Synthesis of (R)-1,3-butanediol by enantioselective oxidation using whole recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase

Article abstract of DOI:10.1271/bbb.66.925

The synthesis of (R)-1,3-butanediol (BDO) from its racemate was studied using whole cells of recombinant Escherichia coli expressing an (S)-specific secondary alcohol dehydrogenase (CpSADH) from Candida parapsilosis by enantioselective oxidation. Under the optimized conditions, the yield of (R)-1,3-BDO reached 72.6 g/l, with a molar recovery yield of 48.4% from a racemate of 15% and an optical purity of 95% ee.

Full text of DOI:10.1271/bbb.66.925

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Synthesis of (R)-1,3-Butanediol by Enantioselective
Oxidation Using Whole 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 (R)-1,3-Butanediol
by Enantioselective Oxidation Using Whole Recombinant Escherichia coli Cells Expressing (S)-Specific Secondary Alcohol
Dehydrogenase, Bioscience, Biotechnology, and Biochemistry, 66:4, 925-927, DOI: 10.1271/bbb.66.925
To link to this article: http://dx.doi.org/10.1271/bbb.66.925
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Biosci. Biotechnol. Biochem., 66 (4), 925–927, 2002
Note
Synthesis of (R)-1,3-Butanediol by Enantioselective Oxidation Using
Whole 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 November 6, 2001; Accepted December 5, 2001
The synthesis of (
racemate was studied using whole cells of recombinant
Escherichia coli expressing an ( )-speciˆc secondary al-
cohol dehydrogenase (CpSADH) from Candida parap-
silosis by enantioselective oxidation. Under the op-
R)-1,3-butanediol (BDO) from its
shaker (140 rpm) to the optical density at 600 nm
(OD600) of 3–4; after the addition of 2 lactose as an
z
S
inducer, the culture medium was further shaken for
11 h. The cells harvested by centrifugation were used
as a catalyst.
timized conditions, the yield of (
R)-1,3-BDO reached
The synthesis of (
the standard reaction mixture (25 ml) containing
100 m potassium phosphate buŠer (pH 6.8), racem-
9
R)-1,3-BDO was done at 30 C in
72.6 g l, with a molar recovery yield of 48.4
%
%
from a
ee
W
racemate of 15
%
and an optical purity of 95
.
M
ic 1,3-BDO, and cultured cells obtained from 25 ml
of the medium with shaking at 145 strokes per min in
a 500-ml Sakaguchi ‰ask. The amounts of 1,3-BDO
and 4H2B were measured by gas chromatography
under the following conditions: column, PoraPak PS
(Waters Corporation, Milford, MA, U.S.A.);
Key words: secondary alcohol dehydrogenase; (
1,3-butanediol;
NAD+
enantioselective oxidation
R
)-
-regeneration;
(R)-1,3-butanediol (BDO) is an optically active al-
cohol used as an intermediate for the production of
pharmaceuticals such as azetidinone derivatives.1) We
are studying a method for the practical synthesis of
9
column temperature, 165 C; carrier gas, N2; detec-
tion, ‰ame ionization detector. The optical purity of
1,3-BDO was measured as 1,3-BDO diacetyl by chiral
(
R
)-1,3-BDO from the more inexpensive racemate by
HPLC with a Chiralcel OB packed column (4.6
×
enantioselective oxidation, rather than from the cor-
responding ketone, 4-hydroxy-2-butanone (4H2B) by
asymmetric reduction. In our previous reports, we
showed that Candida parapsilosis IFO 1396 cells pro-
250 mm, Daicel Chemical Industries, Ltd., Tokyo,
9
Japan) at 40 C, eluted with n-hexane:2-propanol
(19:1) at a ‰ow rate of 1 ml min, and detected at
W
220 nm.
duced (
of (
R
)-1,3-BDO by the enantioselective oxidation
The pH of a reaction mixture containing cultured
cells obtained from 3.8 ml of the medium was op-
timized in the range of 6.5–8.0, as lysis and foaming
in reaction mixtures at pH above 8.0 were observed
and prohibited exact analyses. As shown in Fig. 1,
after the incubation of 17 h, there was little diŠerence
in the optical purities of residual 1,3-BDO among pH
6.5–8.0; after an incubation of 40 h, alternatively,
the reaction at pH 6.8 gave the highest optical purity
S
)-1,3-BDO2) and that the enantioselective oxida-
tion was catalyzed by an (S)-speciˆc secondary alco-
hol dehydrogenase (CpSADH).3) Furthermore, we
reported the cloning and expression of the CpSADH
gene and the preliminary synthesis of (R)-1,3-BDO
with recombinant E. coli cells expressing CpSADH.4)
In this paper, we describe the practical production of
(R)-1,3-BDO under optimal conditions.
E. coli cells expressing CpSADH for the synthesis
(88.6z ee (R)). These ˆndings suggested that the
of (
R
)-1,3-BDO were prepared by the following
rate-limiting step of enantioselective oxidation was a
method. E. coli W3110 (IFO 12713) cells, harboring
regeneration step of the coenzyme, NAD+, and the
a high expression plasmid for CpSADH, pSE-C-
optimal pH and or the stablest pH for the regenera-
W
PA1,5) were grown in 100 ml of a 2 YT medium
tion of the coenzyme was around pH 6.8, since the
×
(Bacto-Tryptone, 20 g l; Bacto-Yeast extract, 10 g l;
optimal pH of CpSADH for the oxidation of (S)-1,3-
W
W
W
NaCl, 10 g l; pH 7.2) containing ampicillin (50 mg l)
BDO was 9.0 and CpSADH was stable within the
W
in a 500-ml baŒed shake ‰ask at 30
9C on a rotary
range of 8.0–11.0.3)
†
To whom correspondence should be addressed. Tel: +81-298-56-1322; Fax: +81-298-56-1323; E-mail: h-yamamoto
＠
daicel.co.jp
Abbreviations: 4H2B, 4-hydroxy-2-butanone; BDO, butanediol; CpSADH, (
parapsilosis ee, enantiomer excess
S
)-speciˆc secondary alcohol dehydrogenase of Candida
;

