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Short Communication
Highly efficient synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate
and its derivatives by a robust NADH-dependent reductase
from E. coli CCZU-K14
Yu-Cai He
a,b,
⇑
, Zhi-Cheng Tao
a
, Xian Zhang
b
, Zhen-Xing Yang
a
, Jian-He Xu
b
a
Laboratory of Biocatalysis and Bioprocessing, College of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou 213164, PR China
b
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China
highlights
Reductase (CmCR) from Candida magnoliae was discovered by genome mining.
Recombinant E. coli CCZU-K14 was employed for efficiently reducing COBE.
COBE at 3 M was reduced to (S)-CHBE (>99.9% ee; yield > 99%) after 14 h.
E. coli CCZU-K14 could synthesize (S)-CHBE and its derivatives (>99.9% ee).
article info
Article history:
Received 5 February 2014
Received in revised form 21 March 2014
Accepted 24 March 2014
Available online 2 April 2014
Keywords:
Asymmetric reduction
Biotransformation
Ethyl 4-chloro-3-oxobutanoate
Ethyl (S)-4-chloro-3-hydroxybutanoate
Recombinant E. coli CCZU-K14
abstract
An NADH-dependent reductase (CmCR) from Candida magnoliae was discovered by genome mining for
carbonyl reductases. After CmCR was overexpressed in Escherichia coli BL21, a robust reductase-produc-
ing strain, recombinant E. coli CCZU-K14, was employed for the efficient synthesis of ethyl (S)-4-chloro-3-
hydroxybutanoate ((S)-CHBE) from the reduction of ethyl 4-chloro-3-oxobutanoate (COBE). After the
optimization, the optimum reaction conditions were obtained. Notably, E. coli CCZU-K14 had broad sub-
strate specificity in reducing both aliphatic and aromatic substrates, and excellent enantioselectivity of
CCZU-K14 was observed for most of the tested substrates, resulting in chiral alcohols of over 99.9% ee.
Moreover, COBE at a high concentration of (3000 mM) could be asymmetrically reduced to (S)-CHBE in
the high yield (>99.0%) and high enantiometric excess value (>99.9% ee) after 14 h. Significantly, E. coli
CCZU-K14 shows high potential in the industrial production of (S)-CHBE and its derivatives (>99.9% ee).
Ó2014 Elsevier Ltd. All rights reserved.
1. Introduction
Ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] is a key
intermediate for the production of chiral drugs, including choles-
terol-lowering HMG-CoA reductase inhibitors such as Lipitor (Cao
et al., 2011; Cai et al., 2012; He et al., 2006; Yamamoto et al.,
2004). Therefore, a more practical way to synthesize highly optical
active of (S)-CHBE (>99.9% ee) is of great interest. Compared with
conventional chemical synthesis, the asymmetric bioreduction of
ethyl 4-chloro-3-oxobutanoate (COBE), which is inexpensive and
easily synthesized, is an economical approach to the production
of (S)-CHBE (Cai et al., 2012; Ye et al., 2010b). However, the asym-
metric biotransformation of COBE to (S)-CHBE by reductases often
require cofactor NADH or NADPH as an electron donor (Ye et al.,
2009). Because of the high cost of these cofactors, in situ cofactor
regeneration is an effective approach to the economic viability of
industrial-scale biotransformations (Ye et al., 2010a). Enzyme-cou-
pled approach and substrate-coupled system could be employed as
the efficient and cost-effective cofactor recycling systems. For
example, glucose dehydrogenase could be used as enzyme-coupled
system for recycling NAD
+
or NADP
+
(Ye et al., 2010a); isopropanol
could be chosen as cosubstrate for improving the yield of (S)-CHBE
(Wang et al., 2011). Although some co-expression systems have
been used for designing these recycling systems (Kizaki et al.,
2001), few high-level co-expression of the enzymes against
3000 mM COBE were obtained with high enzyme activity and
excellent enantioselectivity in the monophasic aqueous media.
To asymmetrically synthesize (S)-CHBE (>99.9% ee) in the highly
efficient process, it is necessary to screen for the appropriate
http://dx.doi.org/10.1016/j.biortech.2014.03.133
0960-8524/Ó2014 Elsevier Ltd. All rights reserved.
⇑
Corresponding author at: Laboratory of Biocatalysis and Bioprocessing, College
of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou
213164, PR China. Tel.: +86 519 8633 4597; fax: +86 519 8633 4598.
E-mail addresses: heyucai2001@163.com,yucaihe2007@aliyun.com (Y.-C. He).
