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org/acscatalysis Research Article

Rational Design of a De Novo Enzyme Cascade for Scalable

Continuous Production of Antidepressant Prodrugs
Yunbin Wu, Rui Xu, Yuxin Feng, and Heng Song*
Cite This: ACS Catal. 2022, 12, 3767−3775 Read Online

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ABSTRACT: Natural products and their derivatives provide a promising source for drug discoveries. However, several natural
products and derivatives are still difficult to advance to in vivo drug testing due to the requirement of an ample supply of drug
candidates. Herein, we describe an immobilized enzyme cascade for the scalable continuous production of neuropharmaceutical
prodrugs, L-4-chlorokynurenine (L-4-Cl-Kyn) and its non-natural analogue L-4-Br-Kyn. This synthetic route features a highly
efficient one-pot C−H activation/oxidation/hydrolyzation cascade for the assembly of the Kyn core structure, inexpensive
substituted aryl sources for the abundance of Kyn scaffold types, and easily available gram-scale MOF−hydrogel hybrid material for
the immobilization of enzymes to recycle biocatalysts and maximize their catalytic efficiency. As a result, our method accumulates
370 mg of L-4-Cl-Kyn and 365 mg of L-4-Br-Kyn, 250 mL product volume per cycle for 5 cycles, which is nearly twice the yield of
free enzymatic catalysis in the same enzyme amount frame. Therefore, this study establishes the applicability of synthetic biology
strategies and immobilization biotechnology in synthesizing high value-added chemicals and narrows the gap in moving cell-free
immobilized enzyme cascade systems from academic studies to industrial applications. Moreover, animal experiments demonstrate
that L-4-Br-Kyn displays a slightly better antidepressant effect than the phase II drug L-4-Cl-Kyn, which further emphasizes the
importance of our work for the facile scale-up synthesis to provide large amounts of pharmaceutical products in facilitating the
development or discovery of drug candidates. These practical features of our study will undoubtedly be welcomed by academic and
industrial researchers.
KEYWORDS: biocatalysis, scalable continuous production, enzyme immobilization, chlorokynurenine, bromokynurenine,
antidepressant prodrugs, natural products, retrobiosynthesis

N atural products and their derivatives are our greatest

medicine cabinet and have provided humankind with
more than one-third of FDA-approved new molecular
entities.1−3 Extensive efforts have been devoted to discovering
novel structures of natural products with preliminary in vitro
bioactivity tests in the hope of providing improved and potent
drug candidates. However, scaling up the production of newly
discovered natural products is also essential for supplying
substantial amounts required for in vivo tests to verify their
pharmaceutical effects and accelerate the drug discovery
process.4
Since nature has developed sophisticated synthetic plans
over millions of years to construct natural molecules,
mimicking and adopting nature’s ability to manipulate the
serially organized enzymatic cascades can be a feasible way to
construct complicated molecules derived from nature.5 Indeed,
both metabolic engineering6,7 and cell-free biocatalysis (CFB)8
have been utilized to develop multienzyme catalytic cascades
for chemical synthesis in vivo and in vitro, respectively.9
Compared to the metabolic engineering method, CFB is a
promising approach with advantages of facile plug-and-play
cascade assembly, flexible reaction conditions, and direct
access to the reaction environment for monitoring, manipu-
lation, and maintenance.10,11 Although CFB systems have been
driving biological processes in an efficient and adaptable
manner, their industrial applications still face two main
challenges. One is that most important enzymes are delicate
and struggle to achieve continuous catalysis as well as long-
Received: January 29, 2022
Revised: March 3, 2022
Published: March 10, 2022

© 2022 American Chemical Society https://doi.org/10.1021/acscatal.2c00522

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Scheme 1. General Design of Our Worka
a
The whole work is composed of three parts: (1) design of short de novo enzymatic cascades via retrobiosynthetic analysis, (2) enzyme
immobilization strategy for scalable continuous production of high-value natural product pharmaceuticals, and (3) animal experiments to discover
novel leads with optimized pharmaceutical activity.
