Synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate with recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase

Article abstract of DOI:10.1271/bbb.66.481

The synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-ECHB) from ethyl 4-chloroacetoacetate was studied using whole recombinant cells of Escherichia coli expressing a secondary alcohol dehydrogenase of Candida parapsilosis. Using 2-propanol as an energy source to regenerate NADH, the yield of (R)-ECHB reached 36.6 g/l (more than 99% ee, 95.2% conversion yield) without addition of NADH to the reaction mixture.

Full text of DOI:10.1271/bbb.66.481

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Synthesis of Ethyl (R)-4-Chloro-3-hydroxybutanoate
with Recombinant Escherichia coli Cells Expressing
(S)-Specific Secondary Alcohol Dehydrogenase
Hiroaki YAMAMOTOa, Akinobu MATSUYAMAa & Yoshinori KOBAYASHIa
a Tsukuba Research Center, Daicel Chemical Industries, Ltd. 27 Miyukigaoka, Tsukuba,
Ibaraki 305-0841, Japan
Published online: 22 May 2014.
To cite this article: Hiroaki YAMAMOTO, Akinobu MATSUYAMA & Yoshinori KOBAYASHI (2002) Synthesis of Ethyl (R)-4-
Chloro-3-hydroxybutanoate with Recombinant Escherichia coli Cells Expressing (S)-Specific Secondary Alcohol
Dehydrogenase, Bioscience, Biotechnology, and Biochemistry, 66:2, 481-483, DOI: 10.1271/bbb.66.481
To link to this article: http://dx.doi.org/10.1271/bbb.66.481
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Biosci. Biotechnol. Biochem., 66 (2), 481–483, 2002
Note
Synthesis of Ethyl (R)-4-Chloro-3-hydroxybutanoate with Recombinant
Escherichia coli Cells Expressing (S)-Speciˆc Secondary
Alcohol Dehydrogenase
Hiroaki YAMAMOTO,† Akinobu MATSUYAMA, and Yoshinori KOBAYASHI
Tsukuba Research Center, Daicel Chemical Industries, Ltd., 27 Miyukigaoka, Tsukuba,
Ibaraki 305-0841, Japan
Received October 2, 2001; Accepted November 7, 2001
The synthesis of ethyl
(R)-4-chloro-3-hydroxy-
as 4-hydroxy-2-butanone, 2-butanone, acetone, and
acetophenone. Furthermore, we reported the cloning
and expression of the gene coding for CpSADH.14) In
butanoate (( )-ECHB) from ethyl 4-chloroacetoacetate
R
was studied using whole recombinant cells of Es-
cherichia coli expressing a secondary alcohol de-
hydrogenase of Candida parapsilosis. Using 2-propanol
as an energy source to regenerate NADH, the yield of
this report, we describe the synthesis of (R)-ECHB
by asymmetric reduction of ECAA without the addi-
tion of NADH, using whole E. coli cells overproduc-
ing CpSADH.
A high-expression plasmid for CpSADH, pSE-
CPA1, was constructed based on the pSE420 vector
containing the trc promoter (Invitrogen Corporation,
Carlsbad, California), as illustrated in Fig. 1. E. coli
cells harboring pSE-CPA1 were grown in 100 ml of 2
(R)-ECHB reached 36.6 g l (more than 99% ee, 95.2%
W
conversion yield) without addition of NADH to the
reaction mixture.
Key words:
(
S
)-speciˆc secondary alcohol dehydro-
genase; ethyl )-4-chloro-3-hydroxy-
(
R
butanoate; NADH-regeneration; asym-
metric reduction
Ethyl
(R)-4-chloro-3-hydroxybutanoate ((R)-
ECHB) is a chiral compound useful for the synthesis
of biologically and pharmacologically important
materials: (
R
)-carnitine,1,2)
(
R
)-4-amino-3-hydroxy-
butyric acid,3,4) and (
R
)-4-hydroxy-2-pyrrolidone.5,6)
Several microorganisms and enzymes have been
found to catalyze the reduction of ethyl 4-chloroa-
cetoacetate (ECAA) to (R
)-ECHB.7–9) In particular,
10)
Kataoka et al
.
reported the production of (
and an optical
enantiomer excess (ee), using Es-
R)-
ECHB, with a molar yield of 94
purity of 92
z
z
cherichia coli cells co-expressing an aldehyde reduc-
tase I11) from Sporobolomyces salmonicolor and a
glucose dehydrogenase12) from Bacillus megaterium
as catalyst, in an
system.
n-butyl acetate water biphasic
W
In our previous report,13) we showed that an
Fig. 1. Construction of pSE-CPA1.
The cloning vector pSE420 was digested with NcoI and
BamHI, treated with the Klenow fragment, and self-ligated to
give pSE420B. A 1,024-bp DNA fragment containing the entire
CpSADH gene was ampliˆed by PCR with the primers CPA-
NAD+-dependent
dehydrogenase (CpSADH) produced by Candida
parapsilosis IFO 1396 could catalyze the enan-
tioselective oxidation of several secondary alcohols,
such as (S)-1,3-butanediol, (S)-2-butanol, and (S)-1-
phenylethanol, and also the oxidation of achiral 2-
propanol, and the reduction of several ketones, such
(S)-speciˆc secondary alcohol
ATG2
(TTTGGATCCAACAATGTCAATTCCATCAAGC)
and CPA-TAG2 (CCTCTCGAGTTACTATGGATTAAAAA-
CAACTC) from pKK-CPA1,14) and was inserted at the BamHI
and XhoI sites of pSE420B to give the ˆnal construct pSE-
CPA1.
†
To whom correspondence should be addressed. Tel: +81-298-56-1322; Fax: +81-298-56-1323; E-mail: h-yamamoto
＠
daicel.co.jp
Abbreviations: CpSADH, secondary alcohol dehydrogenase from Candida parapsilosis; ECAA, ethyl 4-chloroacetoacetate; ee, enan-
tiomer excess; ECHB, ethyl 4-chloro-3-hydroxybutanoate

