Highly stereoselective reduction of prochiral ketones by a bacterial reductase coupled with cofactor regeneration

Article abstract of DOI:10.1039/c1ob05285c

A carbonyl reductase gene (yueD) from Bacillus sp. ECU0013 was heterologously overexpressed in Escherichia coli, and the encoded protein (BYueD) was purified to homogeneity and characterized. The NADPH-dependent reductase showed a broad substrate spectrum towards different aromatic ketones, and α- and β-ketoesters. Although the enantioselectivity was high to moderate for the reduction of α-ketoesters, all the tested β-ketoesters and aromatic ketones were reduced to the corresponding chiral alcohols in enantiomerically pure forms. Furthermore, the practical applicability of this enzyme was evaluated for the reduction of ethyl 4-chloro-3-oxobutanoate (1a). Using Escherichia coli cells coexpressing BYueD and glucose dehydrogenase, 215 g L^-1 (1.3 M) of 1a was stoichiometrically converted to ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-1b) in an aqueous-toluene biphasic system by using a substrate fed-batch strategy, resulting in an overall hydroxyl product yield of 91.7% with enantiomeric purity of 99.6% ee.

Full text of DOI:10.1039/c1ob05285c

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PAPER
Highly stereoselective reduction of prochiral ketones by a bacterial reductase
coupled with cofactor regeneration†
Yan Ni, Chun-Xiu Li,* Li-Juan Wang, Jie Zhang and Jian-He Xu*
Received 22nd February 2011, Accepted 28th April 2011
DOI: 10.1039/c1ob05285c
A carbonyl reductase gene (yueD) from Bacillus sp. ECU0013 was heterologously overexpressed in
Escherichia coli, and the encoded protein (BYueD) was purified to homogeneity and characterized. The
NADPH-dependent reductase showed a broad substrate spectrum towards different aromatic ketones,
and a- and b-ketoesters. Although the enantioselectivity was high to moderate for the reduction of
a-ketoesters, all the tested b-ketoesters and aromatic ketones were reduced to the corresponding chiral
alcohols in enantiomerically pure forms. Furthermore, the practical applicability of this enzyme was
evaluated for the reduction of ethyl 4-chloro-3-oxobutanoate (1a). Using Escherichia coli cells
-
1
coexpressing BYueD and glucose dehydrogenase, 215 g L (1.3 M) of 1a was stoichiometrically
converted to ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-1b) in an aqueous-toluene biphasic system by
using a substrate fed-batch strategy, resulting in an overall hydroxyl product yield of 91.7% with
enantiomeric purity of 99.6% ee.
-
1
Introduction
at an impressive substrate concentration of 300 g L , however,
20
with only 92% enantiomeric excess. Several other synthetic
Chiral secondary alcohols with additional functional groups
are frequently required as important and valuable intermedi-
ates for pharmaceuticals and other fine chemicals. Asymmetric
bioreduction of prochiral ketones to (S) or (R)-enantiomers of
alcohols using isolated enzymes or whole cell systems has attracted
great attention and has been extensively investigated due to its
high stereoselectivity, mild reaction conditions and environmental
21–23
procedures have been developed to obtain (R)-1b;
nevertheless,
the relatively low substrate loadings do limit their potential
for practical application. The discovery of oxidoreductases with
the capability of producing optically pure (R)-1b efficiently is
therefore of great interest. Apart from conventional screening
approaches, increasing availability of genomic information seems
to offer an enormous potential to provide biocatalysts with great
1
compatibility. Although the requirement of expensive cofactors
24,25
applicability.
represents an inherent disadvantage of redox enzymes, several
remarkable achievements have been made in the development of
efficient and cost-effective cofactor recycling systems, such as the
Previously, we reported the isolation and identification of a
ketone reductase-producing Bacillus sp., strain no. ECU0013.
26
This paper deals with the cloning and heterologous expression of
a yueD gene encoding reductase (BYueD) from the strain. The
YueD protein from Bacillus subtilis, which shows 41% amino acid
identity to Bacillus cereus benzil reductase, has been identified to
2–5
enzyme-coupled approach and substrate-coupled system.