term use.12,13 The other challenge is that applying CFB to
industries cannot be implemented directly, as they are
susceptible to the inhibition of substrates14,15 or products16,17
at elevated concentrations, which means that the velocity curve
of the reaction rises to a maximum as the substrate or product
concentration increases and then descends either to zero
(complete inhibition) or a nonzero asymptote (partial
inhibition) due to the predefined active site geometry of
enzymes and the similarity of substrates and products.18
Therefore, the integration and even cooperation of CFB
systems and immobilization biotechnology have a tendency to
improve the stability of enzymes and enable the recycling of
biocatalysts to maximize the catalytic efficiency for the
continuous accumulation of products.19
The current enzyme immobilization techniques mainly
exploit covalent bonds20 and noncovalent bonds21 to
immobilize enzymes. Covalent bond immobilization can
strengthen the link between enzymes and solid carriers but
usually decreases enzyme stability during covalent bond
formation.21 For noncovalent bond immobilization, both
metal−organic frameworks (MOFs)22,23 and hydrogels24
have been used for immobilization of enzymes to minimize
biocatalyst cost contributions, especially the biotechnology of
MOF-entrapped enzymes, which has attracted great interest
for their potential applications. Although the pioneering work
in this field has been widely reported in recent years, there still
exists a significant gap in moving immobilization biotechnol-
ogy from academic studies to industrial applications. This gap
can be largely attributed to the limited immobilization capacity
and the inability to economically produce desired high-quality
biocarriers at an industrially relevant scale.25,26 Thus, current
efforts to immobilize cell-free enzyme systems have mainly
focused on the operation of some durable enzymes (e.g., CAT,
GOx, HRP, etc.) and short enzymatic cascades (generally two
enzymes).27,28 However, natural product biosynthesis path-
ways are normally quite complex and need the cooperation of
multiple enzymes.29,30 Therefore, integration of immobiliza-
tion biotechnology with CFB systems to produce high-value
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pharmaceuticals is relatively rare, let alone industrial-scale
production.
Retrobiosynthesis,31 a promising approach for de novo
pathway design, has underpinned the process for the
production of high value-added chemicals and pharmaceut-
icals. Moreover, retrobiosynthetic analysis,32,33 with the aims of
shrinking the complicated enzymatic cascades to be compatible
with the current capacities of immobilization carriers, is
expected to accelerate the practical applications of immobilized
enzyme cascades in industrial bioproduction.
In this work, we report a cell-free immobilized enzyme
cascade for the scalable continuous production of neuro-
pharmaceutical prodrugs (e.g., L-4-Cl-Kyn and its non-natural
analogue L-4-Br-Kyn) for optimization of pharmaceutical
activities and treatment of major depressive disorder34
(Scheme 1). First, we adopt retrobiosynthetic strategy to
design a shortened two-enzyme biosynthesis pathway to
produce L-halokynurenines with readily available indole
analogues and L-Ser, which avoids using different regioselec-
tive halogenases35 to develop halogen-substituted units and
enriches the Kyn scaffold types with low-cost sources. Also,
this is currently the most efficient and economical way to
synthesize halogenated kynurenine in both biocatalysis36 and
organic synthetic chemistry.37 Then, elegant integration of our
two-step bioreaction with immobilization biotechnology was
achieved to recycle enzymes and maximize their catalytic
efficiency for continuous production purposes. The specially
designed MOF−hydrogel hybrid carrier not only shows
unparalleled advantage in gram-scale preparation but also
possesses relatively stable crystallinity to resist collapse during
the recycling process. As a result, this allows recycling enzymes
for 5 cycles (250 mL product volume per cycle), affording 370
mg of L-4-Cl-Kyn and 365 mg of L-4-Br-Kyn, which is
approximately double the overall productivity of free enzymatic
catalysis in the same enzyme amount frame. With enough L-4-
Cl/Br-Kyn in hand, we finally test their antidepressant effect in
vivo. Also, the animal experimental results show that L-4-Br-
Kyn is also effective for the treatment of depression.
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Figure 1. (A) Design of short de novo enzymatic cascades via retrobiosynthetic analysis. (B) (1) Pf 0A9-catalyzed coupling reaction of 6-Cl-indole
and L-serine, (2) Tar13-catalyzed oxidative reaction of L-6-Cl-Trp, (3) Tar13-catalyzed oxidative reaction after treatment with TFA, (4) Pf 0A9/
Tar13-catalyzed tandem reaction initiating with 6-Cl-indole and L-serine, and (5) Pf 0A9/Tar13-catalyzed tandem reaction after treatment with
TFA. m/z [M + H]+ 271.0480 corresponds to N-formyl-L-4-Cl-Kyn, and m/z [M + H]+ 243.0526 corresponds to L-4-Cl-Kyn. Note: due to the
influence of TFA introduced during post-treatment, the peak positions of N-formyl-L-4-Cl-Kyn and L-4-Cl-Kyn were reversed.

