Kinetic Resolution of Nearly Symmetric 3-Cyclohexene-1-carboxylate Esters Using a Bacterial Carboxylesterase Identified by Genome Mining

Article abstract of DOI:10.1021/acs.orglett.1c00714

A new bacterial carboxylesterase (CarEst3) was identified by genome mining and found to efficiently hydrolyze racemic methyl 3-cyclohexene-1-carboxylate (rac-CHCM) with a nearly symmetric structure for the synthesis of (S)-CHCM. CarEst3 displayed a high substrate tolerance and a stable catalytic performance. The enantioselective hydrolysis of 4.0 M (560 g·L-1) rac-CHCM was accomplished, yielding (S)-CHCM with a >99% ee, a substrate to catalyst ratio of 1400 g·g-1, and a space-time yield of 538 g·L-1·d-1.

Full text of DOI:10.1021/acs.orglett.1c00714

pubs.acs.org/OrgLett
Letter
Kinetic Resolution of Nearly Symmetric 3‑Cyclohexene-1-
carboxylate Esters Using a Bacterial Carboxylesterase Identified by
Genome Mining
Zhe Dou, Xuanzao Chen, Satomi Niwayama, Guochao Xu,* and Ye Ni*
Cite This: Org. Lett. 2021, 23, 3043−3047
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sı Supporting Information
ABSTRACT: A new bacterial carboxylesterase (CarEst3) was
identified by genome mining and found to efficiently hydrolyze
racemic methyl 3-cyclohexene-1-carboxylate (rac-CHCM) with a
nearly symmetric structure for the synthesis of (S)-CHCM.
CarEst3 displayed a high substrate tolerance and a stable catalytic
−
1
performance. The enantioselective hydrolysis of 4.0 M (560 g·L )
rac-CHCM was accomplished, yielding (S)-CHCM with a >99%
−
1
ee, a substrate to catalyst ratio of 1400 g·g , and a space-time yield
−
1
−1
of 538 g·L ·d .
he optically active molecule methyl 3-cyclohexene-1-
carboxylate (CHCM) is an important building block for
Scheme 1. (A) Pharmaceutical or Natural Products
Containing a Chiral CHCA Moiety As a Key Building Block
and (B) Enantioselective Resolution by Lipases or Esterases
T
the synthesis of a wide range of pharmaceuticals and high-value
products that confer significant biological activities. For
example, (S)-CHCM has been applied to the synthesis of
1
FK-506, the aglycone of the antitumor drug (+)-phyllantho-
2
3
cin, the sex pheromone components periplanone A and B,
4
5
the toxin pumiliotoxin C, the repellent SS220, the unnatural
amino acid β-homoglutamic acid, carbacyclic-(1 → 3)-glucan
mimetics, and the inhibitor of coagulant factor Xa (fXa),
6
7
which is commercially known as Edoxaban and used for the
treatment of cancer-associated venous thromboembolism due
to its superior oral adsorption and lower bleeding risk8
compared to those of other anticoagulants (Scheme 1A).
(
R)-CHCM has also been applied to the synthesis of the
9
immunosuppressant FK-506 (Tacrulimus), the anti-influenza
drug oseltamivir phosphate (Tamiflu), the natural products
leustroducsin B and phyllanthocin, potential antibacteri-
als, and E-selectin antagonists.
Chiral CHCM can be synthesized through either asym-
10
1
1
12
1
3
14
15a,b
metric Diels−Alder reactions
the corresponding racemic 3-cyclohexene-1-carboxylic acid
rac-CHCA) at the expense of stoichiometric chiral resolution
reagents such as (S)-α-methylbenzylamine and (R)-α-
or the optical resolution of
(
1
5c,16
phenethylamine.
The drawback of these synthesis routes
is that these processes are often cumbersome, requiring
expensive chiral reagents and excessive hazardous organic
solvents. Recently, developing biocatalysts for the preparation
of chiral building blocks has drawn special attention for “green”
enantioselective chemical conversions. Biocatalytic approaches
are generally acceptable as highly enantioselective and
environmentally benign alternatives, but few enzymes display
desirable activities and enantioselectivities toward CHCM
Received: March 1, 2021
Published: April 2, 2021
©
2021 American Chemical Society
https://doi.org/10.1021/acs.orglett.1c00714
3
043
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Organic Letters
pubs.acs.org/OrgLett
Letter
Table 1. Genome Mining of Carboxylesterases for the Enantioselective Resolution of rac-CHCM
a
a
b
c
c
d
ΔG
ΔG
ΔΔG
act. to pNPS
act. to CHCM
ee /config. (%/R
E
value
R
S
p
−1
−1
e
enzyme
accession no.