Bioresource Technology 161 (2014) 461–464
Contents lists available at ScienceDirect
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
reductases. Recently, genome mining can be effectively employed
to search gene data bases for sequences similar to those of known
reductases (Wang et al., 2011).
In this study, an NADH-dependent carbonyl reductase (CmCR)
from Candida magnoliae was employed to synthesize (S)-CHBE
(>99.9% ee) from COBE in the monophasic aqueous media. Using
glucose as cosubstrate, NADH or/and NADPH needn’t be extra
added into reaction system. To increase the yield of (S)-CHBE
through the asymmetric reduction of COBE, various parameters
(cosubstrate, glucose concentration, NAD
+
concentration, reaction
pH, reaction temperature, additives, cell dosage and substrate
loading, etc.) on the asymmetric bioreduction of COBE were inves-
tigated. Furthermore, the biocatalysts were used for the production
of chiral alcohols from the reduction of prochiral ketones. More-
over, asymmetric bioreduction of 3000 mM COBE was successfully
demonstrated.
2. Methods
2.1. Chemicals
COBE and racemic CHBE were obtained from Aladdin Chemistry
Co. Ltd (Shanghai, China). All other chemicals were also from local
commercial sources and of analytical grade.
2.2. Cloning and expression of CmCR gene in Escherichia coli
Using a TIANamp Bacteria DNA Kit from Tiangen (Shanghai),
genomic DNA was extracted from C. magnoliae provided by Dr.
Xian Zhang at East China University of Science and Technology
(Shanghai, China). Oligonucleotide primers with NdeI and BamHI
restriction sites were designed according to the CmCR gene se-
quence (GenBank Accession No. AB036927.1). The DNA fragment
of CmCR gene was amplified and double-digested with NdeI and
BamHI and then inserted into the expression vector pET-28a
(Novagen, Shanghai). The resulting plasmid, pET-28a-CmCR, was
transformed into E. coli BL21 (DE3) cells. The cells of were culti-
vated at 37 °C in LB medium containing 50
l
g/mL of kanamycin.
When the OD
600
of the culture reached 0.60, IPTG was added to a
final concentration of 0.50 mM, and cultivation was continued at
37 °C for a further 10 h. The cells were harvested by centrifugation
(8000g, 10 min) at 4 °C, and washed twice with potassium phos-
phate buffer solution (PBS) (100 mM, pH 7.0). The enzyme activity
was assayed according to the reference (Ye et al., 2010b).
2.3. Optimization of reaction conditions
Various reaction parameters on the effects of bioreduction reac-
tion were investigated. The effects of cosubstrate on the reductase
activity were performed at 30 °C and 180 rpm by adding 0.1 g
E. coli CCZU-K14 wet cells into 2 mL PBS buffer (100 mM, pH 7.0)
containing 1 mmol COBE, 1 mmol potential cosubstrate (ethanol,
fructose, glucose, glycerol, mannitol, mannose, methanol, isopropa-
nol or sucrose) and 0.1
l
mol NAD
+
in 10 mL Erlenmeyer flask
capped with a septum. To investigate the effects of cosubstrate con-
centration on the reductase activity, biotransformation was per-
formed with 0.1 g E. coli CCZU-K14 wet cells, 1 mmol COBE,
0.1
l
mol NAD
+
and certain concentration of cosubstrate (0.5–
2.5 mmol glucose/(mmol COBE)). To investigate the effects of
NAD
+
on the reductase activity, biotransformation was performed
by adding 0.1 g wet cells, 1 mmol COBE, 1.5 mmol glucose and cer-
tain concentration of NAD
+
(0.05–0.6
l
mol NAD
+
/(mmol COBE)). To
investigate the effect of reaction temperature and pH on the reduc-
tion, bioconversions were performed at various temperature (25–
45 °C) and various pH (6.0–9.0) by adding 0.1 g wet cells, 1 mmol
COBE, 1.5 mmol glucose and 0.1
l
mol NAD
+
into the reaction media.