■ RESULTS AND DISCUSSION

Design of Short De Novo Enzymatic Cascades via
Retrobiosynthetic Analysis. To shorten the enzymatic
cascade for the production of L-4-Cl-Kyn, we first adopted
retrobiosynthetic analysis to design a novel and shortened
biosynthesis pathway requiring only two enzymes, as illustrated
in Figure 1. Instead of using tryptophan (Trp) as the starting
substrate in the previously reported pathway,36 our route
intended to use haloindoles and L-serine as original sources to
form L-halotryptophan, followed by enzymatic ring opening
and chemical hydrolysis to obtain L-halokynurenines (Figure
1A). In this design, halogen-substituted components are
derived from haloindole substrates, which are easily obtained
at a low cost. Thus, this de novo route avoids using halogenase
(e.g., Tar14), which requires two extra coupling reaction steps
and NADPH as redox input in the enzymatic coupling
reaction.36 Moreover, the final hydrolysis step could be
completed during the purification process with acid treatment,
as the ring-opening product N-formyl-L-4-Cl-Kyn could be
chemically converted to L-4-Cl-Kyn under acidic conditions.38
Therefore, the retrobiosynthesis of L-halokynurenines cuts
down its original biosynthetic pathway to two enzymatic steps.
More importantly, since the chemical synthesis of L-
halokynurenine analogues is challenging,37 this methodology
has the potential to provide direct access to its structural
analogues for the discovery of potent drug candidates.
To validate our retrobiosynthetic design, we searched the
protein databases for Trp synthases to assemble Trp analogues
from L-serine and the corresponding indole analogue with
retention of enantiopurity. Several engineered Trp synthases
from Pyrococcus furiosus have been reported to perform the
required conversion, including Pf 2B9, Pf5G8, Pf 0A9, etc.39
Since Pf 0A9 provides a relatively broader spectrum of Trp
analogues and a high conversion rate, we decided to further
investigate the activity of Pf 0A9. The Pf 0A9 enzyme was
readily overexpressed in E. coli and purified by Ni-NTA affinity
chromatography (Figure S1). Incubation of Pf 0A9 with
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substrates 6-Cl-indole (0.25 mM) and L-serine (0.275 mM)
produced L-6-Cl-Trp at pH 8.0 and 30 °C (Figure 1B), which
was characterized by NMR spectroscopy and mass spectrom-
etry after isolation (Table S1, Figures S2, and S3). At 30 °C,
the kinetic parameters of Pf 0A9 were measured by monitoring
the formation of the product (L-6-Cl-Trp) using HPLC at 280
nm (Table S2, Figure S4). The apparent kinetic parameters Km
and kcat toward 6-Cl-indole were 53.89 ± 4.13 μM and 0.0468
± 0.0008 s−1 (kcat,app/Km,app ∼ 8.68 × 10−4 s−1·μM−1), and the
corresponding values for L-serine were 2.98 × 103 ± 152.00
μM and 3.804 ± 0.050 s−1 (kcat,app/Km,app ∼ 1.28 × 10−3
s−1·μM−1). Furthermore, Pf 0A9 showed broad substrate
tolerance and produced various L-Trp analogues in moderate
to excellent yields, as reported in Table S3. Therefore, Pf 0A9
was selected as the Trp analogue synthase in our designed
cascade for further study.
In the second step, Trp analogues need to be transformed
into corresponding N-formyl-L-Kyn analogues. Tar13 enzyme
from the marine bacterium Saccharomonospora sp. CNQ-490
can catalyze this ring-opening reaction,36 and it is the only
tryptophan 2,3-dioxygenase selective for halogenated sub-
strates up to date. Tar13 provided excellent performance
toward L-6-Cl-Trp (>90% conversion determined by HPLC)
under conditions compatible with Pf 0A9, i.e., pH 8.0 and 30
°C (Figure 1B), which was confirmed by NMR structural
elucidation and mass spectrometry (Figure S6). The Km and
kcat for Tar13 toward L-6-Cl-Trp were respectively 124.30 ±
10.68 μM and 0.031 ± 0.001 s−1 (kcat,app/Km,app ∼ 2.49 × 10−4
s−1·μM−1) (Table S4, Figure S7), which were obtained by
monitoring the formation of the product (L-4-Cl-Kyn) with
HPLC at 280 nm. Since N-formyl-L-Kyn analogues can be
converted into L-halokynurenines under acidic conditions
during the purification process, the use of kynurenine
formamidase in the reported pathway can be eliminated, and
a two-step one-pot enzymatic cascade was then established
using Pf 0A9 and Tar13. To completely obtain the final
product L-4-Cl-Kyn, we explored the acid ratio during the acid
treatment for the chemical hydrolysis of N-formyl-L-4-Cl-Kyn.
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Figure 2. (A) Synthesis of ZHHM for the immobilization of Pf 0A9/Tar13 enzyme cascade. (B) (1) Pf 0A9/Tar13@MS-hydrogel-catalyzed
tandem reaction after treatment with TFA (boxed signals correspond to the background absorption of MS-hydrogel) and (2) Pf 0A9/Tar13@
ZHHM-catalyzed tandem reaction after treatment with TFA (boxed signals correspond to the background absorption of ZHHM). (C) SEM
images of (1) Pf 0A9/Tar13@MS-hydrogel and (2) Pf 0A9/Tar13@ZHHM. (D) PXRD patterns of Pf 0A9/Tar13@MS-hydrogel, ZHHM, and
Pf 0A9/Tar13@ZHHM before and after the reaction. (E) (a−d) Laser scanning confocal microscopy images of the two-enzyme-loaded ZHHM.
