Asymmetric synthesis of (S)-4-chloro-3-hydroxybutanoate by sorbose reductase from Candida albicans with two co-existing recombinant Escherichia coli strains

Article abstract of DOI:10.1080/09168451.2015.1012145

An NADPH-dependent sorbose reductase from Candida albicans was identified to catalyze the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE). The activity of the recombinant enzyme toward COBE was 6.2 U/mg. The asymmetric reduction of COBE was performed with two coexisting recombinant Escherichia coli strains, in which the recombinant E. coli expressing glucose dehydrogenase was used as an NADPH regenerator. An optical purity of 99% (e.e.) and a maximum yield of 1240 mM (S)-4-chloro-3-hydroxybutanoate were obtained under an optimal biomass ratio of 1:2. A highest turnover number of 53,900 was achieved without adding extra NADP+/NADPH compared with those known COBE-catalytic systems.

Full text of DOI:10.1080/09168451.2015.1012145

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Asymmetric synthesis of (S)-4-chloro-3-
hydroxybutanoate by sorbose reductase from
Candida albicans with two co-existing recombinant
Escherichia coli strains
Ping Caia, Mingdong Ana, Sheng Xua, Ming Yana, Ning Haoa, Yan Lia & Lin Xua
a State Key Laboratory of Materials-Oriented Chemical Engineering, College of
Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, P.R.
China
Published online: 13 Mar 2015.
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To cite this article: Ping Cai, Mingdong An, Sheng Xu, Ming Yan, Ning Hao, Yan Li & Lin Xu (2015): Asymmetric synthesis
of (S)-4-chloro-3-hydroxybutanoate by sorbose reductase from Candida albicans with two co-existing recombinant
Escherichia coli strains, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1012145
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Bioscience, Biotechnology, and Biochemistry, 2015
Asymmetric synthesis of (S)-4-chloro-3-hydroxybutanoate by sorbose reductase
from Candida albicans with two co-existing recombinant Escherichia coli
strains
Ping Cai, Mingdong An, Sheng Xu, Ming Yan, Ning Hao, Yan Li and Lin Xu*
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical
Engineering, Nanjing Tech University, Nanjing, P.R. China
Received October 20, 2014; accepted January 9, 2015
http://dx.doi.org/10.1080/09168451.2015.1012145
An NADPH-dependent sorbose reductase from
Candida albicans was identified to catalyze the
asymmetric reduction of ethyl 4-chloro-3-oxobutano-
ate (COBE). The activity of the recombinant enzyme
toward COBE was 6.2 U/mg. The asymmetric
reduction of COBE was performed with two coexis-
ting recombinant Escherichia coli strains, in which
the recombinant E. coli expressing glucose dehydro-
genase was used as an NADPH regenerator. An
optical purity of 99% (e.e.) and a maximum yield of
1240 mM (S)-4-chloro-3-hydroxybutanoate were
obtained under an optimal biomass ratio of 1:2. A
highest turnover number of 53,900 was achieved
without adding extra NADP+/NADPH compared
with those known COBE-catalytic systems.
because of its high stability and cheap substrate. The
biocatalysts were usually obtained by coexpressing the
reductases and glucose dehydrogenase in one strain,
but the order of the genes, Shine-Dalgarno (SD)
regions, or aligned spacing between the SD sequence
and the translation initiation codon may affect the
expression level of either enzyme.9) In addition, the
velocities of synthesis reaction and coenzyme regenera-
tion may be difficult to regulate in a coexpression sys-
tem.10) Thus, a two-strain coexisting system was
proposed based on the principle that the coenzyme
regeneration system in one strain can supply the bio-
synthesis in another different strain.11,12) The two-strain
system is more flexible than the coexpression system
because the balance between synthesis reaction and
coenzyme regeneration can be easily achieved by
adjusting the biomass of the two strains.
Key words: NADPH-dependent sorbose reductase;
Candida
albicans;
(S)-
4-chloro-
In this study, we reported an NADPH-dependent
3-hydroxybutanoate; coenzyme regenera-
tion
sorbose reductase (SOU1) from C. albicans to cata-
13)
lyze the reduction of COBE. Greenberg et al.
first
reported SOU1 as an important reductase involved in
sugar metabolism. However, the amino acid sequence
analysis revealed that SOU1 shared high sequence
homology with known COBE catalytic-enzymes.
SOU1 exhibited a reduction activity toward COBE.
Thus, a two-strain coexisting system for (S)-CHBE
production was constructed using SOU1 and glucose
dehydrogenase. The recombinant strain Escherichia
coli Rosseta (pET-22b-SOU1) was mixed with E. coli
Rosseta (pET-22b-GDH) expressing glucose dehydro-
genase for both COBE reduction and coenzyme
regeneration (Fig. 1).
Ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] is
a promising chiral intermediate in the synthesis of