(kcal·mol−1)
(kcal·mol−1)
(kcal·mol−1)
(U·mg
)
(U·mg )
or S)
CarEst1
CarEst2
CarEst3
CarEst4
CarEst5
CarEst6
CarEst7
CarEst8
CarEst9
CarEst10
ANF82944
CAG70279
AXY61874
AMW77467
AWL30387
OUY06386
ENV84364
KEZ78936
BAB65028
AOA56957
−19.2 ± 1.1
−21.6 ± 0.9
−20.5 ± 1.2
−18.9 ± 0.8
−18.3 ± 0.9
−16.3 ± 0.8
−17.5 ± 1.0
−21.9 ± 1.1
−19.7 ± 0.5
−22.3 ± 1.0
−17.0 ± 0.8
−18.9 ± 0.8
−17.6 ± 1.0
−16.3 ± 0.9
−16.5 ± 1.0
−15.4 ± 0.6
−15.8 ± 0.7
−20.8 ± 0.8
−19.1 ± 1.1
−19.8 ± 1.3
−2.2**
−2.7**
−2.9**
−2.6**
−1.8**
−0.9**
−1.7**
−1.1*
11.7 ± 0.1
14.0 ± 0.1
12.7 ± 0.1
4.70 ± 0.01
6.10 ± 0.06
3.10 ± 0.08
5.70 ± 0.10
22.5 ± 0.7
11.2 ± 0.8
230 ± 3
367 ± 5
376 ± 5
114 ± 3
317 ± 6
42.5 ± 1.2
405 ± 8
1.5 ± 0.1
2.1 ± 0.1
90.5 ± 0.8
77.2 ± 0.3/S
88.0 ± 0.3/S
90.1 ± 0.4/S
72.0 ± 0./S
72.2 ± 0.4/S
43.1 ± 0.3/S
67.3 ± 0.5/S
13.5 ± 0.8/S
17.2 ± 0.6/R
83.0 ± 0.4/S
13.1
17.6
22.8
7.1
7.1
2.7
6.7
1.4
0.7
13.8
−0.6
−2.5**
26.8 ± 1.0
a
b
ΔG , binding free energy to (R)−CHCM; ΔG , binding free energy to (S)-CHCM. ΔΔG = ΔG − ΔG . Student’s t test was performed with p <
R
S
R
S
c
0
.1 (denoted as *) and p < 0.05 (denoted as **). One unit (U) of activity was defined as the amount of enzyme required to catalyze the hydrolysis
d
of 1 μmol CHCM per minute at 30°C; act., specific activity toward either p-nitrophenyl 3-cyclohexene-1-carboxylate or rac-CHCM. ee , ee value
p
e
24
on rac-CHCM; config., configuration. The E value was calculated according to Chen et al.
a
Table 2. Optimization of Biocatalytic Conditions for the Synthesis of (S)-CHCM
substrate
(mol·L−1
biocatalyst
(kU·L−1
S/C
(g·g−1
temperature
time
(h)
conversion
(%)
ees
space-time yield
(g·L−1·d−1
)
entry
(g·L−1)
)
(g·L−1
)
)
)
(°C)
(%)
1
2
3
4
5
6
7
140
140
140
280
420
560
700
1.0
1.0
1.0
2.0
3.0
4.0
5.0
0.08
0.08
0.08
0.08
0.2
4
4
4
1750
1750
1750
3500
2100
1400
500
30
20
10
30
30
30
30
3.3
6.0
6.0
8.0
10.0
9.0
63.3
60.3
58.8
64.2
64.3
64.3
64.3
99.4
99.0
98.7
99.7
99.4
99.3
99.6
275
222
231
300
363
538
500
4
10
20
70
0.4
1.4
12.0
aReactions were performed in 100 mL of PBS buffer (100 mM, pH 8.0) with 1.0−5.0 M rac-CHCM and 0.08−1.4 g·L−1 lyophilized cell powder.
because of its nearly symmetric cyclohexene structure (Scheme
B).
Commercial pig liver esterase (PLE) and horse liver esterase
HLE) have been reported to catalyze the enantioselective
designated as AcEst1 exhibited the highest enantioselectivi-
20b
1
ty.