(F) Comparison of reaction rates of tandem reactions catalyzed by free enzyme and Pf 0A9/Tar13@ZHHM. (G) Diagram showing that the
formation rate of product (L-4-Cl-Kyn) varies with the concentration of substrate (6-Cl-indole). (H) HPLC yields of L-4-Cl/Br-Kyn in catalyst
reuse experiments (1 mL scale).

Trifluoroacetic acid (TFA) was found to be efficient for acid
treatment, and 10 equiv of TFA was required for complete
hydrolysis to provide the final product, which was confirmed
by mass spectrometry (Table S5). In summary, the initial
cascade reaction was developed using 6-Cl-indole (0.25 mM)
and L-serine (0.275 mM) as substrates, giving L-4-Cl-Kyn in
>90% conversion as determined by analytical HPLC (Figure
1B).
Considering that substrate promiscuity was observed with
both Pf 0A9 and Tar13, we further investigated the cascade
tolerance toward different indole analogues. Interestingly, 6-Br-
indole (0.25 mM) was readily accepted, with >90% conversion
yield to give the corresponding L-4-Br-Kyn (Figure S8).
However, no significant conversions were observed for other
substituted positions of halogenated indoles. This substrate
scope may be limited by the selectivity of Tar13.36 Overall, the
above reactions demonstrate that our designed retrobiosyn-
thetic cascade provides a novel and two-step route to produce
L-4-halokynurenine, establishing a short enzymatic cascade
foundation for the following continuous cell-free production.
Enzyme Immobilization Strategy for Scalable Con-
tinuous Production of High-Value Natural Product
Pharmaceuticals. Considering the practical circular produc-
tion requirements, developing a simple, green, low-cost, and
large-scale preparation method for the enzyme cascade
immobilization is highly desirable. For this purpose, we
selected hydrogels based on their properties like easy scale-
up, biocompatibility, biodegradability, and multifunctionality.
A hydrogel in high concentration (Figures S10 and S11)
consisting of salicylic acid and melamine (MS-hydrogel) was
first selected as the solid carrier for enzyme immobilization out
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of several materials due to its biocompatibility, hydrophilic
nature able to preserve protein bioactivity upon immobiliza-
tion, and easy gram-scale preparation. The catalytic activity of
immobilized enzymes was first evaluated by immobilizing the
two enzymes separately. Pf 0A9 and Tar13 were then incubated
with MS-hydrogel, respectively, in pH 8.0 buffer at 4 °C for 20
h (Figure 2A). In addition, the corresponding Pf 0A9@MS-
hydrogel and Tar13@MS-hydrogel were recovered by
centrifugation and used directly without further treatment.
After confirming that the yield of the single immobilized
reaction was almost equivalent to that of free enzyme, we next
tested the reactive activity of the enzyme cascade immobilized
on MS-hydrogel. Expectedly, L-4-Cl-Kyn was obtained in
∼66% HPLC yield (Table S6, Figure 2B). To obtain the
optimum composition for hydrogel preparation, we also tested
other combinations of different compounds capable of forming
hydrogen bonds (VC + melamine and trimesic acid +
melamine) using the heating−cooling method. The activity
of each resulting Pf 0A9/Tar13@hydrogel was assessed using
6-Cl-indole and L-serine as substrates to synthesize L-4-Cl-
Kyn. The product formation monitored by HPLC at 280 nm
(Table S6, Figure S12) indicated that the two-component
hydrogel obtained from melamine and salicylic acid provided
superior activity as the solid carrier for enzyme immobilization.
Therefore, the MS-hydrogel was utilized for immobilization
purposes in the following tests. However, further inspection of
MS-hydrogel and Pf 0A9/Tar13@MS-hydrogel using scanning
electron microscopy (SEM) exhibited a flaky structure before
and after enzyme immobilization (Figures 2C and S13),
implying that it was not sturdy enough for practical continuous
production. Even though the SEM image of recycled Pf 0A9/
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Tar13@MS-hydrogel showed a sticky amorphous state (Figure
S13), it crashed after two reaction cycles (Figure S14), which
was not satisfactory for the recycling demands. To resolve the
stiffness issue of MS-hydrogel, we turned to MOFs, which are
porous materials with excellent crystallinity and are able to
support the MS-hydrogel and increase its rigidity.40 Among
MOFs, ZIF-8 (consists of Zn2+ ions and 2-methylimidazole)
has been found biocompatible and capable of protein
immobilization.41 Thus, we combined MS-hydrogel with
ZIF-8 to form a hybrid material (Figure S15) with increased
crystallinity and to protect MS-hydrogel from collapsing during
the recycling process.