482
H. YAMAMOTO et al.
Table 1. EŠects of Energy Source on the Synthesis of (
Using Whole E. coli Cells Harboring pSE-CPA1
R
)-ECHB
Yield
×
YT medium (Bacto-Tryptone, 20 g l; Bacto-Yeast
W
extract, 10 g l; NaCl, 10 g l; pH 7.2) containing
W
W
ampicillin (50 mg l) in a 500-ml baŒed shake ‰ask at
W
(R)-ECHB
ECAA
30
9
C on a rotary shaker (140 rpm) to the optical den-
Energy source
(g l)
W
(g l)
(
ee
)
W
sity at 600 nm of 3–4; after addition of 2
z
lactose as
an inducer, the culture medium was further shaken
for 11 h. The cells harvested by centrifugation were
5
z
Glucose
4.28
ND
1.40
9.83
À
À
99
99
13.8
97.1
z
z
5
z
2-Propanol
suspended in 50 m
M
z
Tris-HCl (pH 9.0) and 0.02 2-
The reaction mixture (25 ml) containing 200 m
buŠer (pH 6.5), 1 ECAA, cultured cells obtained from 25 ml of the medi-
um, and 5 (w v) of the indicated compound for energy source (glucose or
2-propanol) was shaken in a 500-ml Sakaguchi ‰ask at 140 strokes per min
and 30 C for 17 h. The amounts of ECAA and ECHB, and the optical puri-
M potassium phosphate
mercaptoethanol, and disrupted with a Bioruptor
UCD-200 (Cosmo BIO Co., Ltd., Tokyo, Japan).
The supernatant was obtained by centrifugation at
z
z
W
9
16,000
trophotometrically at 30
mixture contained 50 mol of (
2.5 mol of Tris-HCl buŠer (pH
mol of NAD+, 50
9), and enzyme in a ˆnal volume of 1 ml. One unit of
the enzyme is deˆned as the amount of enzyme that
g
for 10 min. CpSADH was assayed spec-
C. The standard assay
)-1,3-butanediol,
ty of ECHB in the reaction mixture were measured by gas chromatography
and chiral HPLC. ND, not detected.
9
m
m
S
m
Table 2. EŠects of the Ratio of 2-Propanol to ECAA on the Syn-
thesis of (R)-ECHB
catalyzes the formation of 1
at 30 C.
mmol of NADH per min
Reaction mixture
ECAA 2-Propanol
4 eq.
(
R
)-ECHB
ECAA
9
(g l)
W
(g l)
ee Yield
(
z
)
W
E. coli W3110 cells harboring pSE-CPA1 prepared
4
z
13.4
10.6
4.4
26.9
29.1
31.9
À
À
À
99
99
99
66.4
71.9
78.8
z
z
z
by the method mentioned above showed a CpSADH
(243 m
M
)
3 eq.
2 eq.
activity of 43.2 U ml-medium, which was 78-fold
W
higher than that of Candida parapsilosis. ECAA
Reaction conditions were the same as those in Table 1 except for the use of
z ECAA and the amounts of 2-propanol added. Eq., molar excesses.
served as an about 3.4-times better substrate for
4
CpSADH than 4-hydroxy-2-butanone (212 U mg-
W
protein at pH 6.0 and 30
Using the whole E. coli W3110 cells harboring
pSE-CPA1, the synthesis of ( )-ECHB was investi-
9
C).
R
gated. At ˆrst, the energy source for the microbial
reduction was examined. The reduction of ECAA
was done at 30
(25 ml) containing 200 m
buŠer (pH 6.5), 1 ECAA, cultured cells obtained
from 25 ml of the medium, and 5 (w v) of a com-
9
C for 17 h in the reaction mixture
M
potassium phosphate
z
z
Fig. 2. Enzymatic Reduction of ECAA and Regeneration of a
Coenzyme, NADH, Using CpSADH.
W
pound as an energy source (either glucose or 2-
propanol) with shaking at 140 strokes per min in a
500-ml Sakaguchi ‰ask. These reactions were done at
pH 6.5, as ECAA was unstable and was degraded at
more than pH 7.0, and the optimal pH for asymmet-
ric reduction of ECAA was 6.5 as a result of prelimi-
nary experiments. 2-Propanol was expected to func-
tion as a co-substrate for CpSADH to regenerate
NADH, as it was a good substrate for oxidation by
CpSADH and an inexpensive compound. The
amounts of ECAA and ECHB in the reaction
mixture were measured by gas chromatography
(Shimadzu GC-14A, Kyoto, Japan) under the follow-
of (
z
R)-ECHB (13.8 conversion) from 10 g l of
W
ECAA, while the use of 2-propanol aŠorded 9.83 g l
W
of ECHB (97.1
quantitative conversion with very high optical selec-
tivity (more than 99 ee). These ˆndings suggested
z
conversion), resulting in an almost
z
that NADH regeneration using glucose as an energy
source was ine‹cient in E. coli, or alternatively
CpSADH itself could regenerate its cofactor e‹cient-
ly by oxidation of 2-propanol as a co-substrate as
shown in Fig. 2.
ing conditions: column, Thermon 3000 5
mosorb W (Shinwa Chemical Industries Ltd., Kyoto,
Japan); column temperature, 150 C; carrier gas, N2;
z
Chro-
Secondly, the ratio of 2-propanol as a co-substrate
to ECAA as a substrate was optimized in the reaction
mixture. As shown in Table 2, two molar excesses of
9
detection, ‰ame ionization detector. The optical
purity of ECHB was measured by chiral HPLC
2-propanol over ECAA gave the best result (31.9 g l
W
of (
addition of 2-propanol to the reaction mixture gave a
lowered amount of )-ECHB accumulated,
R)-ECHB; conversion yield, 78.8z). Further
with a Chiralcel AS packed column (4.6
×
250 mm,
Daicel Chemical Industries Ltd., Tokyo, Japan) at
(
R
0
9C, eluted with
n
-hexane:ethanol:2-propanol:cyclo-
presumably due to an inhibitory eŠect of 2-propanol
on CpSADH.
Finally, the temperature for the conversion
9
reaction was optimized in the range of 20–30 C. As
hexane (92:2.5:1.25:0.25) at a ‰ow rate of 1 ml min,
W
and detected at 220 nm. As shown in Table 1, the use
of glucose as an energy source aŠorded only 1.40 g l
W