Optically active ethyl 4-chloro-3-hydroxybutyrate (1b) is a
useful chiral building block in the synthesis of biologically and
pharmacologically important compounds. For example, (R)-1b
27
reduce benzil to benzoin. Since then, however, there has been no
6
is a precursor for L-carnitine and (R)-4-amino-3-hydroxybutyric
report about any further characterization of this reductase, which
renders it an interesting candidate for us to explore, especially
regarding its substrate spectrum. In this paper, the substrate-
specificities and stereo-specificities of BYueD were evaluated for
the reduction of a variety of ketones. Furthermore, we showed its
applicability in the production of (R)-1b coupled with a NADPH-
regenerating system by glucose dehydrogenase (GDH).
7
acid (GABOB), while (S)-1b is a chiral intermediate for HMG-
8
,9
10
CoA reductase inhibitors and 4-hydroxypyrrolidone. Several
enzymes from various microorganisms have been found to be
1
1–17
able to produce (S)-1b,
whereas (R)-1b is in great demand yet
18,19
less readily available.
A recombinant aldehyde reductase from
Sporobolomyces salmonicolor has been utilized to produce (R)-1b
Results & discussion
Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Lab-
oratory of Bioreactor Engineering, East China University of Science and
Technology, Shanghai, 200237, China. E-mail: chunxiuli@ecust.edu.cn,
jianhexu@ecust.edu.cn; Fax: +86-21-6425-0840
Protein over-expression and purification
BYueD was successfully expressed in E. coli cells with standard
cloning techniques and the genomic DNA of Bacillus sp. ECU0013
†
1
Electronic supplementary information (ESI) available. See DOI:
0.1039/c1ob05285c
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as template. A BLAST search revealed that the protein showed
8% amino acid identity to that from Bacillus subtilis 168
Genbank accession no. AAC44308) and 41% identity to Bacillus
9
(
cereus benzil reductase (Genbank accession no. BAB86009). The
His-tagged recombinant protein was mainly present in the soluble
fraction and was purified through affinity chromatography. The
-
1
specific activity of BYueD after purification was 0.86 U mg
corresponding to a 1.6 fold improvement in activity compared
to the crude extract. Protein separation by SDS-PAGE resulted
in a single band corresponding to a molecular mass of 30.7 kDa
(
Fig. 1), in agreement with its theoretical value. Additionally, the
purified protein eluted from the TSK G2000 SWxl column as a
single peak with an elution volume corresponding to an apparent
molecular mass of 59.3 kDa, which indicated that BYueD was a
homodimeric enzyme consisting of two identical subunits.
Fig. 1 SDS-PAGE analysis of the purified reductase. Lane 1, crude
extract; Lane 2, flow through; Lane 3, purified enzyme; Lane 4, protein
markers. Protein bands were visualized by silver staining.
Fig. 2 Effects of temperature and pH on activity of the purified enzyme.
Dependence of enzyme activity on pH and temperature
(A) Activity–temperature profile was estimated at various temperatures
◦
(
20–60 C) in phosphate buffer (50 mM, pH 7.0) using standard assay. (B)
The effects of temperature and pH on the reductase were
investigated by monitoring the change in enzyme activity over a
range of 20–60 C (Fig. 2a), and a pH range of 5.0–11.0 (Fig. 2b).
Activity–pH profile was determined using standard assay in the following
50 mM buffers: (i) citrate (pH 5.0–6.0); (ii) phosphate (pH 6.0–8.5) and (iii)
Gly-NaOH (pH 8.5–11.0). Relative activity was expressed as a percentage
of the maximum activity under experimental conditions.
◦
According to the activity–temperature profile, the maximum
◦
enzyme activity was observed at a temperature of about 45–50 C
◦
and then reaction rate decreased rapidly over 50 C due to thermal
ketones. Substrate specificity of the enzyme was assessed rapidly
by using spectrophotometric assays and enantioselectivity was
analyzed in aqueous phosphate buffer using an external NADPH
regeneration system consisting of glucose dehydrogenase (GDH)
and glucose. As shown in Table 1, the reductase efficiently
catalyzed the reduction of a- and b-ketoesters, with a dramatically
high activity towards ethyl pyruvate. Interestingly, the reductase
exhibited excellent prochiral selectivity in the reduction of b-
ketoesters (>99% ee), while a-ketoesters were reduced with high to
moderate enantioselectivity. It seems that the size of the R group
had an impact on determining enzyme activity and selectivity for
the reduction of a-ketoesters. As shown in Table 2, the reductase
was also active for the reduction of acetophenone derivatives
and acetyl pyridines, giving corresponding alcohols in excellent
enantiomeric purity (>99% ee). R groups of acetophenone
derivatives exerted a relatively significant effect on the enzyme
activity, with 2,2,2-trifluoroacetophenone being the most active
substrate. On the whole, the enantioselectivity data indicates that
the enantiopreference of this enzyme generally follows Prelog’s
rule. Some of the chiral products have been used as important
building blocks for pharmacologically active compounds or fine
inactivation. The activity–pH profile showed that the enzyme
activity exhibited a pH optimum at 7.5–8.0. An approximate thirty
percent decrease in activity was obtained using Gly-NaOH buffer
instead of phosphate buffer at the same pH values (pH 8.5). These
results are in agreement with benzil reductase from B. cereus which
◦
showed an optimum temperature of 50 C and an optimum pH of
27
6.0–8.0.