Figures S13 and S16 show the SEM images of MS-hydrogel
and ZIF-hydrogel hybrid material (ZHHM), where the
monodispersed crystalline structure of ZHHM is evident
with an average diameter of 10−15 μm. The powder X-ray
diffraction (PXRD) characterizations of ZHHM also indicated
a more crystalline pattern than the original hydrogel (Figure
S17). The enzymatic cascade of Pf 0A9/Tar13 can be
conveniently and efficiently immobilized with ZHHM after
incubation in the mixed buffer at 4 °C for 20 h. Also, SDS-
PAGE of the supernatant and Pf 0A9/Tar13@ZHHM
exhibited that most of the proteins were immobilized on
ZHHM (Figure S24). The SEM images and PXRD patterns
(Figure 2C,D) of ZHHM after Pf 0A9/Tar13 immobilization
confirm that the crystallinity and morphology of ZHHM are
retained. The confinement of the two enzymes on ZHHM was
confirmed by fluorescence confocal microscopy imaging with
fluorescein isothiocyanate (FITC)-labeled Pf 0A9 and rhod-
amine B-labeled Tar13 incorporated on ZHHM. Figure 2E
shows a bright-field image of Pf 0A9/Tar13@ZHHM (a), a
merged fluorescent image of the two-enzyme-loaded ZHHM
(b), and individual fluorescent images of the two labeled
enzymes (c and d). Excitation of Pf 0A9/Tar13@ZHHM at
488 nm yielded green fluorescence of FITC-labeled Pf 0A9 (c),
while excitation at 561 nm afforded red fluorescence of
rhodamine B-functionalized Tar13 (d). The merged image (b)
indicates that the two enzymes were confined on the ZHHM
carriers.
The effect of immobilization on enzyme cascade activity was
explored by monitoring the catalytic activity of the resulting
Pf 0A9/Tar13@ZHHM using HPLC at 280 nm (Table S7).
The blue curve in Figure 2F shows the time-dependent
formation of L-4-Cl-Kyn by the Pf 0A9/Tar13 biocatalytic
system immobilized on ZHHM nanoreactors. With the
progress of the reaction, the formation of the product
intensified and reached a saturation value around 6 h. The
red curve depicts the time-dependent formation of L-4-Cl-Kyn
by a homogeneous mixture with equal concentrations of two
enzymes. The initial slower activity of Pf 0A9/Tar13@ZHHM
indicates that substrates may take time to diffuse and access the
enzyme loaded on ZHHM carriers. To validate which enzyme
in the cascade was more affected, we next carried out the
kinetic experiments of immobilized enzymes by measuring the
formation of the product using HPLC at 280 nm. The kinetic
parameters Km and kcat for Pf 0A9@ZHHM towards 6-Cl-
indole were 27.97 ± 1.83 μM and 0.0067 ± 0.0001 s−1
(kcat,app/Km,app ∼ 2.40 × 10−4 s−1·μM−1), and the correspond-
ing values for L-serine were 1.04 × 103 ± 66.03 μM and 0.081
± 0.001 s−1 (kcat,app/Km,app ∼ 7.79 × 10−5 s−1·μM−1) (Table
S10, Figure S18). The Km and kcat for Tar13@ZHHM towards
L-6-Cl-Trp were 50.44 ± 4.27 μM and 0.0102 ± 0.0003 s−1
(kcat,app/Km,app ∼ 2.0 × 10−4 s−1·μM−1) (Table S11, Figure
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S19). Thus, the reduced reaction rate of the immobilized
cascade is mainly due to the slowed catalytic rate of Pf 0A9@
ZHHM, which is 2 orders of magnitude lower than free
enzyme in kcat/Km value of L-serine. The optimum operational
conditions for immobilized enzymes were also evaluated by
testing their residual activity compared with free enzymes in
solution after incubation at varied conditions, including
temperature (25, 30, 37, 40, and 45 °C) and pH (6.0, 7.0,
8.0, and 9.0) (Tables S12−S15, Figures S20 and S21). The
immobilized enzymes gave the highest conversion at 30 °C and
pH 8.0. These results confirmed that Pf 0A9/Tar13@ZHHM
maintained high conversion under the optimum conditions
since the ZHHM support provided a favorable environment to
preserve protein folding and thus retained the enzyme activity.
