Coupled (R)-carbonyl reductase and glucose dehydrogenase catalyzes (R)-1-phenyl-1,2-ethanediol biosynthesis with excellent stereochemical selectivity

Article abstract of DOI:10.1016/j.procbio.2015.08.002

The biotransformation of 2-hydroxyacetophenone to (R)-1-phenyl-1, 2-ethanediol (PED) by NADH-dependent (R)-carbonyl reductase (RCR) from Candida parapsilosis is slow and gives low yields, probably as a result of insufficient cofactors. To improve the biotransformation efficiency of (R)-PED from 2-hydroxyacetophenon, an enzyme-coupling system containing RCR and glucose dehydrogenase (GDH) was constructed to strengthen NADH-recycling pathway in Escherichia coli, in which the Shine-Dalgarno sequence and the aligned spacing sequence were used as linkers between them. The introduction of glucose dehydrogenase had little affects on the cell-growth. The co-expression conditions of RCR and glucose dehydrogenase was optimized to rebalance their catalytic functions. The ratio of k_cat/K_M for enzyme-coupling system catalyzing 2-HAP and glucose was about 1.0, suggesting the good balance between the functions of RCR and GDH. The rebalanced system gave excellent performance in (R)-PED biotransformation: an optical purity of 99.9% and a yield of 99.9% at optimal conditions: 35°C and pH 7.0. The introduction of glucose dehydrogenase stimulated increases of 23.8% and 63.8%, in optical purity and yield of (R)-PED, and simultaneously reduced the reaction time two-fold. This work provided a valuable method for efficient chiral alcohol production through protein-expression and biotransformation optimization to rebalance cofactor pathways.