Genome mining is a relatively new, inexpensive, and
readily available bioinformatic tool that can aid in the
(
identification of new enzymes and natural products based on
−
1
21
hydrolysis of racemic CHCM (rac-CHCM). About 10 g·L
the homologies of preidentified genomic information.
rac-CHCM could be converted to (S)-CHCA by either 2 mL·
Herein, we performed genome mining to obtain robust
carboxylesterases (CarEsts) and established an efficient
−
1
22
L
PLE at a 43% isolation yield and a >99% enantiomeric
−
1
excess (ee) or 0.2 g·L HLE at a 41% isolation yield and a 97%
biocatalytic process for the synthesis of chiral (S)-CHCM.
Ten putative carboxylesterases originating from different
microorganisms, including Acinetobacter, Serratia, Salinisphaera,
and Sulfolobus species (Table S1), were identified by BLASTp
searches to have a 35−70% sequence identity of AcEst1 and
were ligated into pET28a with an N-terminal His-tag and
heterologously expressed in E. coli BL21(DE3) (Figure S1).
The binding free energies (ΔG) of CarEst1−10 toward (R)-
CHCM (ΔG ) and (S)-CHCM (ΔG ) were calculated using
1
7
ee. However, commercial enzymes are generally expensive,
and the reproducibility of their outcomes depends on their
commercial availability. Recently, the carboxylesterase BioH,
which is involved in biotin synthesis in Escherichia coli, was
reported to hydrolyze rac-CHCM; however, it had an ee of
18
only 32.3% and an enantioselectivity value (E value) of 2.1.
Through the combinatorial modulation of steric and aromatic
interactions, the best-performing variant of BioH, Mu3 (L24A/
W81A/L209A), reached a 70.9% ee and an E value of 7.1 at 40
mM rac-CHCM. However, high substrate loads resulted in a
decreased enantioselectivity. Therefore, the challenges of a low
substrate tolerance, a low enantioselectivity, and a low
volumetric productivity together limit the industrial biosyn-
thesis of enantiomerically enriched CHCA. Moreover, as most
of the commercial esterases or lipases are from yeast or animal
sources, the identification of bacterial esterases is of special
interest due to the low cost of feasible and scalable prokaryotic
R
S
the molecular mechanics Poisson−Boltzmann surface area
23
(MM-PBSA) method. The preference for the generation of
(R)- or (S)-CHCM can be indicated by the difference between
the ΔG and ΔG values (ΔΔG = ΔG −ΔG ). As shown in
R
S
R
S
Table 1, these recombinant carboxylesterases displayed
variable ΔG values toward (R)- or (S)-CHCM. The lowest
−
1
ΔΔG was found in CarEst3, with a ΔΔG of −2.9 kcal·mol .
Further screening was carried out to evaluate the ability of
these CarEsts to generate enantioselective products from rac-
CHCM. All of them showed hydrolytic activity toward 3-
cyclohexene-1-carboxylate esters (Table 1 and Figure S2).
Recombinant CarEst10 exhibited the highest specific activity of
19
expression systems.
In previous work, we have isolated the esterase-producing
strain Acinetobacter sp. JNU9335 with the ability to
2
0a
−1
enantioselectively hydrolyze rac-CHCM.
Four putative
26.8 U·mg toward p-nitrophenyl 3-cyclohexene-1-carboxy-
esterases were identified from the genome of JNU9335 that
demonstrated activity toward rac-CHCM, and one esterase
late, which was 5.74-fold higher than that of AcEst1. However,
the ee value of CarEst10 was 83.0% lower than the value of
p
3
044
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Letter
Table 3. Kinetic Parameters of Recombinant CarEst3 Toward CHCM
substrate
KM (mM)
Vmax (μmol·(min·mg)−1)
kcat (s−1
)
k /K (s−1·mM−1
)
cat
M
rac-CHCM
12.4 ± 1.4
13.8 ± 2.3
33.0 ± 8.6
1650 ± 120
1502 ± 97
679 ± 99
1100 ± 81
1001 ± 64
452 ± 66
89.0 ± 6.5
72.3 ± 4.6
13.7 ± 2.0
(R)−CHCM
S)−CHCM
(
−
1
8
9.1% from AcEst1. CarEst3 displayed the highest enantiose-
g·L . Within 12.0 h, the ee reached 99.6%, with an S/C of 500
s
−
1
−1
−1 −1
lectivity and specific activity at 90.1% ee and 12.7 U·mg ,
g·g and a space-time yield of 500 g·L ·d . Recombinant
1
p
−
respectively, the latter of which is significantly higher than the
CarEst3 tolerated a load as high as 700 g·L rac-CHCM. For
value of 4.67 U·mg− from AcEst1. The high ee value of
1
feasible biocatalytic manufacturing processing standards, the
p
−
1
CarEst3 was consistent with its low ΔΔG value, demonstrating
the utility of binding affinity analyses when mining
enantioselective biocatalysts from genomic databases. Consid-
ering that it had the highest enantioselectivity and a relatively
high activity, CarEst3 was selected as the best-identified
biocatalyst for the enantioselective resolution of rac-CHCM
for the synthesis of (S)-CHCM.
