Efficient synthesis of an ε-hydroxy ester in a space-time yield of 1580 g L-1 d-1 by a newly identified reductase RhCR

Article abstract of DOI:10.1016/j.tetasy.2014.10.011

A new NADH-dependent carbonyl reductase RhCR capable of efficiently reducing the ε-ketoester ethyl 8-chloro-6-oxooctanoate (ECOO) to give ethyl (S)-8-chloro-6-hydroxyoctanoate [(S)-ECHO], an important chiral precursor for the synthesis of (R)-α-lipoic acid, was identified from Rhodococcus sp. ECU1014. Using recombinant Escherichia coli cells expressing RhCR and glucose dehydrogenase used for the regeneration of cofactor, 440 g L^-1 (2 M) of ECOO were stoichiometrically converted to (S)-ECHO in a space-time yield of 1580 g L^-1 d^-1 without the external addition of any expensive cofactor.

Full text of DOI:10.1016/j.tetasy.2014.10.011

Tetrahedron: Asymmetry 25 (2014) 1501–1504
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Efficient synthesis of an
e-hydroxy ester in a space–time yield of
1580 g LÀ1 dÀ1 by a newly identified reductase RhCR
a
b
a,
Rui-Jie Chen a, Gao-Wei Zheng a, , Yan Ni , Bu-Bing Zeng , Jian-He Xu
⇑
⇑
a Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road,
Shanghai 200237, People’s Republic of China
b School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new NADH-dependent carbonyl reductase RhCR capable of efficiently reducing the e-ketoester ethyl
8-chloro-6-oxooctanoate (ECOO) to give ethyl (S)-8-chloro-6-hydroxyoctanoate [(S)-ECHO], an important
Received 22 March 2014
Revised 9 October 2014
Accepted 10 October 2014
chiral precursor for the synthesis of (R)- -lipoic acid, was identified from Rhodococcus sp. ECU1014. Using
a
recombinant Escherichia coli cells expressing RhCR and glucose dehydrogenase used for the regeneration
of cofactor, 440 g LÀ1 (2 M) of ECOO were stoichiometrically converted to (S)-ECHO in a space–time yield
of 1580 g LÀ1 dÀ1 without the external addition of any expensive cofactor.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
omerically pure hydroxy esters have been reported in the litera-
ture.19–22 However, the development of more efficient and
(R)-
a
-Lipoic acid is a powerful antioxidant and important bio-
practical biocatalysts to synthesize (S)-8-chloro-6-hydroxyocta-
noic acid ester is still highly desirable promising and versatile
reductases enantioselectivity towards or a variety of physiologi-
cally active compounds.
logically active molecule, which is widely used in food supplement
preparations, skin conditioners, and pharmaceuticals for treating
diabetic neuropathy, chronic liver disease, and Alzheimer’s dis-
ease.1–5 As a result, a wide range of methods have been developed
Herein our interests focused on exploring a novel reductase
for the preparation of (R)-a-lipoic acid, including the kinetic resolu-
capable of efficiently reducing the e-ketoester ethyl 8-chloro-6-
tion of the racemate or its intermediates using optically active
oxooctanoate (ECOO) and developing an efficient synthesis of
ECHO with high volumetric productivity.
chemical agents6,7 and lipases,8–10 and asymmetric synthesis using
chiral auxiliaries.11 (R)-
a-Lipoic acid is produced on an industrial
scale via the chemical resolution of racemates synthesized
by chemical processes using the racemic 8-chloro-6-hydroxyocta-
noic acid esters as intermediates.12,13 However at most only 50%
of the racemic intermediates are utilized to obtain enantiomerically
2. Results and discussion
2.1. Enzyme screening
pure
unrewarding.
a-lipoic acid, making this process appear economically
To begin with, several carbonyl reductases newly mined and
stocked in our laboratory were screened. As shown in Table 1, most
reductases exhibited low activity toward ECOO, which is probably
Asymmetric reduction of keto esters using keto reductases or
alcohol dehydrogenases is an ideal process to produce enantiome-
rically pure hydroxy esters due to its inherent advantages, such as
being able to provide up to 100% theoretical yield, excellent stere-
because the substrate is an
accepted by the majority of reductases, versus the readily reduced
-, b-, and -keto esters due to the long side chain of ECOO. Fortu-
e-ketoester, which is hard to be
a
c
oselectivity, superior atom economy, and eco-friendliness.14–18
A
nately, a new reductase RhCR from Rhodococcus sp. ECU1014 can
efficiently covert ECOO into (S)-ECHO with >99% conversion and
good enantioselectivity. After being purified to homogeneity by
single step affinity chromatography, the RhCR showed an extre-
mely high activity in the reduction of ECOO (90.5 U mgÀ1 protein),
for which the specificity constant (kcat/Km) was determined as
few dehydrogenases and microorganism cells that could reduce
8-chloro-6-oxooctanoic acid esters into the corresponding enanti-
⇑
Corresponding authors. Tel.: +86 21 64252498; fax: +86 21 64250840.
E-mail addresses: gaoweizheng@ecust.edu.cn (G.-W. Zheng), jianhexu@ecust.
256 mMÀ1 sÀ1
.
edu.cn (J.-H. Xu).
http://dx.doi.org/10.1016/j.tetasy.2014.10.011
0957-4166/Ó 2014 Elsevier Ltd. All rights reserved.