Full text of DOI:10.1016/j.procbio.2015.08.002

Accepted Manuscript
Title: Coupled (R)-carbonyl reductase and glucose
dehydrogenase catalyzes (R)-1-phenyl-1,2-ethanediol
biosynthesis with excellent stereochemical selectivity
Author: Xiaotian Zhou Rongzhen Zhang Yan Xu Hongbo
Liang Jiawei Jiang Rong Xiao
PII:
S1359-5113(15)30056-8
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.procbio.2015.08.002
PRBI 10491
To appear in:
Process Biochemistry
Received date:
Revised date:
Accepted date:
15-4-2015
10-6-2015
2-8-2015
Please cite this article as: Zhou Xiaotian, Zhang Rongzhen, Xu Yan, Liang Hongbo,
Jiang Jiawei, Xiao Rong.Coupled (R)-carbonyl reductase and glucose dehydrogenase
catalyzes (R)-1-phenyl-1,2-ethanediol biosynthesis with excellent stereochemical
selectivity.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2015.08.002
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Coupled (R)-carbonyl reductase and glucose dehydrogenase
catalyzes (R)-1-phenyl-1,2-ethanediol biosynthesis with excellent
stereochemical selectivity
a
a,b*
a,b*
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a
Xiaotian Zhou , Rongzhen Zhang , Yan Xu , Hongbo Liang , Jiawei Jiang , Rong
c
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Xiao
a
Key Laboratory of Industrial Biotechnology of Ministry of Education & School of
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Biotechnology, Jiangnan University, Wuxi 214122, P. R. China
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National Key Laboratory for Food Science, Jiangnan University, Wuxi 214122, P. R.
China
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Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway,
NJ 08854, USA
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*Corresponding authors: Rongzhen Zhang and Yan Xu
Tel: +86-510-85197760; Fax: +86-510-85864112
Address correspondence to: Rongzhen Zhang, rzzhang@jiangnan.edu.cn; and
Yan Xu, yxu@jiangnan.edu.cn
Present address: School of Biotechnology, Jiangnan University, 1800 Lihu Avenue,
Wuxi 214122, P. R. China
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Graphical abstract
The coupled system based on RCR and GDH was rebalanced through
protein-expression optimization. It gave excellent performance on (R)-PED
biotransformation without exogenous cofactors.
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Highlights:
1. The construction of coupling system contain RCR and GDH
2. The introduction of GDH has little effects on cell-growth
3. The functions of RCR and GDH were rebalanced through
protein-expression optimization
4. The coupling system produced (R)-PED with excellent stereochemical
selectivity
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Abstract:
The biotransformation of 2-hydroxyacetophenone to (R)-1-phenyl-1, 2-ethanediol
(PED) by NADH-dependent (R)-carbonyl reductase (RCR) from Candida parapsilosis
is slow and gives low yields, probably as a result of insufficient cofactors. To improve
the biotransformation efficiency of (R)-PED from 2-hydroxyacetophenon, an
enzyme-coupling system containing RCR and glucose dehydrogenase (GDH) was
constructed to strengthen NADH-recycling pathway in Escherichia coli, in which the
Shine–Dalgarno sequence and the aligned spacing sequence were used as linkers
between them. The introduction of glucose dehydrogenase had little affects on the
cell-growth. The co-expression conditions of RCR and glucose dehydrogenase was
optimized to rebalance their catalytic functions. The ratio of kcat/K
M
for
enzyme-coupling system catalyzing 2-HAP and glucose was about 1.0, suggesting the
good balance between the functions of RCR and GDH. The rebalanced system gave
excellent performance in (R)-PED biotransformation: an optical purity of 99.9% and a
yield of 99.9% at optimal conditions: 35 °C and pH 7.0. The introduction of glucose
dehydrogenase stimulated increases of 23.8% and 63.8%, in optical purity and yield of
(R)-PED, and simultaneously reduced the reaction time two-fold. This work provided a
valuable method for efficient chiral alcohol production through protein-expression and
biotransformation optimization to rebalance cofactor pathways.
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Keywords: (R)-1-phenyl-1, 2-ethanediol, (R)-carbonyl reductase, NADH
regeneration, glucose dehydrogenase, Candida parapsilosis
56
Abbreviations: GDH, Glucose dehydrogenase; 2-HAP, 2-hydroxyacetophenone;
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RCR, (R)-carbonyl reductase; (R)-PED, (R)-1-phenyl-1, 2-ethanediol; (S)-PED,
(S)-1-phenyl-1, 2-ethanediol
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1. Introduction
Optically active alcohols are very useful chiral blocks in the special chemical and
pharmaceutical industries [1, 2]. For example, (R)-1-phenyl-1, 2-ethanediol (PED) can
be used for synthesis of an antidepressant drug, fluoxetine, which extends the effects
of 5-hydroxy tryptamine through selective inhibition for the extraction of 5-hydroxy
tryptamine in central nervous system [3]. With the increasing requirement of chiral
compounds in different fields, people pay more and more attentions to high optical
purity and yield of enantiopure chemicals [4-6]. The efficient preparation of single
enantiomers has become a hot research in recent years [7-9].
The cofactor-dependent asymmetric reduction of ketones catalyzed by alcohol
dehydrogenases, is a valuable method for the synthesis of optically active alcohols
[10-12]. It was reported that carbonyl reductase from Aspergillus niger, Candida
tropicalis, Rhizopus arrhizus, Ralstonia sp., Rhodotorula glutinis, Trichothecium sp. et
al. efficiently catalyzes prochiral substrate into the corresponding chiral products
[13-18]. Candida parapsilosis (S)-carbonyl reductase gives good performance in the
reduction of 2-hydroxyacetophenone (HAP) to (S)-PED. However, with the increase of
substrate concentrations, protons transfer of cofactor NAD(P)(H) will be a restriction
factor during alcohol dehydrogenase-mediated biotransformation [19]. The
construction of enzyme-coupled system to introduce cofactor-regeneration enzyme is
an attractive technique to improve chiral biosynthesis [20].
There are several enzymes involved in NAD(P)(H) recycling, such as formate
dehydrogenase, glucose dehydrogenase (GDH), and alcohol dehydrogenases [21-23].
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The enzyme GDH is one of the most frequently used cofactor-regeneration enzymes
because it can regenerate both NADPH and NADH [24,25]. There are several
advantages in the GDH-mediated cofactor-regeneration system as follows: GDH is
characterized with favourable thermodynamics, and it has high enzyme activity over a
broad pH range and high stability against several organic compounds [24-26]; The
hydride source is cheap glucose; And glucose/gluconic acid produced by the system
does not interfere with the synthesis reaction [23]. For example, Zhang et al.
efficiently produced (S)-PED with optical purity of over 99% in a yield of 96% by
introduction of Bacillus sp. YX-1 GDH into (S)-carbonyl reductase-mediated
biotransformation pathway [27]. Xiao et al. reported a novel whole-cell
+
biotransformation containing GDH for NAD regeneration to prepare chiral chemicals
[28].
Kosjek
et
al.
realized
efficient
preparation
of
4,
4-dimethoxytetrahydro-2H-pyran-3-ol and (S)-4-chloro-3-hydroxybutanoate using
coupled systems consisting of carbonyl reductase and GDH [29-31].
In our previous work, we reported an NADH-dependent (R)-carbonyl reductase
(RCR, also named (R)-alcohol dehydrogenase) from C. parapsilosis catalyzing the
reduction of 2-hydroxyacetophenone (2-HAP) to (R)-PED [32, 33]. Very recently, we
determined crystal structure of RCR and its catalytic mechanism of stereoselectivity
[34]. However, the recombinant Escherichia coli-RCR catalyzed the transformation of
(R)-PED with an optical purity of 81% in a yield of 61% [35]. The low
biotransformation efficiency may be due to insufficient cofactors or weak
cofactor-regeneration pathway [36]. By replacing the protein expression hosts, the
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recombinant RCR still produced (R)-PED with unsatisfied efficiency in
Saccharomyces cerevisiae and Pichia pastoris [33, 37]. Wang et al. significantly
improved the biotransformation efficiency of (R)-PED through the addition of glycerol
and isopropol into the reaction system by a dual-cosubstrate-coupled system, but the
chiral synthesis is not clean process [11]. In this work, to realize the efficient
preparation of (R)-PED with a higher optical purity by RCR, we introduced GDH from
Bacillus sp. YX-1 to strengthen cofactor regeneration during RCR-catalyzed
biotransformation in E. coli. The coupled system containing RCR and GDH gives
excellent performance in producing (R)-PED with almost absolute stereochemical
selectivity.
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15 2. Materials and Methods
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2.1 Microorganisms and chemicals
®
The PrimeSTAR HS, pMD19-T vector, restriction enzymes and T4 DNA ligase
were bought from Takara (Otsu, Japan). 2-HAP was purchased from TCI Development
Co. Ltd. (Shanghai, China). (R)-PED, (S)-PED and NADH were purchased from
Sigma-Aldrich (St. Louis, USA). Hexane and isopropanol used for high performance
liquid chromatography (HPLC) were of chromatographic grade from Sigma-Aldrich
(St. Louis, USA). All other chemicals used were of analytical grade and commercially
available. The organisms were cultivated as described previously [12]. The strains used
in this work are listed in Table 1.
Insert Table 1
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2.2 Construction of co-expression system of RCR and GDH
The co-expression system containing RCR and GDH were constructed using a
Shine–Dalgarno and aligned spacing sequence (GAAGGAGATATACC) as a linker
between them. The fusion gene RCR-SD-AS-GDH was cloned using a modified
overlap-extension technique on plasmid pET-28a(+) [38]. The recombinant plasmid
pET-R-SD-AS-G was transformed into the competent E. coli BL21 (DE3), E. coli RIL
and E. coli Rosetta cells chemically, and the positive E. coli BL21/pET-R-SD-AS-G, E.
coli RIL/pET-R-SD-AS-G and E. coli Rosetta/pET-R-SD-AS-G clones were verified
by DNA determination. The plasmids and primers used were listed in Table 1.
2.3 Protein expression
The recombinant strains E. coli BL21/pET-R-SD-AS-G, E. coli
RIL/pET-R-SD-AS-G and E. coli Rosetta/pET-R-SD-AS-G were grown in LB medium
-
1
containing 50 μgml kanamycin at 37°C. When OD600 value of the culture reached 0.8,
isopropyl-β-D-thiogalactopyranoside (IPTG) of 0.5 mM was added to induce protein
expression. The cultures were cultivated at 37°C for 16 h and then harvested by
centrifugation.
The protein expression of RCR and GDH was optimized under the different
OD600 value (0.4-1.5) of the culture, different IPTG concentrations (0.2-2.0 mM),
different induce temperature (17°C -37°C), different induce times (8-20 h).