To investigate the effects of additive on the reductase activity, bio-
transformation was performed at by adding 0.1 g wet cells, 1 mmol
COBE, 0.2
l
mol additive (AlCl
3
, CaCl
2
, CoCl
2
, EDTA, FeCl
3
, MgCl
2
,
MnCl
2
, NiCl
2
or ZnCl
2
), 1.5 mmol glucose and 0.1
l
mol NAD
+
into
the reaction media. To investigate the effects of cell dosage on the
reductase activity, bioconversion was performed at 30 °C and
180 rpm by adding certain concentration of wet cells (0.02–0.4 g),
1 mmol COBE, 0.2
l
mol Mn
2+
, 1.5 mmol glucose and 0.1
l
mol
NAD
+
into the reaction media. The effects of substrate concentration
on the reduction, biotransformations were performed by adding
0.2 g wet cells, certain concentration of COBE (0.5–6 mmol),
0.2
l
mol Mn
2+
, 1.5 mmol glucose/(mmol COBE), and 0.1
l
mol
NAD
+
/(mmol COBE) into the 2 mL reaction media in 10-mL
Erlenmeyer flask capped with a septum. After pre-incubated in a
180 rpm rotary shaker at selected temperature for 30 min, the
reaction mixture was extracted twice with ethyl acetate, and the
extracts were dried with anhydrous sodium sulfate for the assay.
2.4. Analytical methods
In this study, the reported substrate COBE and product CHBE
concentration refers only to the concentration in the reaction med-
ia based on the total volume. The concentrations of COBE and CHBE
were assayed by gas chromatography (He et al., 2006). The ee value
of (S)-CHBE was assayed according to the literature (Wang et al.,
2011).
3. Results and discussion
3.1. Screening of recombinant reductases
After genome data mining, four oxidoreductases having 40–80%
amino acid identities with known sequences encoding COBE reduc-
tases were selected from the NCBI data base and overexpressed in
E. coli cells. Notably, no overexpression operation of coenzyme
recycling enzyme was performed in this study. The enzyme CmCR
from C. magnoliae showed high activity towards reducing COBE
into (S)-CHBE (>99.9% ee) with excellent enantioselectivity. After
the overexpression, the recombinant strain named E. coli CCZU-
K14 with the highest reductase activity (20 U/mg crude protein),
and it was chosen as a potential biocatalyst for further studies.
The CmCR gene was amplified by PCR (Fig. 1a, lane 2). Sequence
analysis indicated that the CmCR gene (852 bp) encoded 284 ami-
no acids. SDS–PAGE analysis of recombinant protein of CmCR was
shown in Fig. 1b (lane 2).
3.2. Optimization of reaction conditions
During the biotransformation, different reaction conditions have
significant effects on enzyme activity (He et al., 2012, 2013; Wang
et al., 2011). Therefore, it is necessary to optimize the reduction con-
ditions (e.g., cosubstrate, glucose concentration, NAD
+
concentra-
tion, reaction pH, reaction temperature, additives, cell dosage and
substrate loading, etc.) for improving the catalytic efficiency. In this
study, nine potential cosubstrates (ethanol, fructose, glucose, glyc-
erol, mannitol, mannose, methanol, isopropanol and sucrose) were
investigated on the reductase activity, respectively (Data not
shown). Glucose, isopropanol, mannitol, and mannose as cosub-
strate could significantly improve the reductase activity. Probably,
the recombinant E. coli CCZU-K14 had substrate-coupled system.
It was reported that substrate-coupled system could be used as
the efficient and cost-effective cofactor recycling system (Wang
et al., 2011). Using glucose as cosubstrate, the highest reductase
activity was obtained. Therefore, the optimum cosubstrate was
462 Y.-C. He et al. / Bioresource Technology 161 (2014) 461–464
glucose. Furthermore, the effects of cosubstrate concentration
(0.25–10 mmol glucose/mmol COBE) on the reductase activity were
investigated. As shown in Table 1S, it was found that the reductase
activity significantly increased with the cosubstrate concentration
rising up to 1.5 mmol glucose/mmol COBE. When the amount of
cosubstrate was over 1.5 mmol glucose/mmol COBE, the reductase
activity clearly decreased. Probably, the viscosity of reaction media
increased so that the reductase activity decreased. Therefore,
1.5 mmol glucose/mmol COBE was chosen as the optimum amount
of cosubstrate. In contrast to some COBE reductases (Yasohara et al.,
2000; Ye et al., 2010b), CmCR preferred the inexpensive cofactor
NAD(H) instead of NADP(H) as the electron donor. To effectively
synthesize (S)-CHBE by CmCR, the concentration of NAD
+
was also
optimized. As shown in Table 1S, the optimum of NAD
+
concentra-
tion was 0.1
l
mol NAD
+
/(mmol COBE). However, reductase S1 from
C. magnoliae AKU4643 was an NADPH-dependent reductase (Yaso-
hara et al., 2000), and glucose dehydrogenase was required to be
overexpressed. Significantly, it had the enzyme-coupled system.