1502
R.-J. Chen et al. / Tetrahedron: Asymmetry 25 (2014) 1501–1504
Table 1
Table 2
Substrate specificity of RhCR
Screening of reductases using ECOO as a substratea
Entry
Enzyme
Specific activity
(U mgÀ1 protein)
Conversionb (%)
eeb (%)/config
Entry Substrate
Relative activitya (%) eeb (%)
O
O
1
2
3
4
5
6
7
8
RhCR
LbuCR
CgKR1
LbCR
CgKR2
FabG
YtbE
YueD
ScCR
KtCR
40
30
<1
3
99
99
8
93 (S)
79 (S)
79 (R)
11 (R)
n.a.c
n.a.
n.a.
n.a.
n.a.
n.a.
1
0.03
0.2
7.9
OEt
OEt
O
90
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
2
22
O
O
O
O
9
3
1.0
1.9
2.9
39
78
22
10
11
12
13
14
OEt
OEt
DnCR
ClCR
MpCR
ApCR
n.a.
n.a.
n.a.
n.a.
O
4
F3C
O
O
a
Reaction conditions: the reaction mixture consisted of the appropriate amount
5
6
Cl
Cl
of reductase (cell lysate or lyophilized crude enzyme powders), 2 U of GDH from
Bacillus megaterium (lyophilized crude enzyme powders), 10 mM of ECOO, 20 mM
of glucose, 0.5 mM of NAD(P)+ and 100 mM of sodium phosphate buffer (pH 6.0) in
a total volume of 1 mL. The mixtures were incubated at 30 °C for 24 h.
OEt
O
100
93
b
OEt
Conversion and ee were determined by GC.
n.a.: not available.
c
O
a
The activity was determined by standard assay protocol. Activities were nor-
2.2. Dependence of enzyme activity on pH and temperature
malized to that for ECOO (100%).
b
Determined by GC analysis.
The effects of temperature and pH on the reductase activity
were investigated over a temperature range of 25–70 °C (Fig. 1a),
and a pH range of 3.0–10.0 (Fig. 1b). According to Figure 1a, the
enzyme RhCR exhibited maximum activity in the range of 30–
45 °C and then the reaction rate decreased rapidly over 50 °C due
to thermal inactivation. The activity–pH profile showed that the
enzyme activity exhibited a pH optimum at 6.0. Therefore, the
reductase RhCR showed an optimum temperature of 30–45 °C
and an optimum pH of 6.0.
esters (entries 1–3 and 6). Furthermore, both the activity and ste-
reoselectivity increased with the introduction of electron-with-
drawing groups (entries 4–6). The highest activity and
stereoselectivity were obtained during the reduction of ECOO
(entry 6), an
group.
e-ketoester with a strong electron-withdrawing
2.4. Bioconversion of ECOO into (S)-ECHO on an analytical scale
The stability was examined by incubating the purified enzymes
in the optimized buffer (pH 6.0) at 30, 40, and 50 °C for a varied per-
iod of time and the residual activities were determined. The reduc-
tase RhCR gave half-lives of 84, 58, and 3.8 h at 30, 40, and 50 °C,
respectively. Based on the above results, pH 6.0 and 30 °C were cho-
sen as the optimal reaction conditions in the following work.
The newly discovered biocatalyst was utilized for the synthesis
of (S)-ECHO to further demonstrate its potential in industry. It is
well known that the requirement for expensive nicotinamide
cofactors is a major hindrance in the practical application of reduc-
tases.23,24 Therefore, a glucose dehydrogenase (GDH) from Bacillus
megaterium was used for the regeneration of the oxidized cofactor
(NAD+) during the reduction of ECOO catalyzed by recombinant
E. coli cells containing RhCR. Without the external addition of any
expensive cofactor, as much as 132 g LÀ1 of ECOO was completely
converted within 6.5 h (Table 3, entry 1) in the cofactor-regener-
ated catalytic system. Even though the recombinant E. coli cell
loading was decreased to 60 kU LÀ1 (ca. 5.0 g LÀ1, an acceptable
threshold in industry), the substrate was almost completely con-
2.3. Substrate spectrum
The substrate specificity of RhCR for various ketoesters was fur-
ther explored (Table 2). We found that the reduction activity and
stereoselectivity of RhCR was significantly affected by the number
of the insertion group (–CH2–) between the carbonyl and ester
bonds of the substrates, with a tendency of
e- > d- >
c- > b-keto
120
100
80
60
40
20
0
120
B
A
100
80
60
40
20
0
20
30
40
50
60
70
80
2
3
4
5
6
7
8
9
10 11
Temperature (oC)
pH
Figure 1. Effects of temperature and pH on activity of the purified RhCR. (A) Activity–temperature profile was determined at various temperatures (25–70 °C) in a sodium
phosphate buffer (100 mM, pH 6.0). (B) Activity–pH profile was determined using a standard assay in the following buffers (100 mM): ( ) sodium citrate (pH 3.0–6.0); (
)
sodium phosphate (pH 6.0–8.0); ( ) Gly–NaOH (pH 8.5–10.0).