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2.4 Protein purification
The harvested cells were resuspended in 0.1 M potassium phosphate buffer (pH
7.0) and lysed by sonication with an ultrasonic oscillator (Sonic Materials Co., USA),
The cell debris were removed by centrifugation (12000×g, 40 min) at 4°C, and the
supernatant was applied to a HisTrap HP affinity column (GE Healthcare, Piscataway,
NJ, USA) equilibrated with a buffer (20 mM Tris-HCl, 0.3 M NaCl; pH 7.5), and then
was eluted with a buffer (20 mM Tris-HCl, 0.3 M NaCl, 0.3 M imidazole; pH 7.5)
using an ÄKTA purifier system (GE Healthcare, Piscataway, USA). The homogeneity
of purified enzymes was judged by Coomassie brilliant blue staining of SDS-PAGE
gels.
2.5 Enzyme assay
The reduction activities of RCR were measured at 35°C and pH 7.0 by
spectrophotometrically recording the rate of change of NADH absorbance at 340 nm.
One unit of enzyme activity is defined as the amount of enzyme catalyzing the
oxidation of 1 μmol of NADH per minute under the measurement conditions. The
standard assays were performed as described by Nie et al. [39]. The oxidation
activities of GDH were measured at 35°C and pH7.0 by spectrophotometrically
recording the rate of change of NADH absorbance at 340 nm. One unit of enzyme
activity is defined as the amount of enzyme catalyzing the reduction of 1 μmol of
+
NAD per minute under the measurement conditions. The protein concentration was
determined by the Bradford method with bovine serum albumin as the standard.
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2.6 Kinetic parameters
Various concentrations of substrate 2-hydroxyacetophenone (0.05–0.5 mM),
enzyme (10–200 μM), and cofactors NADH (0.05–0.5 mM) in 100 mM phosphate
buffer (pH 7.5) were used for kinetic parameters determination [40]. The data were
fitted to the Michaelis–Menten equation by using a nonlinear least-square iterative
method using KaleidaGraph (Synergy Software, Reading, PA). Three sets of kinetic
parameters were obtained from three independent experiments and then simply
averaged to yield the final estimates. The final estimates are shown with the standard
errors for the three sets.
2.7 Biotransformation and analytical methods
The bioconversion mixture (2 mL) consisted of 0.1 M potassium phosphate buffer
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(pH 7.0), 6 gL 2-HAP, 12 gL glucose, and 0.1 g washed wet cells. The reactions
were carried out at 35°C for 36 h with shaking at 200 rpm. After the cells were
removed by centrifugation, the supernatant was extracted with ethyl acetate., and the
organic layer was filtered through a 0.22 μm PVDF syringe filter (Troody Technology,
Shanghai, China) for analysis. The optical purity and yield of (R)-PED were
determined by HPLC (HP 1100, Agilent, USA) equipped with a Chiralcel OB-H
column (4.6 mm ×250 mm; Daicel Chemical Ind., Ltd., Japan) as described by Nie et
al. [39].
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2.8 Optimal pH and temperature
The pH optimum of (R)-PED transformation was determined between pH 4.0 and
10.0 using 0.1 M citric acid buffer (pH 4.0, 4.5, 5.0, 5.5), 0.1 M potassium phosphate
buffer (pH 6.0, 6.5, 7.0, 7.5), 50 mM Tris-HCl buffer (8.0), and 0.1 M carbonate buffer
(pH 9.0, 10.0).
The temperature optimum of (R)-PED transformation was determined at various
temperatures (20°C–80°C). The biotransformation of (R)-PED was determined with
the standard assay method described above.
01 3. Results and discussion
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3.1 Construction of the coupled-system containing RCR and GDH
The recombinant E. coli-RCR catalyzes the reduction of 2-HAP to (R)-PED with
low optical purity and yield, probably as a result of insufficient cofactors or weak
cofactor-recycling pathway [12]. To improve the biotransformation efficiency of
(R)-PED, the introduction of cofactor-regeneration enzyme GDH into the reaction
system would be a good strategy [24]. In this work, the RCR and GDH genes were
cloned with the Shine–Dalgarno sequence and the aligned spacing sequence
(GAAGGAGATATACC) as
a
linker between them. The fusion gene
RCR-AS-SD-GDH was cloned on pET-28a plasmid to construct the recombinant
plasmid pET-R-SD-AS-G. Since there are a few rare codons in RCR gene, E. coli
BL21, E. coli Rosetta and E. coli RIL were used as hosts for co-expression of RCR and
GDH. The recombinant plasmid pET-R-SD-AS-G was transformed into three kinds of
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hosts: E. coli BL21, E. coli RIL and E. coli Rosetta, and the corresponding strains E.
coli BL21/pET-R-SD-AS-G, E. coli RIL/pET-R-SD-AS-G and E. coli
Rosetta/pET-R-SD-AS-G were obtained and then confirmed by DNA sequencing.
3.2 Co-expression of RCR and GDH
The three recombinant cells E. coli BL21/pET-R-SD-AS-G, E. coli
RIL/pET-R-SD-AS-G and E. coli Rosetta/pET-R-SD-AS-G were induced with 0.5 mM
isopropyl-β-thiogalactopyranoside (IPTG) and supplied for SDS-PAGE analysis.
SDS-PAGE analysis results showed that two apparent bands corresponding to the
expected sizes (44 kDa and 30 kDa) of the target recombinant enzymes, RCR and
GDH, were observed in all recombinant cell extracts (Fig. 1). The cell-free extracts of
recombinant E. coli BL21/pET-R-SD-AS-G, E. coli RIL/pET-R-SD-AS-G and E. coli
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Rosetta/pET-R-SD-AS-G showed reductive activities of 0.6 Umg , 0.6 Umg and 0.4
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Umg with 2-HAP as substrate, and 1.1 Umg , 1.3 Umg and 0.9 Umg with glucose
as substrate, respectively (Table 2). The three recombinant strains catalyzes the
reduction of 2-HAP to (R)-PED with optical purity of 45.1%, 96.1% and 87.9%, in
yields of 20.4%, 63.8% and 60.2%, at 35°C and pH 7.0. From these above results, E.
coli RIL/pET-R-SD-AS-G shows the highest specific activity and biotransformation
efficiency of (R)-PED among the three recombinant strains.
Insert Fig. 1
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Insert Table 2
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3.3 Introduction of GDH had little effect on cell growth
The cofactor regeneration enzyme, GDH, involved in glucose metabolic pathway,
catalyzes glucose to glucono-δ-lactone, which maybe affect the grow properties of
recombinant cells. We determined the cell-growth properties by measuring the
turbidity of the culture at OD600 using a UV–visible spectroscopy system (Fig. 2).
Based on the curves, the recombinant strain E. coli RIL/pET-R-SD-AS-G grew at a
similar rate compared with E. coli RIL/pET-RCR, E. coli RIL/pET-GDH and the
control strain E. coli RIL/pET-28a. The results suggested that the introduction of GDH
into E. coli RIL had almost no effect on cell growth properties, which is similar to the
results reported by Yamamoto et al [26]. A coupled-system E. coli
RIL/pET-R-SD-AS-G based on GDH and RCR involved in the reaction of 2-HAP to
(R)-PED would be preferable for further studies of enzyme-catalyzed
biotransformations. As Pennacchio et al. reported, the introduction of glucose/gluconic
acid into an asymmetric reduction by the cofactor-regeneration enzyme GDH did not
interfere with the chiral synthesis [23].
Insert Fig. 2
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3.4 The function rebalance of RCR and GDH through their protein-expression
optimization
Since the coupled-system E. coli RIL/pET-R-SD-AS-G contains two enzymes,
RCR and GDH, the protein expression conditions will be optimized to improve their
protein expression level and (R)-PED biotransformation efficiency. The results showed
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E. coli RIL/pET-R-SD-AS-G catalyzed the biosynthesis of (R)-PED with a high optical
purity of 96.3% and yield of 68.2% when its OD600 value reached 1.0 (Fig. 3A). With
the increase of IPTG concentration, the optical purity and yield of (R)-PED by E. coli
RIL/pET-R-SD-AS-G were improved and reached the highest level: an optical purity
of 96.5% and a yield of 83.2% with 0.4 mM IPTG (Fig. 3B). When IPTG
concentration over 0.4 mM was added, the recombinant cells produced (R)-PED with a
low optical purity and a low yield, which might be due to the cell-growth inhibition
resulting in low protein expression. After the addition of 0.4 mM IPTG, the
recombinant cells were continue to culture at 37°C for 10 h, the harvested cells
catalyzed the biotransformation of (R)-PED with the highest efficiency with optical
purity of 96.9% in a yield of 94.8% (Fig. 3C, Fig. 3D). Thus, under the optimal
expression conditions: OD600 1.0, 0.4 mM IPTG, induce duration 10 h, and the culture
temperature 37°C after IPTG induction, the recombinant E. coli RIL/pET-R-SD-AS-G
catalyzed the biotransformation of (R)-PED with an optical purity of 96.9% in a yield
of 94.8%.
Insert Fig. 3
3.5 Kinetics demonstrates the good balance between RCR and GDH in coupling
system
Since the recombinant RCR was tagged with N-terminal 6×histine, and GDH was
fused with C-terminal 6×histine, they were both purified by one-step Ni-affinity
chromatography. SDS-PAGE analysis showed that two enzymes: RCR and GDH were
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simultaneously purified from the recombinant strain E. coli RIL/pET-R-SD-AS-G (Fig.
4), with the corresponding molecular of 30 kDa and 44 kDa. The purified proteins
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exhibited specific activity of 17.8 Umg for 2-HAP reduction, and 21.5 Umg for
glucose oxidation. The kinetic parameters were determined for 2-HAP or glucose at
different concentrations (0.05–0.5 mM) and cofactors at various concentrations
(0.05–0.5 mM). As shown in Table 3, the purified RCR and GDH from E. coli
RIL/pET-R-SD-AS-G showed the similar catalytic efficiency (kcat/K
purified RCR from E. coli RIL/pET-RCR towards 2-HAP, suggesting the
co-expression of RCR with GDH had no effects on the function of RCR. The kcat/K
M
) with the
M
values of the enzyme RCR and GDH was 3.2 for the reduction of 2-HAP to release
+
NAD , and 3.9 for the oxidation of glucose to form NADH. The ratio of kcat/K
M
for
RCR and GDH catalyzing 2-HAP and glucose was about 1.0, showing the good
balance between the functions of RCR and GDH. C. parapsilosis RCR and Bacillus sp.
XY-1 GDH are both homotetramer structures and each molecular of RCR or GDH
+
consists of one NADH or NAD .[30, 39]. So, in one reaction, the reduction of NADH
+
and oxidation of NAD by the coupled system containing RCR and GDH almost kept
+
the good rebalance between NAD -NADH recycling. These results further confirmed
the protein expression optimization is available to rebalance cofactor pathway to
improve the biotransformation efficiency of (R)-PED by the coupled system. In
general, the higher initial reaction rate induced an increase of optical purtity of
(R)-PED. The introduction of glucose dehydrogenase stimulated the improved reaction
rate, resulting in an increase of optical purtity of (R)-PED [20, 22].
1
5