To establish an efficient biocatalytic process, reaction
parameters, including temperature, substrate load, and
biocatalyst dosage, were assessed. Initial experiments were
carried out in 100 mL of PBS buffer (100 mM, pH 8.0) with
essential requirements of a ≥100 g·L substrate load, a ≤5 g·
−
1
−1
L
biocatalyst dosage, a ≥20 g·g S/C, a ≤24 h reaction
−
1
−1
time, a ≥99% ee, and a ≥100 g·L ·d space-time yield should
be satisfied. This recombinant CarEst3 exhibits a great
potential for industrial applications.
To explore the intrinsic properties of recombinant CarEst3,
it was purified by nickel-affinity chromatography. The purified
CarEst3 migrated as a single band at 43 kDa according to the
SDS-PAGE analysis (Figure S5). The specific activity of the
purified CarEst3 was 74.3 U·mg , representing a 5.85-fold
increase compared to a value of 12.7 U·mg for the crude
extract. The apparent kinetic parameters of purified CarEst3
25
−
1
−
1
−1
0
.1 mol (140 g·L ) substrate rac-CHCM and 480 U (0.08 g·
1
−
L ) of CarEst3 derived from lyophilized cells of E. coli BL21
at 30°C (Table 2, entry 1). The reaction pH was maintained at
toward CHCM were analyzed (Table S2). The V and kcat
values were calculated to be 1649 μmol·min ·mg and 1100
s , respectively, providing evidence for its prominent
max
−
1
−1
−
1
8
.0 by titrating 2.0 M Na CO . After merely 3.3 h, a 99.4% ee
2
3
s
was achieved at a conversion ratio of 63.3%, with a substrate to
performance, especially at high substrate loads. The K value
M
−
1
biocatalyst ratio (S/C) of 1750 g·g and a space-time yield of
of CarEst3 was 12.4 mM, and the k /K value was calculated
cat
M
−
1
−1
−1
−1
2
75 g·L ·d . Furthermore, lower temperatures, including 20
to be 89.0 s ·mM . The kinetic parameters of CarEst3
toward (R)- and (S)-CHCM were also determined to
understand its enantioselectivity (Table 3). The K and V
and 10°C, were tested (Table 2, entries 2 and 3; Figure S3)
since they might be favorable for the enzymatic enantiose-
lective reaction. However, the temperature had little influence
on the enantioselectivity of recombinant CarEst3. Longer
M
max
values toward (R)-CHCM were 13.8 mM and 1502 μmol·
−
1
−1
min ·mg , respectively, while values of 33.0 mM and 679
−
1
−1
reaction times were required to achieve ee values similar to
μmol·min ·mg were determined for (S)-CHCM, indicating
CarEst3 displays a higher affinity and catalytic efficiency
s
those at 30°C because of the decreased activity at lower
temperatures. Therefore, lower space-time yields were
achieved at 20 and 10°C compared to those at 30°C.
Based on the high performance of CarEst3 at 1.0 M, the
−
1
toward (R)-CHCM. As a result, the k /K value of 72.3 s ·
cat
M
−
1
mM toward (R)-CHCM was much higher than that of 13.7
−
1
−1
s ·mM for (S)-CHCM, agreeing with the (R)-selectivity of
CarEst3.
