enzymes[4,
or enzymes thermostabilized by
protein engineering.[4, 19a, 20] To broaden the spectrum of available
biocatalysts and to identify suitable PAD enzymes, we directly
compared the enzymatic activity and thermostability of four
different enzymes (Table S1) under identical conditions. To this
end, we expressed and purified (Figure 2A) the previously
described EsPAD[21] from Enterobacter spec. and LpPAD[22] from
Lactobacillus plantarum as well as LbPAD from Lactobacillus
brevis, a close homologue of an already described enzyme[23]
(Figure S1), and the not yet described BmPAD from Bacillus
megaterium.
and LpPAD converting caffeic acid 5 twice as fast as BmPAD.
Since the activities of all enzymes are in the same range, we
focused on the enzyme’s thermostability as the essential
parameter for the intended use in the 3D printing process. To this
end, we characterised the enzymes’ temperature-dependent loss
of activity by incubating 1 μM enzyme solutions for 10 min at
variable temperatures in the range between 30 °C - 80 °C. The
remaining enzyme activity was then determined at 25 °C using
p-coumaric acid as substrate. Plots of the resulting activities
against incubation temperature allowed to determine T50-values,
defined as the temperature at which 50 % of the initial enzyme
activity still remained (Figure 2C and Figure S2–S5). From these
analyses, EsPAD emerged as the most thermostable enzyme,
which also showed the highest activity with p-coumaric acid as
substrate.
To enable direct comparison of the four PAD variants, we initially
studied their substrate specificity and activity. To this end, trans-
cinnamic acid, the simplest phenacrylic acid without any aromatic
substitution, and the hydroxysubstituted phenacrylic acids, p-
coumaric acid 1 and caffeic acid 5, were tested as substrates.
Their enzymatic activity at 28°C was determined by the decrease
in absorbance at the substrate-specific wavelengths of 274 nm,
294 nm and 311 nm, respectively. As expected, all four PAD
enzymes only showed activity with the para-hydroxysubstituted
phenacrylic acids, p-coumaric acid and caffeic acid, confirming
that the para-hydroxyfunction is essential for the formation of the
para-quinone
methide
intermediate,
which
leads
to
decarboxylation of the substrate.[14] We found that the activity of
all four enzymes is on the same order of magnitude under the
conditions examined here (Figure 2B and Table S2), with EsPAD
converting p-coumaric acid 1 three times faster than the LbPAD
To confirm that the determined T50 value is a well suited measure
for prediction of the applicability of the enzymes for the 3D printing
process, agarose bioinks were prepared from the four enzymes
(2 µM) and used for printing of reaction modules (0.97 nmol
enzyme per module) with geometries as described previously.[4]
By employment of the flow reactor setup (Figure 3A), the reaction
modules were perfused with 0.125 mM p-coumaric acid in
reaction buffer at a constant flow rate of 12.5 µL ∙ min-1. Samples
of the reactor outflow were taken at regular time intervals and
analysed by HPLC (Figure S6). As expected from the T50
measurements, the reactor modules containing LpPAD, BmPAD
or LbPAD produced no detectable amounts of 4-vinylphenol from
Scheme 1: Synthesis of 4-hydroxystilbene 3 from p-coumaric acid 1 employing
a
phenacrylate decarboxylase (EsPAD) in
a flow reactor setup and a
subsequent Heck reaction using palladium(II) acetate.
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p-coumaric acid, whereas the EsPAD-containing modules (with
38 ± 15 % enzyme activity remaining after the printing process)
converted up to 78 % of the available p-coumaric acid under these
conditions (Figure 3B). The observed time-dependent decrease
of the conversion rate has been observed before and is related to
the leaching of the encapsulated enzyme.[4]
In order to improve the system towards full conversion, we
increased the enzyme concentration inside the bioink to 100 µM
EsPAD. Under these reaction conditions, 30 mL of 1 mM
p-coumaric acid were converted over 40 h with efficiencies of up
to 98 % (Figure S7). After this process optimization, we could
simply parallelize four identical flow reactors, each equipped with
a 100 µM EsPAD reactor module (48.3 nmol enzyme per module,
Figure 3C and S8). This increase in scale by numbering-up led to
a conversion of 35 mg p-coumaric acid 1 to 4-vinylphenol 2 in a
total volume of 211 mL and with an average total turnover of 590
4-vinylphenol molecules per enzyme.
Following to extraction of the collected outflows and a quick
chromatographic purification of the raw product, solid
4-vinylphenol 2 was obtained with an isolated yield of 54 %
(Figure 3D). According to the reaction shown in Scheme 1, the
purified 4-vinylphenol 2 was then coupled with iodobenzene 4 by
using a palladium-catalysed Heck reaction. This led to the
formation of 4-hydroxystilbene 5, obtained as white crystals in a
total yield of about 15 % (Figure 3D).
In summary, we here demonstrate for the first time that
unmodified EsPAD can be used in the production of bioinks,
which are suitable for direct 3D printing of reactor modules using
additive manufacturing processes without post-processing steps.
We also directly compared four different phenacrylate
decarboxylases and identified EsPAD as the enzyme with the
highest thermostability and the greatest turnover of the PAD
enzymes tested. After optimization of reaction parameters, 3D
printed modules were readily prepared and used for continuous
flow production of 4-vinylphenol 2 from p-coumaric acid 1. Scale-
up of the reaction was conveniently achieved by numbering-up of
the reactors without further process optimization. Such processes
can be integrated into chemoenzymatic reaction cascades, as
shown here using the example of the subsequent palladium-
catalysed formation of 4-hydroxystilbene 3. As shown here, new
enzyme classes can be easily screened for enzymes compatible
with the 3D bioprinting presented. The high modularity as well as
scalability of the reactor system and the ease of its manufacturing
can then be utilized to introduce these new enzyme classes into
flow reaction systems.
Experimental Section
Experimental details can be found in the Supporting Information.
Acknowledgements
We thank Manfred Maier for the support, advice and assistance
with the 3D printing process and the fluidic set up.
Keywords: 3D printing • enzyme • biocatalysis • flow chemistry •
hydrogel
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Entry for the Table of Contents (Please choose one layout)
Layout 2:
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Martin Peng, Esther Mittmann, Lukas
Wenger, Jürgen Hubbuch, Martin K. M.
Engqvist, Christof M. Niemeyer, Kersten
S. Rabe*
Page No. – Page No.
3D-Printed phenacrylate
4-Hydroxystilbene is obtained from p-coumaric acid employing a phenacrylate
decarboxylase enzyme immobilized in 3D printed agarose-based flow reactor
modules and a subsequent palladium(II) acetate-catalyzed Heck reaction with a
total yield of 14.7 % on the mg-scale.
decarboxylase flow reactors for the
chemoenzymatic synthesis of 4-
hydroxystilbene
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