926
H. YAMAMOTO et al.
Fig. 2. EŠects of the Concentrations of 1,3-BDO on Enan-
Fig. 1. EŠects of pH on the Synthesis of (
R
)-1,3-BDO.
potassium
1,3-BDO, and cultured cells
obtained from 3.8 ml of the medium, was shaken in a 500-ml
Sakaguchi ‰ask at 140 strokes per min and 30 C for 40 h. The
optical purity of ( )-1,3-BDO after an incubation of 17 ( ) and
40 ( ) h was measured by chiral HPLC.
tioselective Oxidation.
The reaction mixture (25 ml), containing 7–15
The reaction mixture (25 ml), containing 100 m
phosphate buŠer (pH 6.5–8.0), 5
M
z 1,3-BDO and
z
cultured cells obtained from 25 ml of the medium, was incubat-
ed at pH 6.8. Other reaction conditions and analytical methods
were the same as those given in Fig. 1. 1,3-BDO concentrations:
9
R


), 10

#
$

z ( ).
7
z
(
z
(
), 12
z
(
), 15
z
(
), 20

Table 1. EŠects of the Incubation in the Reaction Mixture Con-
taining 10 1,3-BDO on the Residual Activities of CpSADH and
the 1,3-BDO-producing Activity
Secondly, the eŠects of the concentrations of
1,3-BDO in the reaction mixture were studied. At the
z
reaction containing 7
z
1,3-BDO, the optical purity
ee after incubation
Speciˆc 1,3-BDO-produc-
ing activity
of ( )-1,3-BDO reached 97.6
R
z
CpSADH
Treatment
(
z)
of 17 h, as shown in Fig. 2. However, in the reactions
containing higher concentrations of 1,3-BDO, the
optical purities of (R)-1,3-BDO did not increase
(
z
ee OD600 h)
W
W
None
After reaction
After storage
100
64.5
81.4
1.90
0
1.76
from a 17-h to a 40-h incubation. To elucidate the
reason for the cessation of enantioselective oxidation
The reaction mixture (25 ml), containing 10
obtained from 25 ml of the medium, was shaken at 30
z
1,3-BDO and cultured cells
C for 17 h. Other
in the reaction mixtures containing 10
the residual CpSADH activity of the cell-free extract
and the ( )-1,3-BDO-producing activity of E. coli
cells were measured. After a 17-h incubation, E. coli
cells retained a CpSADH activity of 64.5 but had
z
1,3-BDO,
9
reaction conditions were the same as those given in Fig. 1. The cells were col-
lected by centrifugation and washed with saline, and the CpSADH activity
R
of the cell-free extracts and the (
were measured. The CpSADH activity was measured as follows: the collect-
ed cells were suspended in 50 m Tris-HCl (pH 9.0) and 0.02 2-mercap-
toethanol, and disrupted with a Bioruptor UCD-200 (Cosmo BIO Co., Ltd.,
R)-1,3-BDO-producing activity of the cells
z
M
z
no 1,3-BDO-producing activity, as shown in Table 1.
These ˆndings suggested that the cessation of enan-
tioselective oxidation resulted from the loss of NAD+
Tokyo, Japan). The supernatant was obtained by centrifugation at 16,000
g
for 10 min. The CpSADH was assayed spectrophotometrically.3) One unit
of the enzyme is deˆned as the amount of enzyme that catalyzes the forma-
and or the NAD+-regeneration activity of the cell.
tion of 1
was measured as follows:
1,3-BDO and collected cells (45 OD600) was incubated in a 500-ml Sakaguchi
‰ask at 30 for 17 h with reciprocal shaking (245 strokes min). The
speciˆc
change of optical purity, per OD600 at the start of the reaction and per hour
of the reaction time ( ee OD600 h). None, no treatment; after storage, af-
ter storage at 4 C for 17 h instead of the synthetic reaction of (
m
mol of NADH per min at 30
9C. (R)-1,3-BDO-producing activity
W
To overcome the loss of NAD+ and or NAD+-
A
reaction mixture (25 ml) containing
5
z
W
regeneration activity, several adducts were tested,
such as acetone, sodium hydrogen sulˆte, and several
9
C
W
(R)-1,3-BDO-producing activity was expressed as the percent
components of the medium. Acetone (20 g l-reaction
W
z
W
W
mixture) was expected to function as a co-substrate
9
R)-1,3-BDO.
to regenerate NAD+ by CpSADH itself, and sodium
hydrogen sulˆte (57.8 g l-reaction mixture) was ex-
W
pected to reduce the inactivation of NAD+-regenera-
tion activity by 4H2B. The addition of acetone or so-
dium hydrogen sulˆte, against our expectations,
caused a decrease of the optical purity. Alternatively,
YT medium (Bacto-Tryptone, 10 g; Bacto-Yeast ex-
tract, 5 g; NaCl, 5 g in a reaction mixture) was ex-

Synthesis of (
R
)-1,3-Butanediol Using a Recombinant E. coli
927
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W
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increase in the turbidity of the reaction mixture at
600 nm after a 17-h incubation and an increase of the
optical purity of (R)-1,3-BDO from 89.8z to 96.1z
after a 41-h incubation. The addition of Bacto-Yeast
extract alone could replace that of YT-medium with
eŠect, but the identiˆcation of the eŠective compo-
nents in Bacto-Yeast extract and the reasons for the
eŠects upon the addition of YT-medium remains to
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ee
.
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