−
1
substrate load was increased to 2.0 M (280 g·L ) while the
other conditions were kept consistent with those in entry 1 of
Substrate profiling of the purified CarEst3 was also done by
determining its activity toward 3-cyclohexene-1-carboxylate
esters with different alkoxy groups, such as methoxy (CHCM),
ethoxy (CHCE), isopropoxy (CHCP), and butoxy (CHCB)
groups. The specific activities toward CHCM, CHCE, CHCP,
Table 2. The ee value reached 99.7% at 8.0 h. The S/C was
s
−
1
determined to be as high as 3500 g·g , and the space-time
yield was 300 g·L ·d . This newly identified CarEst3 is thus
highly efficient in the enantioselective hydrolysis of rac-
CHCM. A high S/C (>100 g·g ) value is especially important
for industrial applications due to the lower emulsion, better
−
1
−1
−
1
−1
and CHCB were 376, 388, 159, and 182 U·mg , respectively
(Figures 1 and S6). Recombinant CarEst3 displayed a higher
25
mass transfer, and lower downstream costs. Furthermore, to
explore the substrate or product tolerance of recombinant
CarEst3, the substrate loads were increased to 3.0−5.0 M
−
1
(
Table 2, entries 5−7; Figure S4). Using merely 0.2 g·L
−1
recombinant CarEst3, 3.0 M (420 g·L ) rac-CHCM was
enantioselectively hydrolyzed, yielding (S)-CHCM with an ee
s
−
1
of 99.4%, an S/C of 2100 g·g , and a space-time yield of 363
−
1
−1
g·L ·d by 10.0 h. When the substrate load was further
increased to 4.0 M, the biocatalyst dosage was elevated to
complete the reaction within 12 h. Surprisingly, 4.0 M rac-
−
1
CHCM (560 g·L ) could be easily hydrolyzed, producing
−
1
(
S)-CHCM with an ee of 99.3%, an S/C of 1400 g·g , and a
s
1
−
−1
space-time yield of 538 g·L ·d within 9.0 h. Although a
lower S/C should be sacrificed to achieve a higher space-time
yield, the S/C of >100 g·g− is permissible for scale-up
synthesis. As a result, the substrate load was further increased
1
Figure 1. Substrate profiles of recombinant CarEst3 toward various
cyclohexene and piperidine carboxylate esters.
−
1
to 5.0 M rac-CHCM (700 g·L ) at a biocatalyst dosage of 1.4
3
045
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pubs.acs.org/OrgLett
Letter
activity toward esters with short chain lengths, such as CHCM
and CHCE. Interestingly, CarEst3 also exhibited a higher
enantioselectivity toward CHCM and CHCE, with ee values
p
of 90.1% and 81.2%, respectively, that are much higher than
those of 68.1% and 33.5% for CHCP and CHCB, respectively.
The increased chain length of the alcohol group might further
decrease the difference between the cyclohexyl group and the
alcohol group in regard to the steric hindrance and
hydrophobicity, which lead to a poor enantioselective
recognition of substrates. N-Heterocycle piperidine carboxylate
esters were also investigated, including N-methylpiperidine-2-
carboxylate methyl ester (MP2CM), N-methylpiperidine-3-
carboxylate methyl ester (MP3CM), and N-methylpiperidine-
4
-carboxylate methyl ester (MP4CM). CarEst3 could also
catalyze the hydrolysis of piperidine carboxylate esters and
displayed the highest specific activity toward MP2CM (167
−
1
−1
mU·mg ), followed by MP3CM (142 mU·mg ) and finally
−
1
MP4CM (127 mU·mg ) (Figures 1 and S7). CarEst3
demonstrated an ee of 64.2% toward MP2CM, while it almost
p
lost the enantioselectivity toward MP3CM when the ester
group was in the meta-position (Figure 1), indicating the
importance of the positions of the substituents in enantiose-
lective recognition.
3
-Cyclohexene-1-carboxylate esters contain a nearly sym-
metric hexatomic ring, which is difficult for enzymes to
discriminate. Recombinant CarEst3 is an efficient biocatalyst
for the synthesis of chiral 3-cyclohexene-1-carboxylate
derivatives and, to the best of our knowledge, provides the
highest recorded substrate load and enantioselectivity to date.
To elucidate the mechanism underlying the high enantiose-
lectivity for CHCM substrates, the homologous modeling
structure of CarEst3 was constructed, and ligands, either (R)-
or (S)-CHCM, were docked into its active center. Multiple
molecular dynamic simulations (100 ns ×5) were performed.
The ΔG values toward (R)- and (S)-CHCM were −20.5 ± 1.2
Figure 2. Interaction of (A) (R)-CHCM and and (B) (S)-CHCM
with residues in the substrate binding pocket of CarEst3; red stick,
catalytic residues; green stick, oxyanion hole; yellow stick, residues
with interactions; and blue stick, CHCM.