Studies concerning the thermostability were performed at three
◦
different temperature points of 30, 40 and 50 C. The enzyme was
◦
quite labile at higher temperature (50 C) but much more stable
◦
◦
at 30 C and 40 C, giving half-lives of 330, 107 and 0.7 h at
3
◦
0, 40 and 50 C, respectively. It suggests that although higher
temperatures resulted in a rapid loss of activity, BYueD was stable
in mild reaction conditions.
Substrate spectrum
The enzyme showed no activity with NADH and full activity with
NADPH. To find a reductase with a broad substrate spectrum,
specific activity and enantioselectivity of the purified enzyme
were assayed in the reduction of various ketoesters and aromatic
5
464 | Org. Biomol. Chem., 2011, 9, 5463–5468
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Table 1 Reduction of a- and b-ketoesters
a
b
Entry
R
1
R
2
Specific activity
ee (%)
c
1
2
3
4
5
6
7
8
CH
CH
CH
2
2
3
Cl
Br
C
C
C
C
C
C
2
2
2
2
2
2
H
H
H
H
H
H
5
5
5
5
5
5
0.86
0.28
0.11
0.012
58
2.0
1.6
>99 (R)c
>99 (R)
>99 (S)
>99 (R)
>99 (S)
CF
3
CH
3
d
(CH
3
5
5
)
2
CH
82 (S)
d
C
C
6
H
H
CH
C
3
75 (R)
6
(CH
2
)
2
2
H
5
0.20
26 (S)
a
The unit of specific activity was U mg-1 protein. The ee value was measured by chiral GC analysis unless indicated otherwise. Determined by chiral
b
c
d
GC analysis after acetylation of the product. Determined by chiral HPLC analysis.
Table 2 Reduction of aromatic ketones
a
b
Entry
9
R
X
Specific activity
ee (%)
H
H
H
H
H
H
Cl
Br
0.023
0.014
0.12
>99 (S)
>99 (R)
>99 (R)
>99 (R)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
10
11
12
13
14
15
16
17
18
Cl
Br
CF
CH
H
3
1.0
3
0.014
0.010
0.026
0.067
0.018
0.049
H
2-Acetylpyridine
3-Acetylpyridine
4-Acetylpyridine
a
The unit of specific activity was U mg-1 protein. The ee value was measured by chiral GC analysis.
b
6
chemicals, for example, (R)-1b for nutrient L-carnitine; (R)-
reductase are unknown. It is worth mentioning that the enzyme
also exhibited a relatively superior preference for ethyl 4-chloro-
3-oxobutanoate (1a), which can be reduced by the enzyme to
optically pure (R)-1b. Therefore, the asymmetric reduction of 1a
with BYueD was further investigated as a case study for evaluating
the practical applicability of this recombinant reductase.
28
4
b for antidepressant befloxatone; (R)-10b for antidepressants
fluoxetine, tomoxetine, and nisoxetine; and (R)-12b for liquid
29
30
crystals.
Kinetic constants
The kinetic constants of the reductase determined for several
of the most active substrates are shown in Table 3. Based
Coexpression of YueD and GDH genes
on the specificity constant (kcat/K
m
), this enzyme shows the
In order to establish the efficient asymmetric bioreduction of
greatest preference for ethyl pyruvate as compared with other
ketones tested. However, the natural substrate and function of the
1a, a glucose dehydrogenase (GDH) from Bacillus subtilis was
introduced for the regeneration of the oxidized cofactor (NADP ).