Synthesis of Ethyl (
R
)-4-Chloro-3-hydroxybutanoate
483
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102, 6304–6311
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R
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(
R
)-ECHB
Reaction
Temp.
ECAA
(g l)
W
(„)-g-amino-(R)-b-hydroxybutyric acid (GABOB).
(g l)
ee
(
z)
Yield
W
J. Org. Chem., 49, 3707–3711 (1984).
30
25
20
9
C
9.7
2.5
0.5
24.0
35.3
36.6
À
À
À
99
99
99
62.4
91.7
95.2
z
z
z
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C
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Reaction conditions were the same as those in Table 1 except for the tem-
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result for the reaction with 3.8 ECAA and 2 molar
excesses of 2-propanol over ECAA (36.6 g l of ( )-
). These ˆndings sug-
gested lower temperatures resulted in the increased
stability of the substrate, ECAA, and less inhibition
of CpSADH by ECAA.
In conclusion we have established a highly enan-
tioselective and e‹cient method for the enzymatic
synthesis of (R)-ECHB by asymmetric reduction
using whole recombinant cells expressing CpSADH
without addition of an expensive coenzyme, NADH.
The microbial reduction for the synthesis of (R)-
ECHB using the recombinant cells could be scaled up
to a 30-l reactor scale in our laboratory. Surprisingly,
CpSADH had little dehydrogenase activities for (
ECHB as well as ( )-ECHB, although it showed a
high activity for ethyl )-3-hydroxybutanoate
9C gave the best
z
R
W
ECHB; conversion yield, 95.2
z
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Howell, J. M., Robison, R. S., and Szarka, L.,
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