Reaction temperature and pH could significantly affect the biocata-
lytic activity (Data not shown). It was found that the reductase
activity from E. coli CCZU-K14 significantly increased with the reac-
tion temperature rising up to 35 °C. At temperatures above 35 °C,
however, the reductase activity decreased considerably, possibly
due to the thermal deactivation of reductase in the cells of E. coli
CCZU-K14 during the reductase reaction. In view of thermobility,
the optimum reaction temperature was chosen to be 30 °C. More-
over, effects of different reaction pH (6.0–9.0) on the initial reaction
rate were also investigated. Considering the reductase activity and
stability of COBE under the different pHs, it was found that the opti-
mum reaction pH was found to be 7.0. It was reported that the max-
imum activity of reductase from S. coelicolor was observed at pH 6.5
and 45 °C(Wang et al., 2011). EDTA and various metal ions (0.1 mM)
were investigated (Table 2S). It was found that Ni
2+
and EDTA
caused strong inhibition on the reductase activity, other metal ions
(Al
3+
,Mg
2+
and Mn
2+
) could enhance the reductase activity. The me-
tal-chelating reagent EDTA reduced the reductase activity, indicat-
ing that metal ions might be required or beneficial for the
reductase activity. Significantly, Mn
2+
(0.1 mM) was an optimum
metal ion additive. Furthermore, the amounts of cell dosage used
for the reduction of COBE were investigated (Data not shown). All
the ee values were over 99.9%. When the cell dosage was less than
0.1 g (wet weight)/mL, the production of the (S)-CHBE was consid-
erably improved with the increase of cell dosage. When the cell dos-
age was above 0.1 g (wet weight)/mL, the reductase activity was not
increased significantly. Probably, the viscosity of the reaction media
increased so that the low dissolved oxygen (DO) might be promi-
nent in the aqueous phase. Therefore, the optimum amount of cell
dosage was 0.1 g (wet weight)/mL. However, it was reported that
the optimum cell dosage was 0.075 g (dry weight)/mL of Aureoba-
sidium pullulans CGMCC 1244 cells (He et al., 2006). Moreover, the
different substrate COBE concentrations (250–3000 mM) on the
reductase activity were also investigated (Data not shown). The
reductase activity increased with the COBE concentration rising
up to 1000 mM. When the substrate concentration was over
1000 mM, the reductase activity significantly decreased. Therefore,
the optimum substrate concentration was 1000 mM.
Based on the above results, the optimum reaction conditions
were obtained: cosubstrate glucose 1.5 mmol glucose/(mmol
COBE), NAD
+
0.1
l
mol NAD
+
/(mmol COBE), reaction temperature
30 °C, reaction pH 7.0, metal ion additive MnCl
2
(0.1 mM), sub-
strate COBE 1000 mM, and cell dosage 0.1 g (wet weight)/mL.
3.3. Substrate specificity and enantioselectivity
Enantiomerically pure alcohols are known as the important and
valuable chiral synthons for the production of pharmaceuticals and
fine chemicals (Cai et al., 2012; Wang et al., 2011; Ye et al., 2009). To
investigate substrate specificity of recombinant E. coli CCZU-K14
reductase, various substrates (Fig. 1S, 1a–9a, 1000 mM) were tested.
Clearly, reductase from recombinant E. coli CCZU-K14 had broad
substrate specificity in reducing both aliphatic and aromatic sub-
strates (Table 3S). The yields of over 85% were achieved for each
substrate. Excellent enantioselectivity of CCZU-K14 was observed
for most of the tested substrates, resulting in chiral alcohols of over
99.9% ee. Using COBE as substrate, the highest reductase activity
was obtained. Therefore, recombinant E. coli CCZU-K14 reductase
showed high potential in the production of chiral alcohols.
3.4. Asymmetric reduction of COBE
To test the efficiency of the reductase reaction, asymmetric bio-
transformation of COBE by recombinant E. coli CCZU-K14 was per-
formed in monophasic aqueous media under the optimized
reaction conditions. As shown in Fig. 2, biotransformation of
500 mM COBE for 0.5 h, (S)-CHBE was obtained in a high yield
(>99.0%). Moreover, 1000 and 1500 mM of COBE could be com-
pletely reduced into (S)-CHBE after 1 and 2 h, respectively. Notably,
COBE at a high concentration of (3000 mM) could be asymmetri-
Fig. 1. (a) Cloning of the CmCR gene from C. megaterium by PCR. Lane 1: Marker. Lane 2: CmCR gene. (b) SDS–PAGE analysis of recombinant protein of CmCR. Lane 1: Marker.
Lane 2: supernatant (soluble proteins) after sonication of E. coli CCZU-K14.