+
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Table 3 Steady-state kinetic constants
-
1
cat/K
m
(s-1 mM-1)
Substrate
K
m
(mM)
k
cat (s )
k
Ethyl 4-chloro-3-oxobutanoate
Ethyl pyruvate
Ethyl 3-methyl-2-oxobutanoate
Ethyl 2-oxo-4-phenylbutanoate
Methyl benzoylformate
0.70 ± 0.10
0.094 ± 0.002
8.5 ± 1.5
1.08 ± 0.04
48 ± 3
9.0 ± 0.6
0.53 ± 0.03
3.4 ± 0.1
0.29 ± 0.05
1.9 ± 0.1
1.56
513
1.07
0.32
1.31
0.07
0.83
1.7 ± 0.1
2.6 ± 0.1
2
2
-Chloroacetophenone
,2,2-Trifluoacetophenone
4.3 ± 1.3
2.3 ± 0.2
Two E. coli transformant strains were constructed based on
different coexpression styles. On one hand, an expression plasmid
Table 4 Partition coefficients of organic solvents for 1a and 1b and
stabilities of BYueD and GDH in various water/organic solvent biphasic
systems
(
pET-Y-G) with both BYueD and GDH genes were created and
transformed into the E. coli cells (Fig. 3); on the other hand,
two separate plasmids with each of the GDH and BYueD genes
Partition
coefficient
b
c
Remaining activity (%)
(
pET20b-GDH and pET28a-BYueD) were introduced sequen-
a
Organic solvent
Log P
1a
1b
BYueD
GDH
tially into the cells. When each of the two designer cells was
subjected to reduction of 1a in the presence of glucose and
NADP , the higher production of (R)-1b was observed with E. coli
Control
Butyl acetate
Toluene
n-Hexane
Ethyl caprylate
n-Heptane
Isooctane
—
—
—
95.1 ± 1.0
90.7 ± 0.5
0.1 ± 0.0
+
1.7
2.5
3.5
3.8
4.0
4.5
10.6 15.8 0.6 ± 0.3
21.3 2.8
1.5
6.7
1.0
0.9
6.2
4.2 ± 0.0
102.5 ± 0.5
90.6 ± 1.5
52.6 ± 1.8
94.9 ± 0.9
1.4 ± 0.3
cells possessing pET-Y-G, in which BYueD and GDH activities
were 200 U and 1490 U per gram of dry cells, respectively. In
0.3
9.3
-
1
contrast, relatively lower BYueD activity (50 U g dry cells)
0.25 97.2 ± 2.0
0.2
4.2
95.2 ± 4.2
94.6 ± 1.4
95.8 ± 0.5
-
1
98.2 ± 2.5
62.0 ± 1.0
but higher GDH activity (1630 U g dry cells) were observed
in the E. coli cells containing two plasmids, which probably
resulted in the moderate reducing power. Obviously, recombinant
cells with higher BYueD activity would be an ideal choice, since
BYueD activity tended to be the limiting factor of the reaction.
Consequently, E. coli cells harboring pET-Y-G were used as
catalysts for the subsequent experiments.
Dibutylphthalate 5.4
a
b
Ref. 31. Partition coefficient is the molar ratio of each compound found
in the organic phase to the same compound in the aqueous phase (data
from ref. 32, 33 and 34). 0.1 M phosphate buffer (pH 7.0) containing
BYueD (2 U mL ) and GDH (16 U mL ) was combined with an equal
volume of each test organic solvent in a closed vessel. The mixture was
shaken at 30 C for 12 h and the residual enzyme activities in the aqueous
c
-1
-1
◦
solution were assayed.
n-hexane, n-heptane and isooctane, 1a and 1b could not be
efficiently extracted into the organic layer because of the poor
partition efficiencies. Due to the too high boiling point of ethyl
caprylate and dibutylphthalate which would increase the difficulty
of product separation, toluene was eventually selected as the
organic phase throughout the experiments.
Subsequently, a substrate fed-batch strategy was carried out to
mitigate its toxicity and deactivation effect on the enzymes, since
preliminary experiments showed that the reductase activity was
repressed in the presence of high substrate concentration. The
reaction was performed in a water–toluene biphasic system with
Fig. 3 Schematic presentation of the plasmid containing BYueD and
GDH genes and analysis of expression level by SDS-PAGE. (A) Structure
of the coexpression plasmid pET-Y-G. (B) SDS-PAGE analysis of the
expression. Lane 1, soluble fraction of cell-free extract from E. coli with
pET20b-GDH; Lane 2, soluble fraction of cell-free extract from E. coli
with pET28a-BYueD; Lane 3, soluble fraction of cell-free extract from E.
coli with pET-Y-G; Lane 4, soluble fraction of cell-free extract from E. coli
with pET20b-GDH and pET28a-BYueD; Lane 5, protein markers.