R.-J. Chen et al. / Tetrahedron: Asymmetry 25 (2014) 1501–1504
1503
Novagen (Shanghai, China). 1H NMR and 13C NMR were measured
on a Bruker Avance 400 MHz and 500 MHz spectrometer with
chemical shifts reported as ppm. The substrate conversion and
product ee value were determined by GC analysis using a CP-Chira-
sil-DEX CB (Varian, USA).
Table 3
Asymmetric reduction of ECOO with E. coli cells of RhCR and GDHa
Entry
RhCR
ECOO
(g LÀ1
)
NAD+
(mM)
Time
(h)
eeb
(%)
Conv.b
(%)
(kU LÀ1
)
1
2
3
120
60
240
360
240
132
132
440
660
440
0
0
0
0
0
6.5
9
8
24
5
93
93
93
93
93
>99
99
>99
95
4.2. Cloning and expression and purification
4
5c
>99
The reductase genes were amplified by polymerase chain reac-
tion (PCR) from the genomic DNAs, which were extracted and puri-
fied from various bacteria using a TIANamp Bacteria DNA Kit
(Tiangen, Shanghai). The amplified DNAs were digested with two
restriction enzymes and ligated into expression plasmid pET-28a
(+) or pET-43.1a (+) digested with the same enzymes. The con-
structs were ultimately transformed into E. coli BL21 (DE3) compe-
tent cells. Cells were inoculated in 100 ml LB medium (10 g LÀ1
tryptone, 5 g LÀ1 yeast extract and 10 g LÀ1 NaCl) at 37 °C and
200 rpm. When the OD600 reached 0.6–0.8, the enzyme expression
was induced with 0.5 mM IPTG at 25 °C for another 12 h. Cells
were harvested by centrifugation at 8800 rpm (4 °C for 10 min).
Cells were resuspended in buffer A (20 mM of Na2HPO4–NaH2-
PO4 buffer, pH 7.4, 500 mM of NaCl, 10 mM of imidazole) and son-
icated. The cell lysate was centrifuged at 8800 rpm (4 °C for
20 min). The supernatant was transferred into an Ni-NTA column
equilibrated with buffer A. Stepwise elution with imidazole was
completed in 10 mM increments up to 500 mM. The purity of the
fractions was analyzed by SDS–PAGE.
a
Reaction conditions: ECOO (0.6–3.0 M), D-glucose (1.5 equiv with respect to
ECOO), lyophilized cells of RhCR (60 kU are equals to approximately 5 g of lyophi-
lized cells), lyophilized GDH powders (1 equiv activity with respect to RhCR), 10 mL
of sodium phosphate buffer (pH 6.0, 100 mM), 30 °C. pH was controlled at 6.0 with
2 M NaOH.
b
Determined by GC.
100 mL of sodium phosphate buffer.
c
verted after 9 h (entry 2). Hence, this system was used for subse-
quent experiments.
Further optimization was accomplished toward higher sub-
strate loading. Increasing the substrate loading to 440 g LÀ1 still
gave >99% conversion within 8 h (entry 3). However, further
improvement to 660 g LÀ1 resulted in incomplete conversion
(95%) (entry 4). In all cases, the enantiomeric excess of the product
was always kept at 93%.
2.5. Preparative scale synthesis of (S)-ECHO
Next, the bioreduction of ECOO was carried out at a 10-fold lar-
ger scale under the optimized reaction conditions to further con-
firm the feasibility of the process (entry 5). All of the substrate
(ECOO, 44 g) was completely transformed within 5 h. After extrac-
tion and normal work-up, 34 g of (S)-ECHO (93% ee) was isolated.
Although the ee of ECHO is not satisfactory, the enantiomeric pur-
4.3. Enzyme assays
The reductase activity was analyzed spectrophotometrically at
30 °C by monitoring the decrease in the absorbance of NADH at
340 nm. The standard analysis mixture (1 mL) was composed of
100 mM Na2HPO4–NaH2PO4 buffer (pH 6.0), 2.0 mM ketoesters,