To maximize the catalytic capacity of the enzyme and
demonstrate the necessity to recycle the enzyme cascade for
continuous CFB, we further explored the catalytic limit of the
two enzymes separately. With the elevated concentration of
substrates under the homogenous conditions, the formation
rate of the product reached a plateau after adding 19 mM 6-Cl-
indole for Pf 0A9 and 12 mM L-6-Cl-Trp for Tar13 (Tables
S17 and S18, Figures S25 and S26). Considering that our
ultimate purpose was to design a two-enzyme cascade reaction,
we next explored the catalytic limit of the Pf 0A9/Tar13-
catalyzed cascade reaction. According to the experimental
results of one-step reaction presented above, the catalytic limit
of the cascade reaction is at least 12 mM 6-Cl-indole. However,
unexpectedly, only 4 mM 6-Cl-indole can be completely
transformed in this cascade (Table S19, Figure 2G). When the
substrate concentration was more than 4 mM, the formation
rate of L-4-Cl-Kyn decreased with increasing concentration of
6-Cl-indole. To find out which enzyme in the cascade was
inhibited, an empirical study was conducted. With the
increased substrate (6-Cl-indole) concentration, the amount
of dimethyl sulfoxide (DMSO) that dissolved organic 6-Cl-
indole also increased. Although 15% of DMSO had no effect
on Pf 0A9 (DMSO accounts for 25% in it own catalytic limit
test), it could reduce the bioactivity of Tar13 by half (Table
S20, Figure S27). When the reactions catalyzed by Tar13
reacted under the same DMSO level (15%), the formation rate
of product (L-4-Cl-Kyn) decreased with increasing concen-
tration of 6-Cl-indole (Table S21, Figure S28). In summary,
the inhibition of the enzymatic activity was mainly caused by
the increased concentrations of DMSO and substrate (6-Cl-
indole) in the cascade reaction. Although it is impossible to
eliminate the influence of DMSO, the substrate inhibition
issue, which limits the formation of the product to 4 mM under
homogeneous conditions, can be resolved by employing the
Pf 0A9/Tar13@ZHHM biocomposite for maximizing the
catalytic efficiency of enzymes through repetitive use to
cumulate products. More specifically, for free enzymatic
catalysis, because the proteins are completely soluble in buffer
and cannot be recovered, the maximum conversion of 6-Cl-
indole will not exceed 4 mM. However, since the
heterogeneous Pf 0A9/Tar13@ZHHM biocomposite can be
recovered by centrifugation and then recycled for multibatch
catalysis, the maximum conversion of 6-Cl-indole will be
higher than 4 mM. This again emphasized the importance and
necessity of recycling biocatalysts for continuous catalysis to
maximize their catalytic power.
The reusability of immobilized enzymes was first tested by
repeatedly catalyzing the tandem reaction of the 1 mL solution
using 4 mM 6-Cl/Br-indole and 4.4 mM L-serine as substrates
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Figure 3. (A) 250 mL scaled enzyme cascade system immobilized on ZHHMs. (B) HPLC yields of L-4-Cl/Br-Kyn obtained from each catalytic
cycle in catalyst reuse experiments (250 mL scale). (C) Comparison of product (L-4-Cl/Br-Kyn) yields of the 250 mL scaled cascade system
catalyzed by free enzyme and Pf 0A9/Tar13@ZHHM.
(Tables S22 and S23, Figure 2H). In the first feeding, indole
analogues were almost completely converted into L-4-Cl/Br-
Kyn in 24 h. Although the productivity was halved in the
second reaction cycle due to partially inactivated enzymes, the
yield of the immobilized biocascade increased to nearly double
that of the free enzymatic catalysis after five reaction cycles,
demonstrating that the Pf 0A9/Tar13@ZHHM biocomposite
could be efficiently recovered and reused. Interestingly, when
the concentration of 6-Cl-indole was reduced to 0.25 mM, the
immobilized biocatalysts still maintained close to 50%
productivity after five reaction cycles (Table S16, Figure
S22). This suggested that the excessive use of the biocatalysts
was the main reason for enzyme inactivation at the catalytic
limit, while the prolonged reaction time was the main cause of
enzyme deactivation under low substrate concentrations. In
addition, the stability of the ZHHM shell after the reaction was
also explored, as indicated by the SEM and PXRD character-
izations (Figures S16 and S17) as well as the images of
recycled biocatalysts (Figure S23). The characterization results
revealed that ZHHM has structural stability compared to the
crashed structure of hydrogel after several recycling steps. All
of the above-mentioned results displayed the feasibility of
extending the catalytic potential and enhancing the stability of
the Pf 0A9/Tar13 cascade by ZHHM as a biocarrier.