3
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3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
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02
03
04
05
06
07
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11
12
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14
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17
18
19
20
21
22
23
Insert Table 3
Insert Fig.4
3.6 Improving (R)-PED biotransformation through pH and temperature
optimization
In enzyme-mediated reaction, with the increase of temperature, the activity of
protein is reduced, resulting in the decrease of reaction rate [37]. On the other hand,
temperature sometimes changes the product stereoselectivity during enzyme-catalyzed
chiral biosynthesis [40]. As showed in Fig.5A, with the increase of temperature lower
than 35°C, the bioconversion efficiency of (R)-PED from 2-HAP by the coupled
system was increased and reached the highest level: an optical purity of 98.2% and a
yield of 97.4% at 35°C.
The pH value affects the dissociation degree of the groups in the enzyme active
center, substrate and coenzyme, thus changes the binding strength between enzyme
and substrate [41]. When the pH is 7.5, the recombinant strain E. coli
RIL/pET-R-SD-AS-G catalyzes the biotransformation of (R)-PED with the highest
optical purity of 99.0% and yield of 98.5% (Fig.5B).
During the biotransformation process by the coupled system E. coli
RIL/pET-R-SD-AS-G, the reaction duration was also investigated targeting the highest
optical purity and yield of (R)-PED under optimal temperature and pH. The results
showed that the optical purity and yield of (R)-PED both reached 99.9% in 24 h
(Fig.5C). Compared to E. coli/RCR, the reaction duration was significantly reduced
1
6