-1
a total 1a addition of 1.3 M (215 g L ) that was fed in three
steps, during which the resulting gluconic acid was neutralized
with Na
2
CO to maintain an initial pH of 6.8. As shown in
3
Fig. 4, at 5 h, 1.1 M 1b was produced in the organic phase
with a satisfactory enantiomeric purity of 99.6% ee for the (R)-
isomer and the remaining 1a substrate was not detectable. The
aqueous solution was extracted because of the moderate partition
efficiency of 1b in the water–toluene system. After normal work-
up, the desired hydroxyl product was obtained with an isolated
yield of 91.7%. As far as we know, this is the first report of BYueD
used as a biocatalyst for highly stereoselective reduction of 1a to
(R)-1b. This asymmetric reduction is promising from a practical
viewpoint, since only one example of a bioreduction system has
been reported that can produce (R)-1b with a concentration higher
Bioconversion of 1a to (R)-1b in a biphasic system
As 1a is unstable in an aqueous solution, an water/organic solvent
biphasic system was applied for the bioreduction of 1a. The
stabilities of BYueD and GDH in the presence of various organic
solvents were first determined. From Table 4, it can be seen that
both BYueD and GDH were strongly inactivated in butyl acetate
which had been frequently introduced as the non-aqueous phase
20,32,35–38
-1
20
in the reduction of 1a.
Although the enzymes were stable in
than 200 g L but with enantiopurity of 92% ee.
5
466 | Org. Biomol. Chem., 2011, 9, 5463–5468
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CTACAAAAACTCTTTAATATCATAAATGCG-3¢). The am-
plified DNA was directly cloned into the TA cloning site of
pMD-18T and the desired gene was subcloned into expression
plasmid pET28a (+) between the BamHI and SalI restriction sites
using standard methods, which was subsequently transformed into
Escherichia coli BL21(DE3) cells. The yueD nucleotide sequences
identified in this study have been submitted to GenBank (accession
no. HM590487). The recombinant protein was expressed and
39
purified as described.
Enzyme assays
Reductase and dehydrogenase activities were assayed spectropho-
◦
tometrically at 30 C, by monitoring the decrease or increase
in the absorption of NADPH at 340 nm. The standard assay
mixture (1 mL) for reductase consisted of 50 mM phosphate buffer
Fig. 4 Production of (R)-1b using E. coli BL21 harboring pET-Y-G in a
water–toluene system with a substrate fed-batch strategy. Reduction of 1a
◦
using 0.5 g dry cells was conducted at 30 C in 10 mL 0.1 M phosphate
(
pH 7.0), 2.0 mM carbonyl substrate and 0.05 mM NADPH. The
+
buffer (pH 6.8) and 10 mL toluene containing 10 mmol NADP , 10.0 mmol
assay mixture (1 mL) for GDH activity was 50 mM phosphate
glucose and 5.0 mmol 1a. 5.0 mmol and 3.0 mmol 1a together with 2
equiv. glucose were added to the system at 1 h and 2 h, respectively. (᭜)
Concentration of 1b in organic phase; (᭡) ee.
+
buffer (pH 7.0), 2.0 mM glucose and 0.05 mM NADP . One unit
of activity was defined as the amount of enzyme which catalyzes
the oxidation of 1 mmol NADPH per minute (BYueD) or reduction
+
of 1 mmol NADP per minute (GDH). Kinetic parameters were
Conclusions
determined by altering the concentration of various substrates at
a constant NADPH concentration (0.05 mM) and calculated by
Lineweaver–Burk plots.
In this work, a carbonyl reductase (BYueD) from Bacillus sp.
ECU0013 was overexpressed in E. coli and purified to homogene-
ity, and its enzymatic properties were characterized, especially with
respect to its substrate specificity and stereospecificity. BYueD
served as a versatile reductase with a broad substrate spectrum
accepting a variety of aromatic ketones, and a- and b-ketoesters.
Although the enantioselectivity was high to moderate for the
reduction of a-ketoesters, all the tested b-ketoesters and aromatic
ketones were reduced to the corresponding chiral alcohols with
excellent enantiomeric purity. Furthermore, a tailor-made whole-
cell biocatalyst containing BYueD and GDH was successfully
created and practically used to catalyze the reduction of 1a.