−
1
−1
g·L ·d (Table S3). The variant Mu3 of BioH was previously
constructed to catalyze the kinetic resolution of rac-
1
7,18
CHCM.
However, the substrate loads are relatively low
−
1
−1
(<10 g·L ), and the S/C ratio of 2 g·g and the low space-
time yield are not acceptable for industrial applications. Among
biocatalysts capable of the enantioselective hydrolysis of rac-
CHCM to (S)-CHCM, CarEst3 identified in this study
−
1
and −17.6 ± 1.0 kcal·mol , respectively (Table 1). CarEst3
displayed a lower ΔG value and a higher binding affinity
toward (R)-CHCM, which is consistent with its low K value.
M
−
1
Average snapshots of (R)- and (S)-CHCM were retrieved from
molecular dynamic simulation trajectories. (R)-CHCM was
well-accommodated into the active center by a π−π interaction
between the phenyl ring of F78 and the double bond of the
substrate, a hydrophobic interaction between L249 and the
cyclohexene of the substrate, and multiple interactions
between H139, D200, H325, and the CHCM methyl group
exhibits the highest substrate tolerance (700 g·L ), S/C
1
−
−1 −1
value (3500 g·g ), and space-time yield (538 g·L ·d ),
demonstrating its great potential in industrial manufacturing.
Taken together, the novel bacterial carboxylesterase CarEst3
identified by genome mining enriches the currently available
biocatalytic toolbox for the synthesis of chiral carboxylic acids.
ASSOCIATED CONTENT
sı Supporting Information
(Figure 2A). With respect to (S)-CHCM, the ester chain
■
rotated to the other side to form a reactive conformation due
to steric hindrance (Figure 2B). This conformational switch
likely resulted in the breakage of the π−π interaction between
the substrate and F78, although the methyl group of CHCM
could also be stabilized by L249 and H325. All these changes
contribute to the lower binding affinity of (S)-CHCM to
CarEst3 compared to that of (R)-CHCM. It should be noted
that CarEst3 is the most efficient biocatalyst identified so far
for discriminating nearly symmetric (R)- and (S)-CHCM.
Rational engineering is in progress to further increase its
enantioselectivity.
Commercial enzymes, including PLE, HLE, and porcine
pancreatic lipase (PPL), have been reported with relatively
high enantioselectivities. However, they are not reliably
reproducible because they consist of mixtures of enzymes;
for example, PLE consists of six isoenzymes. Furthermore, the
space-time yields of the commercial enzymes are lower than 20
*
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.1c00714.
General experimental procedures and spectral data
(PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Guochao Xu − The Key Laboratory of Industrial
Biotechnology, School of Biotechnology, Jiangnan University,
Wuxi 214122, P. R. China; orcid.org/0000-0001-9784-
5648; Email: guochaoxu@jiangnan.edu.cn
Ye Ni − The Key Laboratory of Industrial Biotechnology,
School of Biotechnology, Jiangnan University, Wuxi 214122,
P. R. China; orcid.org/0000-0003-4887-7517;
Email: yni@jiangnan.edu.cn
3
046
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Organic Letters
Authors
pubs.acs.org/OrgLett
Letter
(11) (a) Miyashita, K.; Tsunemi, T.; Hosokawa, T.; Ikejiri, M.;
Imanishi, T. Tetrahedron Lett. 2007, 48, 3829−3833. (b) Miyashita,
K.; Tsunemi, T.; Hosokawa, T.; Ikejiri, M.; Imanishi, T. J. Org. Chem.
Zhe Dou − The Key Laboratory of Industrial Biotechnology,
School of Biotechnology, Jiangnan University, Wuxi 214122,
P. R. China; orcid.org/0000-0002-6131-7314
Xuanzao Chen − The Key Laboratory of Industrial
Biotechnology, School of Biotechnology, Jiangnan University,
Wuxi 214122, P. R. China
2
(
008, 73, 5360−5370.
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Satomi Niwayama − Graduate School of Engineering,
Muroran Institute of Technology, Muroran, Hokkaido 050-
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ACKNOWLEDGMENTS
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We are grateful to the National Key Research and Develop-
ment Program (2018YFA0901700 and 2019YFA0906400), the
Postgraduate Research & Practice Innovation Program of
Jiangsu Province (KYCX20_1808), the National Natural
Science Foundation of China (22077054, 22078127, and
1776112), the national first-class discipline program of Light
Industry Technology and Engineering (LITE2018-07), and the
Program of Introducing Talents of Discipline to Universities
2057−2060.
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