Furthermore, the practical utility of our study was shown by
establishing the scale-up biocatalysis. To fully exhibit the
advantages of immobilized enzyme catalysis, we also
conducted the enlargement reaction catalyzed by free enzyme
as a comparison. Incubation of Pf 0A9/Tar13 with substrates
6-Cl/Br-indole (4 mM) and L-serine (4.4 mM) in 250 mL
scale produced L-4-Cl-Kyn and L-4-Br-Kyn in 190 mg and 185
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mg, respectively (Figure 3C). Meanwhile, the immobilized
biocatalyst Pf 0A9/Tar13@ZHHM could be utilized for five
continuous cycles (250 mL product volume per cycle),
accumulating 370 mg of L-4-Cl-Kyn and 365 mg of L-4-Br-
Kyn (Figure 3A,C). The experimental results showed that the
productivity of the immobilized biocatalysis is nearly double
the yield of the free enzymatic cascade in the same enzyme
amount frame. In addition, the trends of L-4-Cl/Br-Kyn yields
obtained from each catalytic cycle in large-scale catalyst reuse
experiments coincided well with the 1 mL reaction system
(Figures 2H and 3B). Overall, the results of large-scale
experiments exhibited that our Pf 0A9/Tar13@ZHHM has
technical and economic advantages for the biosynthesis of L-4-
Cl-Kyn and its analogue. Also, the recyclability of the
biocomposite has been proved beneficial for improving the
efficiency of the enzymatic preparation of L-4-Cl/Br-Kyn and
reducing the production cost.
Animal Experiments to Discover Novel Leads with
Optimized Pharmaceutical Activity. With enough supply
of L-4-Cl/Br-Kyn by our integrated approach, we then
demonstrated the feasibility of our work for animal tests. We
were prompted to test the pharmaceutical effects of L-4-Cl/Br-
Kyn on the depression model, especially of L-4-Br-Kyn, which
has not been investigated before. Chronic unpredictable mild
stress (CUMS) was used to induce depression-like symptoms
(e.g., weight loss, decreased sucrose preference, reduced
norepinephrine (NE) and 5-hydroxytryptamine (5-HT)
indices, helpless-like and anxiogenic-like behaviors in open-
field tests) in mice and was considered to be a suitable model
for antidepressant evaluation.42 Therefore, the depression
model was established with 5 week CUMS mice (Figure
https://doi.org/10.1021/acscatal.2c00522
ACS Catal. 2022, 12, 3767−3775
ACS Catalysis pubs.acs.org/acscatalysis Research Article

Figure 4. (A) Schematic timeline of experimental procedures. (B) Levels of NE in mice sera. (C) Levels of 5-HT in mice sera. All experiments were
represented as mean ± SEM (n = 5). GraphPad Prism 8.0.1 was used as statistical software to analyze data. ***P < 0.001, significantly different
from the control group; ###P < 0.001, significantly different from the model group (one-way ANOVA followed by Tukey’s test). Panel (A) was
made with the support of smart.servier.com.

4A). Compared with the unstressed control mice, the mice
after the 5 week CUMS protocol displayed (i) slight weight
loss (P < 0.05), indicative of anorexia; (ii) obviously reduced
sucrose preference (P < 0.001), indicative of anhedonia; (iii)
significantly decreased NE (P < 0.001) and 5-HT (P < 0.001)
levels; and (iv) relatively less time staying in the central area (P
< 0.05), indicative of an anxiogenic state without affecting the
locomotor activity (total distance, P = 0.18) in an open-field
task (Figure S34). Overall, our depression animal model was
validated by these behavioral and biochemical alterations. After
successful modeling, both L-4-Cl-Kyn and L-4-Br-Kyn were
used to treat our established CUMS mice model with the
current drug fluoxetine hydrochloride as a positive control. For
preliminary evaluation of the antidepressant activity of L-4-Cl/
Br-Kyn, the effects on behavior were first tested in all of the
mice. The mice were subjected to two typical types of
behavioral tests, e.g., sucrose preference test and open-field test
(Figure S35). After 14 days of administration, L-4-Cl-Kyn (50
mg/kg injection group) and L-4-Br-Kyn (25 and 50 mg/kg
injection groups) both enhanced the percentage of sucrose
consumption as compared to the model group. More
specifically, L-4-Br-Kyn-injected rats (P < 0.01) exhibited a
better recovery effect in sucrose preference than L-4-Cl-Kyn-
injected rats (P < 0.05) at 50 mg/kg dose compared to the
model mice. In the open-field test, although the low
concentration treatment group of L-4-Br-Kyn showed an
increased amount of time spent in the central area compared to
the model mice, however, the difference was not statistically
significant between the two groups (P = 0.54). Also, the total
distance, peripheral time, and internal edge time in all
3773
treatment groups were also not significantly different from
those in the CUMS model. Similarly, although the L-4-Br-Kyn
treatment group at 25 mg/kg dose revealed a slight increase in
body weight, there was still no statistically significant difference
when compared to the model mice (P = 0.49). To further
investigate the biochemical effects of L-4-Cl/Br-Kyn on
depression, we next measured the concentrations of NE and
5-HT in mice sera using ELISA Kits (Figure 4B,C). All
treatment groups displayed significantly (P < 0.001) good
performance in increasing the levels of NE and 5-HT
compared with model mice. However, L-4-Cl-Kyn at 50 mg/
kg dose achieved a slightly better recovery effect in NE and 5-
HT levels than L-4-Br-Kyn when compared to the depression
model. Moreover, hematoxylin and eosin (H&E) staining was
used to observe the antidepressant effect of L-4-Cl/Br-Kyn on
the morphological changes of hippocampal neurons (Figure
S36). Compared with the control group, a large number of
neurons in the hippocampus were severely degenerated and
necrosed (at the arrowhead) after the 5 week CUMS protocol.