3
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3
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3
24
25
26
27
28
29
30
31
32
33
34
35
36
two fold: from 48 h to 24 h [34,38]. So the coupled system based on RCR and GDH
efficiently catalyzes (R)-PED biosynthesis with almost absolute stereochemical
selectivity. The reduced yield of (R)-PED at 30 h may be due to the slightly higher
catalytic efficiency of GDH (kcat/K
M
is 3.9) than that of RCR (kcat/K is 3.2), resulting
M
+
more NAD for RCR catalyzing (R)-PED to 2-HAP. As reported by Xiao et al., the
introduction of cofactor-recycling enzyme into the asymmetric reaction system
significantly improves the chiral biosynthesis efficiency [24]. Pennacchio et al.
recently evaluated asymmetric reduction of α-keto esters with Thermus thermophilus
NADH-dependent carbonyl reductase using GDH for cofactor-regeneration enzyme
[22]. The strengthened cofactor regeneration can overcome coenzyme restriction in the
asymmetric reduction and accelerate the initial reaction rate, resulting in improved
chiral biosynthesis efficiency [23-25, 27].
Insert Fig. 5
3
37
3
3
3
3
3
3
3
3
38 4. Conclusions
39
40
41
42
43
44
45
The NADH-dependent E. coli-RCR catalyzes the reduction 2-HAP to (R)-PED
with low efficiency. To improve the biotransformation efficiency of (R)-PED, the
cofactor-regeneration enzyme GDH was introduced into (R)-PED biosynthesis
pathway in three different E. coli hosts. Among three coupled systems, E. coli
RIL/pET-R-SD-AS-G showed the higher specific activity with 2-HAP and
biotransformation efficiency of (R)-PED than E. coli BL21/pET-R-SD-AS-G and E.
coli Rosetta/pET-R-SD-AS-G. The two enzymes’ functions were rebalanced through
1
7