Strategies such as an aqueous/organic biphasic system as well
as substrate fed-batch mode were adopted to alleviate the decom-
position of 1a and its inhibition of the biocatalyst. Consequently,
the designer cells gave a substrate conversion efficiency of 100%
and an enantiomeric purity of the (R)-isomer of 99.6% ee when
subjected to the reduction of 1a at a total concentration of 1.3 M
Effect of pH and temperature on activity
The enzyme activity was estimated using a standard assay protocol
with 1a as substrate. The optimum pH value was determined
within a pH range of 5.0–11.0, using citrate for a pH range from 5.0
to 6.0, phosphate for a pH range from 6.0 to 8.5 and Gly-NaOH
for a pH range from 8.5 to 11.0. The optimum temperature was
◦
determined at different temperatures in the range of 20–60 C. The
stability was examined by incubating the purified enzymes in the
◦
same phosphate buffer (pH 7.0) at 30, 40 or 50 C for a varied
period of time and the residual activities were assayed as described
above.
Enantioselectivity
The enantioselectivity was determined by examining the reduction
of carbonyl substrates using an NADPH regeneration system
consisting of the purified reductase and externally added glucose
dehydrogenase (GDH). The reaction was carried out in a reaction
mixture (0.4 mL) comprising 50 mM potassium phosphate buffer
-
1
(
215 g L ). This study not only provides useful guidance for further
application of BYueD in biocatalytic production of chiral alcohols
but also establishes an efficient process for the direct synthesis of
(
R)-1b with excellent enantiopurity.
(
0
pH 7.0), 10 mM keto-substrate, 0.2 U of the purified reductase,
+
.4 U of GDH, 20 mM glucose and 0.5 mM NADP with shaking
Experimental section
◦
for 12 h at 30 C. After the reaction, each reaction mixture
was extracted twice with ethyl acetate. The substrate conversion
and product ee value were determined by GC analysis using a
CP-Chirasil-DEX CB (Varian, USA) or HPLC analysis using a
Chiralcel OD-H column (Daicel Co., Japan; 4.6 ¥ 250 mm).
Cloning, expression and purification
Bacillus sp. ECU0013, currently deposited in China General
Microbiological Cultures Center with an accession number of
CGMCC No. 2549, was used as the DNA donor. The strain was
26
cultivated as described previously. The genomic DNA of Bacillus
sp. ECU0013 was extracted and purified using the TIANamp
Bacteria DNA Kit (Tiangen, Shanghai, China). DNA fragments
containing yueD were amplified by polymerase chain reaction
Coexpression of reductase and GDH genes
Construction of the expression vector pET20b-GDH was de-
40
scribed by Zhang et al. To construct plasmid pET-Y-G, the
primers with 15 bases of sequence homology to linearized vectors
were used for PCR with the GDH gene as the template. The
(
PCR) using primers based on the yueD sequence of Bacillus sub-
tilis 168 (5¢-ATGGAACTTTATATCATCACCGGAG-3¢ and 5¢-
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resulting fragment GDH gene was then cloned into the SalI/XhoI
8 D. S. Karanewsky, M. C. Badia, C. P. Ciosek, J. A. Robl, M. J. Sofia,
L. M. Simpkins, B. Delange, T. W. Harrity, S. A. Biller and E. M.
Gordon, J. Med. Chem., 1990, 33, 2952–2956.
TM
site of pET28a-BYueD using CloneEZ
PCR Cloning Kit
(
GenScript, Nanjin, China). The resulting plasmids with reductase
9
M. M u¨ ller, Angew. Chem., Int. Ed., 2005, 44, 362–365.
and GDH genes were transformed into E. coli BL21(DE3). To
construct the two-plasmid system, E. coli BL21 (DE3) harboring
pET20b-GDH was transformed with pET28a-BYueD and grown
10 E. Santaniello, R. Casati and F. Milani, J. Chem. Res., Synop., 1984,
132–133.
1
1
1 W. Shieh, A. S. Gopalan and C. J. Sih, J. Am. Chem. Soc., 1985, 107,
2
993–2994.
-
1
on an LB plate containing ampicillin (100 mg L ) and kanamycin
2 M. Wada, M. Kataoka, H. Kawabata, Y. Yosohara, N. Kizaki, J.
-
1
(
50 mg mL ). The transformation was confirmed by agarose gel
Hasegawa and S. Shimizu, Biosci., Biotechnol., Biochem., 1998, 62,
2
80–285.
electrophoresis and enzymatic activity assay.