Y.-C. He et al. / Bioresource Technology 161 (2014) 461–464 463
cally reduced to (S)-CHBE in the high yield (97.7%) and high
enantiometric excess value (>99.9% ee) after 12 h. Prolonging the
reaction time for another 2 h, (S)-CHBE was obtained in a high
yield (>99.0%) (Data not shown). However, it was reported that
554 mM CHBE was produced in the organic phase with the yield
of 91% after 24 h (Ye et al., 2010a). Significantly, recombinant
E. coli CCZU-K14 showed high activity towards high concentration
of COBE.
To effectively synthesize (S)-CHBE from 3000 mM COBE in a
small scale, a 100 mL reaction mixture of PBS buffer (100 mM,
pH 7.0), 49.38 g COBE, 0.45 mol glucose, 10 g wet cells, NAD
+
(30
l
mol), MnCl
2
(10
l
mol) in a 500-mL flask was used for the bio-
transformation. The pH was adjusted to 7.0 with 2 M NaOH. After
stirred at 300 rpm for 14 h, the reaction was obtained in the con-
version of 100%, and the ee value of the product was excellent
(>99.9% ee). An external NAD
+
concentration of 0.3 mM was suffi-
cient to secure a conversion with a high TTN of 10,000, which
was in a practical range for production of chiral alcohols (Ye
et al., 2010b). The reductase from S. coelicolor A3 could biotrans-
form high concentration (30 g, 600 g/L) of COBE into (S)-CHBE
(>99%, ee) with a high TTN of 12,100 in a water-toluene biphasic
system with the conversion of 99% (Wang et al., 2011). In water-
ethyl caprylate biphasic system, the TTN of CHBE forming NADP
+
was 12,600 (Ye et al., 2010b). Using the resting cells of recombi-
nant E. coli co-expressing S1 from C. magnoliae and GDH from Bacil-
lus megaterium,(S)-CHBE could be obtained at 430 g/L (2581 mM)
in the organic phase of biphasic system with the yield of 85% (Ki-
zaki et al., 2001). Biotransformation of COBE by reductase S1 for
30 h in n-butyl acetate-water biphasic system, the (S)-CHBE in
the organic solvent reached 1060 mM (Yasohara et al., 2000) with
the yield of >99.0%. Clearly, these systems required large amount of
organic solvent, and the biocatalytic efficiencies were still not
ideal. Therefore, these factors limited the industrial production of
(S)-CHBE. In this study, a highly stereoselective bioreduction of
3000 mM COBE into (S)-CHBE by E. coli CCZU-K14 was successfully
demonstrated in the monophasic aqueous system (Fig. 2S), and
large amounts of organic solvents could be avoided. Moreover,
the reduction of COBE is a more economical way for the production
of (S)-CHBE (>99.9% ee) because COBE is relatively inexpensive and
easily synthesized (Ye et al., 2009). Significantly, E. coli CCZU-K14
has the high potential in the industrial production of (S)-CHBE
(>99.9% ee). It is likely that this performance could be enhanced
further by using the strategies of cell immobilization or bioreactor,
and the continuous and larger scale production of (S)-CHBE by the
aid of these immobilized beads in different reactors is of great
interest (Wang et al., 2011).
4. Conclusions
The reductase (CmCR) from recombinant E. coli CCZU-K14
displayed high reductase activity and excellent stereoselectivity
for the bioreduction of COBE and its derivatives using glucose as
cosubstrate. After the reaction optimization, the optimum cosub-
strate concentration, NAD
+
concentration, reaction temperature,
reaction pH, additive, substrate concentration and cell dosage were
1.5 mmol glucose/(mmol COBE), 0.1
l
mol NAD
+
/(mmol COBE),
30 °C, 7.0, Mn
2+
(0.1 mM), 0.1 g (wet weight)/mL, and 1000 mM,
respectively. Furthermore, high concentration of COBE (3000 mM)
could be asymmetrically reduced to (S)-CHBE with high yield
(99.0%) and excellent ee (>99.9%) after 14 h.
Acknowledgements
All authors gratefully acknowledge support from the National
Natural Science Foundation of China (No. 21102011) and the Open
Project Program of the State Key Laboratory of Bioreactor Engineer-
ing (Shanghai, China).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2014.03.
133.
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0
500
1000
1500
2000
2500
3000
3500
4000
024681012
Time (h)
S-CHBE (mM)
500mM 1000mM 1500mM
2000 mM 3000 mM
Fig. 2. Asymmetric reduction of COBE in monophasic aqueous system. All exper-
iments were performed in triplicate.
464 Y.-C. He et al. / Bioresource Technology 161 (2014) 461–464