3
3
3
3
3
3
3
3
3
46
47
48
49
50
51
52
53
54
protein-expression optimization. The kinetic demonstrated the good balance between
the functions of RCR and GDH. At optimum 35°C and pH 7.5, the rebalanced system
gave excellent performance in preparation of (R)-PED with an optical purity of 99.9%
and a yield of 99.9% at 24 h. The introduction of glucose dehydrogenase in E. coli
RIL/pET-R-SD-AS-G stimulated increases of 23.8% and 63.8%, respectively, in
optical purity and yield, and simultaneously reduced the reaction time two-fold (from
48 h to 24 h). This work provides a valuable enzyme-coupled method for efficient
chiral biosynthesis through protein-expression and biotransformation optimization to
rebalance cofactor pathways.
3
55 Acknowledgements
3
3
3
3
3
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3
56
57
58
59
60
61
62
This project was supported by the National Science Foundation of China
(31370100), Jiangsu Province Basic Research Program (BK20131101), the Program
for New Century Excellent Talents in Universities (NCET-13-0833), the Fundamental
Research Funds for the Central Universities (JUSRP51409B),, the National Key Basic
Research and Development 973 Program of China (2011CB710800), and the Program
of Introducing Talents of Discipline to Universities (111-2-06), a Project Funded by the
Priority Academic Program Development of Jiangsu Higher Education Institutions.
3
63 References
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3
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3
64
65
66
67
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1