1
3 M. Wada, M. Kataoka, H. Kawabata, M. Kataoka, Y. Yasohara, N.
Kizaki, J. Hasegawa and S. Shimizu, J. Mol. Catal. B: Enzym., 1999, 6,
3
33–339.
Bioreduction of 1a to (R)-1b
1
1
4 H. Yamamoto, N. Kimoto, A. Matsuyama and Y. Kobayashi, Biosci.,
Biotechnol., Biochem., 2002, 66, 1775–1778.
Bioconversion of 1a to (R)-1b was carried out by whole-cell
reaction of E. coli harboring pET-Y-G. The reaction mixture
consisted of 1.0 mmol potassium phosphate buffer (pH 7.0),
5 H. Yamamoto, K. Mitsuhashi, N. Kimoto, A. Matsuyama, N. Esaki
and Y. Kobayashi, Biosci., Biotechnol., Biochem., 2004, 68, 638–649.
16 M. Amidjojo and D. Weuster-Botz, Tetrahedron: Asymmetry, 2005, 16,
+
1
0.0 mmol of glucose, 10 mmol NADP , 0.5 g of dry cells in 10 mL
899–901.
1
7 Q. Ye, M. Yan, Z. Yao, L. Xu, H. Cao, Z. J. Li, Y. Chen, S. Y. Li, J. X.
Bai, J. Xiong, H. J. Ying and P. K. Ouyang, Bioresour. Technol., 2009,
1
of aqueous solution, to which 1a (830 mg, 5.0 mmol; dissolved
in 10 mL of toluene) was added. The two phase bioreaction was
performed by magnetic agitation at 30 C for 5 h. 1a (5.0 and
00, 6022–6027.
◦
18 G. Wang and R. I. Hollingsworth, Tetrahedron: Asymmetry, 1999, 10,
895–1901.
9 A. Forni, E. Caselli, F. Prati, M. Bucciarelli and G. Torre, Arkivoc,
1
3
.0 mmol) and glucose (10.0 and 6.0 mmol) were supplemented
to the reaction system at 1 h and 2 h. The pH was automatically
adjusted to 6.8 by titrating 2 M Na CO . The reaction mixture
1
2
2
002, 11, 45–53.
2
3
0 M. Kataoka, K. Yamamoto, H. Kawabata, M. Wada, K. Kita, H.
was centrifuged (10 000 ¥ g for 5 min) to promote the phase
separation; the aqueous phase was saturated with NaCl and then
extracted three times with ethyl acetate. The organic solution was
Yanase and S. Shimizu, Appl. Microbiol. Biotechnol., 1999, 51, 486–
4
90.
2
1 T. Suzuki, H. Idogaki and N. Kasai, Enzyme Microb. Technol., 1999,
2
4, 13–20.
combined, dried over anhydrous Na
2
SO
4
and evaporated under
22 H. Yamamoto, A. Matsuyama and Y. Kobayashi, Biosci., Biotechnol.,
Biochem., 2002, 66, 481–483.
vacuum. Chemical yield of the product was determined by GC
analysis and the enantiomeric purity of (R)-1b was determined
by chiral GC analysis after acetylation. (R)-1b was afforded as a
2
3 M. A. Yu, Y. M. Wei, L. Zhao, L. Jiang, X. B. Zhu and W. Qi, J. Ind.
Microbiol. Biotechnol., 2007, 34, 151–156.
4 A. Beloqui, P. Dom ´ı nguez de Mar ´ı a, P. N. Golyshin and M. Ferrer,
Curr. Opin. Microbiol., 2008, 11, 240–248.
25 D. J. Pollard and J. M. Woodley, Trends Biotechnol., 2006, 25, 67–73.
6 Y. Xie, J. H. Xu and Y. Xu, Bioresour. Technol., 2010, 101, 1054–1059.
7 R. Maruyama, M. Nishizawa, Y. Itoi, S. Ito and M. Inoue, J. Biotech-
nol., 2002, 94, 157–169.
2
2
5
colorless liquid, with a yield of 1.97 g (91.7%). [a] : +22.3 (c 5.0,
D
4
1
25
D
CHCl
(
2
3
1
), 99.6% ee, (R) {lit. [a] : +20.1 (c 8.24, CHCl
3
), 96% ee,
): d 1.29 (t, 3H, J = 7.1 Hz),
2
2
R)}; H NMR (500 MHz, CDCl
3
.59–2.67 (m, 2H), 3.20 (br s, 1H), 3.59–3.64 (m, 2H), 4.19 (q, 2H,
1
3
J = 7.1 Hz), 4.24–4.29 (m, 1H). C NMR (100 MHz, CDCl
4.1, 38.6, 48.2, 60.5, 67.9, 171.8.