0.1 mM NADH and an appropriate amount of enzyme. One unit
was defined as the amount of enzyme leading to the consumption
ity of the final product (R)-a-lipoic acid could be improved by 99%
by final product crystallization.
3. Conclusion
of 1 lmol of NADH per min.
In conclusion, a more efficient and practical biocatalyst RhCR for
the synthesis of (S)-8-chloro-6-hydroxyoctanoic acid ester via
asymmetric reduction was developed. The reductase RhCR is the
only one that can reduce a substrate of >200 g LÀ1 in a space–time
yield as high as 1580 g LÀ1 dÀ1 (Table 4), which is greatly superior
to the literature records, thus demonstrating its great potential for
industrial application.
4.4. Effect of pH and temperature on the activity
The effects of pH and temperature on the activity of RhCR were
examined within a pH range of 3.0–10.0 (sodium citrate 3.0–6.0;
sodium phosphate 6.0–8.0; Gly–NaOH 8.5–10.0) and a tempera-
ture range of 25–70 °C. The stability was examined by incubating
the purified enzymes in the optimized buffer (pH 6.0) at 30, 40
and 50 °C for a varied period of time and the residual activities
were determined as described above.
4. Experimental
4.1. General
4.5. Preparative scale enzymatic production of (S)-ECHO
Chemical reagents were purchased from Aladdin Chemicals Co.
Ltd. (Shanghai, China) with >97% purity. Other chemicals were of
reagent grade or better. Plasmid pMD18-T for the cloning of PCR
products was obtained from TaKaRa (Dalian, China) and plasmid
pET-28a (+) for the heterogeneous expression was obtained from
A first, ECOO (44 g, 0.2 mol) was placed in 100 mL of sodium
phosphate buffer (100 mM, pH 6.0) containing 24,000 U of lyophi-
lized cells of RhCR (2.0 g), 24,000 U of lyophilized GDH (2.0 g), and
D-glucose (5.4 g, 0.3 mol) at 30 °C. The pH was adjusted to 6.0 by
Table 4
Comparison of RhCR with other reported biocatalysts
Biocatalyst
Substrate (g LÀ1
)
Cofactor (mM)
Time (h)
Conv. (%)
ee (%)/config
STYa (g LÀ1 dÀ1
)
Refs.
Mr cell
Gc cell
LBADH
NGADH
RhCRb
5.0
5.0
2.0
45
440
0
0
0.5
4
0
24
24
n.a.
24
5
66
62
>25
>95
>99
92 (S)
88 (R)
>65 (S)
n.a.
3
3
n.a.
<45
1580
19
19
20
22
93 (S)
This study
a
STY is an abbreviation of space–time yield.
Reaction conditions ECOO (2 M), D-glucose (1.5 equiv with respect to ECOO), lyophilized cells of RhCR (24 kU), lyophilized GDH powders (24 kU), 100 mL of sodium
b
phosphate buffer (pH 6.0, 100 mM), 30 °C. pH was controlled at 6.0 with 2 M NaOH.

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R.-J. Chen et al. / Tetrahedron: Asymmetry 25 (2014) 1501–1504
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[a]
22 = +19.8 (c 1.0, CHCl3); 1H NMR (CDCl3, 400 MHz): d 4.13 (q,
D
J = 7.1 Hz, 2H), 3.93–3.81 (m, 1H), 3.77–3.62 (m, 2H), 2.32 (t,
J = 7.3 Hz, 2H), 1.97–1.80 (m, 2H), 1.77–1.58 (m, 3H), 1.55–1.45
(m, 3H), 1.44–1.35 (m, 1H), 1.26 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3,
400 MHz): d 14.3, 24.7, 25.0, 34.2, 37.1, 39.7, 42.0, 60.4, 68.5,
173.8; HRMS (CI, N2 as the reagent gas) m/z calcd for C10H20O3-
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Financial support by National Natural Science Foundation of
China (Nos. 31200050 and 21276082), Ministry of Science and
Technology, China (Nos. 2011AA02A210 and 2011CB710800), is
gratefully acknowledged.
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