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85 Figure captions:
86
87
88
89
90
Fig. 1 SDS-PAGE analysis of protein expression in the recombinant strains. 1. E.
coli RIL/pET-R-SD-AS-G induced with 0.1 mM IPTG; 2. E. coli
RIL/pET-R-SD-AS-G without IPTG; 3. E. coli Rosetta/pET-R-SD-AS-G induced with
0.1 mM IPTG; 4. E. coli BL21/pET- R-SD-AS-G induced with 0.1 mM IPTG; 5. E.
coli BL21/pET-28a induced with 0.1 mM IPTG; M. Protein marker.
4
4
91
92
Fig. 2 Cell-growth curve of the recombinant strains.
4
4
4
93
94
95
The recombinant strains were cultivated at 37°C in 2.5-L flask bottles, with an initial
working volume of 1.0 L, and then 0.1 mM IPTG was added at 4 h to induce protein
expression. Error bars represent standard deviations (n = 3).
2
2

4
4
96
97
Fig. 3 The effects of expression conditions on transformation efficiency of
(R)-PED
4
4
5
5
5
5
5
98
99
00
01
02
03
04
The recombinant E. coli RIL/pET-R-SD-AS-G were cultured under the different
protein expression conditions of RCR and GDH: OD600 value (0.4–1.5) of the culture,
IPTG concentrations (0.2–2.0 mM), induce temperature (17°C–37°C), induce times
(8–20 h). The recombinant cells were collected for the biotransformation of (R)-PED
in 0.1 M potassium phosphate buffer (pH 7.0) at 35°C.
2
3

5
5
05
06
5
5
5
07
08
09
Fig. 4 SDS-PAGE analysis of purified RCR and GDH from the coupled system. 1.
Elution with 300 mM imidazole; 2. Cell-free extracts of E. coli RIL/pET-R-SD-AS-G;
3. Whole-cells of E. coli RIL/pET-R-SD-AS-G with 0.5 mM IPTG; M. Protein marker.
2
4

5
5
10
11
Fig.5 Optimal pH and temperature for whole-biocatalyst biotransformation.
The pH optimum of (R)-PED transformation was determined between pH 4.0 and 10.0
using 0.1 M citric acid buffer (pH 4.0, 4.5, 5.0, 5.5), 0.1 M potassium phosphate buffer
(pH 6.0, 6.5, 7.0, 7.5), 50 mM Tris-HCl buffer (8.0), and 0.1 M carbonate buffer (pH
9.0, 10.0). The temperature optimum of (R)-PED transformation was determined at
various temperatures (20°C–80°C).
5
5
5
5
5
12
13
14
15
16
2
5