3
): d
28 X. Rabasseda, L. A. Sorbera and J. Castaner, Drugs Future, 1999, 24,
057–1067.
9 R. Ramu, G. B. R. Khanna and A. Kamal, Tetrahedron: Asymmetry,
002, 13, 2039–2051.
0 T. Fujisawa, K. Ichikawa and M. Shimizu, Tetrahedron: Asymmetry,
993, 4, 1237–1240.
1 C. Laane, S. Boeren, K. Vos and C. Veeger, Biotechnol. Bioeng., 1987,
1
1
2
3
3
3
2
1
Acknowledgements
3
0, 81–87.
This work was financially supported by the National Natural
Science Foundation of China (Nos. 20902023 & 31071604), Min-
istry of Science and Technology, P. R. China (Nos. 2011CB710800
2 S. Shimizu, M. Kataoka, M. Katoh, T. Morikawa, T. Miyoshi and H.
Yamada, Appl. Environ. Microbiol., 1990, 56, 2374–2377.
33 J. Y. He, Z. H. Sun, W. Q. Ruan and Y. Xu, Process Biochem., 2006, 41,
&
2009ZX09501-016), and China National Special Fund for State
244–249.
3
4 Q. Ye, H. Cao, M. Yan, F. Cao, Y. Y. Zhang, X. M. Li, L. Xu, Y. Chen,
Key Laboratory of Bioreactor Engineering (No. 2060204).
J. Xiong, P. K. Ouyang and H. J. Ying, Bioresour. Technol., 2010, 101,
6
761–6767.
3
5 Y. Yasohara, N. Kizaki, J. Hasegawa, S. Takahashi, M. Wada, M.
Kataoka and S. Shimizu, Appl. Microbiol. Biotechnol., 1999, 51, 847–
References
8
51.
1
2
R. N. Patel, Adv. Synth. Catal., 2001, 343, 527–546.
W. Kroutil, H. Mang, K. Edegger and K. Faber, Curr. Opin. Chem.
Biol., 2004, 8, 120–126.
K. Goldberg, K. Schroer, S. L u¨ tz and A. Liese, Appl. Microbiol.
Biotechnol., 2007, 76, 237–248.
F. Hollmann, I. W. C. E. Arends and K. Buehler, ChemCatChem, 2010,
36 N. Kizaki, Y. Yasohara, J. Hasegawa, M. Wada, M. Kataoka and S.
Shimizu, Appl. Microbiol. Biotechnol., 2001, 55, 590–595.
37 H. Engelking, R. Pfaller, G. Wich and D. Weuster-Botz, Tetrahedron:
Asymmetry, 2004, 15, 3591–3593.
38 Q. Ye, H. Cao, L. Mi, M. Yan, Y. Wang, Q. T. He, J. Li, L. Xu, Y. Chen,
J. Xiong, P. K. Ouyang and H. J. Ying, Bioresour. Technol., 2010, 101,
8911–8914.
3
4
5
6
7
2
, 762–782.
G. W. Huisman, J. Liang and A. Krebber, Curr. Opin. Chem. Biol.,
010, 14, 122–129.
39 Y. Ni, C. X. Li, H. M. Ma, J. Zhang and J. H. Xu, Appl. Microbiol.
2
Biotechnol., 2011, 89, 1111–1118.
B. N. Zhou, A. S. Gopalan, F. Vanmiddlesworth, W. R. Shieh and C. J.
Sih, J. Am. Chem. Soc., 1983, 105, 5925–5926.
H. C. Kolb, Y. L. Bennani and K. B. Sharpless, Tetrahedron: Asymme-
try, 1993, 4, 133–141.
40 J. D. Zhang, A. T. Li, H. L. Yu, T. Imanaka and J. H. Xu, J. Ind.
Microbiol. Biotechnol., 2011, 38, 633–641.
41 J. P. Marino, M. S. McClure, D. P. Holub, J. V. Comasseto and F. C.
Tucci, J. Am. Chem. Soc., 2002, 124, 1664–1668.
5
468 | Org. Biomol. Chem., 2011, 9, 5463–5468
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