Antidepressant treatment is helpful to the recovery of injured
nerve functions and promotion of regeneration and plasticity
of neurons. Thus, the neuronal injury and apoptosis were
ameliorated in all treatment groups, especially in the high-dose
injection group of L-4-Br-Kyn, which exhibited normal
morphology and no obvious degeneration as compared to
the control. In summary, multiple comparisons suggested that
L-4-Br-Kyn displayed slightly better antidepressant effects than
L-4-Cl-Kyn, which may be able to free mice from depression
faster according to the behavioral test and the recovery
efficiency of neuronal injury.
https://doi.org/10.1021/acscatal.2c00522
ACS Catal. 2022, 12, 3767−3775
ACS Catalysis
■
pubs.acs.org/acscatalysis
CONCLUSIONS
In conclusion, we presented an immobilized enzyme cascade
for the scalable continuous production of neuropharmaceutical
prodrugs, L-4-Cl-Kyn and its non-natural analogue L-4-Br-Kyn,
for the treatment of the major depressive disorder. We first
design a shortened de novo enzyme cascade to assemble the
Kyn core structure with high efficiency. Also, the biggest
highlight in this part is that it is currently the most efficient and
economical way to synthesize halogenated kynurenine in both
biocatalysis and organic synthetic chemistry. To be specific, the
interdisciplinary background of synthetic biology and organic
chemistry prompts us to enrich Kyn scaffold types with readily
available indole analogues and L-Ser as low-cost sources, which
has not been reported before, to avoid using expensive even
noncommercial halotryptophan as the substrate or the
complicated operations caused by the use of different
regioselective halogenases when utilizing Trp as the original
source. As such, this shortened one-pot two-step enzymatic
cascade is compatible with the current immobilization
capacities of biocarriers. Then, the integration of our CFB
system with immobilization biotechnology was successfully
achieved with facile scale-up MOF−hydrogel hybrid material
to recycle enzymes and maximize their catalytic efficiency for
continuous production purposes. Our specially designed
MOF−hydrogel hybrid material with an unparalleled advant-
age in gram-scale preparation not only well matches to high
value-added enzymes but also allows translating reaction
parameters from the 1 mL scale to multimilliliter scale (250
mL) without any reoptimization of the biosynthesis. As a
result, 370 mg of L-4-Cl-Kyn and 365 mg of L-4-Br-Kyn were
easily accumulated after five reaction cycles, which was nearly
double the yield of free enzymatic catalysis in the same enzyme
amount frame. The yield advantage of this immobilized
cascade indicated that our method was capable of providing
enough supply of L-4-Cl-Kyn and its analogue L-4-Br-Kyn for
CUMS animal testing to facilitate the development of L-4-Br-
Kyn for effective treatment of depression. Moreover, animal
experiments did show that L-4-Br-Kyn displayed a slightly
better antidepressant effect than the phase II drug L-4-Cl-Kyn.
Overall, this study establishes the applicability of synthetic
biology strategy and immobilization biotechnology in synthe-
sizing high value-added pharmaceuticals and narrows the gap
in moving cell-free immobilized enzyme cascade systems from
academic studies to industrial applications. It also provides
novel ways to scale up the production of pharmaceutical
products and afford large amounts of nature pharmaceuticals
to accelerate the discovery and development of drug
candidates. These practical features of our study will
undoubtedly be welcomed by academic and industrial
researchers.
■
ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscatal.2c00522.
General procedures, experimental details, character-
ization data of all reactions, and NMR spectra (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Heng Song − College of Chemistry & Molecular Science,
Wuhan University, Wuhan, Hubei Province 430072, China;
3774
Research Article
orcid.org/0000-0002-0049-5053; Email: hengsong@
whu.edu.cn
Authors
Yunbin Wu − College of Chemistry & Molecular Science,
Wuhan University, Wuhan, Hubei Province 430072, China
Rui Xu − College of Chemistry & Molecular Science, Wuhan
University, Wuhan, Hubei Province 430072, China
Yuxin Feng − College of Chemistry & Molecular Science,
Wuhan University, Wuhan, Hubei Province 430072, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acscatal.2c00522
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The authors thank the National Key Research and Develop-
ment Program of China (2018YFA0903200 to H.S.) and the
National Natural Science Foundation of China (No. 31900033
to H.S.) for the financial support.
■
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