5
17
2
6

5
5
18
19
Table1
Strains, plasmids and primers
Strains, plasmids
Characteristics
and primers
Source
Strains
E. coli JM 109/
T-R-SD-AS-G
E. coli BL21/
pET-R-SD-AS-G
E. coli
E. coli JM 109 harboring pET-R-SD-AS-G
This work
This work
This work
E. coli BL21 harboring pET-R-SD-AS-G
E. coli RIL harboring pET-R-SD-AS-G
RIL/pET-R-SD-AS
-
G
E. coli Rosseta/
E. coli Rosseta harboring pET-R-SD-AS-G
This work
pET-R-SD-AS-G
Plasmids
pET-RCR
The donor of RCR gene
The donor of GDH gene
This lab
pET-GDH
This lab
r
pMD19-T
Cloning plasmid, 2.7 kb, Amp
Takara Co.
This work
This work
T-R-SD-AS-G
pET-R-SD-AS-G
Primers
R-SD-AS-G genes on pMD19-T, 4.5 kb
R-SD-AS-G gene on pET-28, 7.5 kb
Sequence (5’→3’)
R-SD-AS-G-f
R-SD-AS-G-r
RCR-r
ATCCTGCTAGCATGTCAATTCCATCAAGCCAGTAC（NheI）
TGACTCTCGAGACCGCGGCCTGCCTG（XhoI）
CGGATACATGGTATATCTCCTTCCTATGGATTAAAAACAACTCTACCTT
AATCCATAGGAAGGAGATATACCATGTATCCGGATTTAAAAGGAAAA
GTC
GDH-f
5
5
20
21
Notes: The sequence of SD-AS is bold; the restriction endonuclease sites are underlined.
2
7

5
5
22
23
Table 2
Enzyme activities in cell-free extracts of recombinant strains
-
1
Specific activities (Umg )
Biotransformation
Reductive
activities with
Oxidative
Recombinant strains
Optical
Yield
(%)
activities with
purity (%)
87.9±0.9
96.1±1.2
45.1±1.2
-
1
-1
2
-HAP (Umg )
glucose (Umg )
E. coli
0
0
0
.6±0.02
.6±0.01
.4±0.01
1.1±0.01
60.2±0.2
63.8±0.7
20.4±0.3
BL21/pET-R-SD-AS-G
E. coli
1.3±0.02
0.9±0.01
RIL/pET-R-SD-AS-G
E.coli
Rosetta/pET-R-SD-AS-G
5
5
24
25
Notes: The biotransformations were carried out at 0.1 M potassium phosphate buffer (pH 7.0),
-
1
35°C with 6 gL 2-HAP as substrate.
5
26
2
8

5
5
5
27
28
29
Table 3
Kinetic parameters for 2-HAP and glucose by the purified RCR and GDH in E. coli
a
RIL
Specific
k
cat/K
M
b
-1
Enzymes
activity
K
M
(μM)
kcat (s )
6
-1 -1
(
×10 s M )
-1
(
Umg )
E. coli RIL/RCR2-HAP
E. coli RIL/RCRglucose
18.5
ND
5.3±0.2
18.1±0.3
ND
3
.4
c
ND
ND
3.2
3.9
E. coli RIL/RCR-GDH2-HAP
E. coli RIL/RCR-GDHglucose
17.8
21.5
4.5±0.1
14.2±0.2
31.4±0.3 122.5±0.7
5
5
5
5
5
5
5
5
5
5
30
31
32
33
34
35
36
37
38
39
Notes: The recombinant RCR was purified from E. coli RIL/RCR, and the coupled
enzyme RCR-GDH was purified from E. coli RIL/pET-R-SD-AS-G using His Trap
HP affinity chromatography.
a
All reactions involved in the calculation of kinetic constants calculation were
assayed at 0.1 M potassium phosphate buffer (pH 7.0) and 35°C. All experiments
were repeated three times.
b
RCR2-HAP (RCRglucose) means the purified RCR with 2-HAP (glucose) as substrate;
RCR-GDH2-HAP (RCRglucose) means the purified coupled RCR-GDH with 2-HAP
(glucose) as substrate.
c
ND, not detected.
2
9