pharmacologically active compounds, such as hydrox-
ymethylglutaryl-CoA
reductase
inhibitors
and
4-hydroxypyrrolidone.1) The asymmetric reduction of
ethyl 4-chloro-3-oxobutanoate (COBE) by enantioselec-
tive oxidoreductases has been proved one of the most
efficient methods for the production of (S)-CHBE.2)
However, two major limitations apply to the
biotransformation process, namely, the preparation of
high-performance oxidoreductase for COBE reduction
and the efficient recycling of coenzymes NAD(P)+/
NAD(P)H.
Materials and methods
Enzymes and chemicals.
COBE, (R)-CHBE, and
Several reductases have been reported to perform the
(S)-CHBE synthesis, including CaCR from Candida
albicans,3) KaCR from Kluyveromyces aestuarii,4)
PsCR5) and PsCRII6) from Pichia stipitis, S1 from
Candida magnoliae,7) and CpCR from Candida par-
apsilosis.8) Glucose dehydrogenase was coupled with
all of these reductases for coenzyme regeneration
(S)-CHBE were obtained from Acros Organics.
NADPH was supplied by Sigma-Aldrich Chemie
GmbH. All restriction endonucleases, Taq polymerase,
and T4 ligase were purchased from TaKaRa (Dalian)
Co. All other chemicals used were of analytical grade
purity and commercially available.
*Corresponding author. Email: xulin@njtech.edu.cn
© 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
P. Cai et al.
and washed with 100 mM potassium phosphate buffer
(pH 6.2). The bioconversion of COBE to (S)-CHBE
was performed in an aqueous–butyl acetate (4:1, v/v)
biphase system containing 100 mM potassium phos-
phate buffer (pH 6.2), 1500 mM COBE, 1550 mM glu-
cose, 1550 mM Na2CO3, Triton X-100 (1‰, v/v),
0.1 g of dry cell weight (DCW) of E. coli Rosseta
(pET-22b-SOU1), and E. coli Rosseta (pET-22b-GDH),
respectively, at a total volume of 25 mL. The reaction
was performed at 30 °C and 220 rpm for 5 h. The
organic layer was isolated to determine the product
concentration and optical purity.
Fig. 1. Two-strain coexisting system for the production of
(S)-CHBE.
To study the effect of cell mass ratio between E. coli
Rosseta (pET-22b-SOU1) and E. coli Rosseta (pET-
22b-GDH) on (S)-CHBE yield, we adjusted the mass
ratio of the two strains under the same amount of total
biomass, which was 200 mg of the gross dry cell
weight in the same reaction system.
Strains and vectors.
The gdh gene was cloned
from Bacillus megaterium DSM 2894. The E. coli
Rosseta (pET-22b-GDH) constructed earlier to produce
recombinant GDH was used for the cofactor regenera-
tion in (S)-CHBE biosynthesis.3) C. albicans was pre-
served in our laboratory. E. coli Rosseta (DE3) and
pET-22b were used for the overexpression of the
recombinant SOU1.
Analytical methods.
The amount of COBE and
CHBE was measured by gas chromatography and the
optical purity of (S)-CHBE was determined by HPLC
on a Chiralcel OB-H column (4.6 mm × 250 mm;
Daicel Chemical Industries, Japan) as described previ-
ously.9) The intracellular level of NADP+/NADPH was
measured after extracting the reaction strain sample
following a previously described method.14)
Cloning and expression of the SOU1 gene.
The
genome of C. albicans was extracted using a TIANamp
yeast DNA kit (TIANGEN, China). The SOU1 gene
from C. albicans was amplified by PCR with designed
primers of 5′-GGAATTCCATATGATGAGTGAAGA-
AATCATTTCA-3′ (NdeI site is underlined) and
5′-CCGGAATTCTTATGGACATGTATAACCCCCAT-
3′ (EcoRI site is underlined). The PCR conditions were
as follows: 35 cycles of 60 s at 94 °C, 30 s at 56 °C,
and 60 s at 72 °C. The 846 bp DNA fragment was
inserted into pET-22b, resulting in recombinant plasmid
pET-22b-SOU1, with its structure verified by double-
enzyme cleavage and gene sequence analysis. The
recombinant E. coli Rosseta (DE3)/pET-22b-SOU1 was
obtained by heat shock transformation.
Results
SOU1 activity toward COBE
SOU1 activity was determined using COBE as the
substrate and NADPH as the electron donor. SOU1
exhibited an activity of 6.2 U mg−1 toward COBE.
Meanwhile, the crude enzyme extract from control cells
E. coli Rosseta (pET-22b) showed no activity on the
substrate.
The recombinant strains were grown to an optical
density (OD 600 nm) of 0.6 at 37 °C and 200 rpm in
LB medium containing 100 mg/L ampicillin and
20 mg/L chloramphenicol. The temperature was then
changed to 30 °C, and expression was induced by
0.8 mM IPTG. After 10 h, the cells were harvested by
centrifugation (5000 g, 5 min, 4 °C), washed with
100 mM potassium phosphate buffer (pH 6.2), and
used for biotransformation.
Bioconversion of COBE to (S)-CHBE with two
coexisting E. coli strains
The SOU1 expressed in E. coli Rosseta (pET-22b-
SOU1) functioned as main catalyst for the synthesis of
COBE to (S)-CHBE, whereas the GDH expressed in
E. coli Rosseta (pET-22b-GDH) catalyzed the reaction
from NADP+ to NADPH to satisfy the needs of
NADPH for the former reaction. Their DCW ratio was
1:1, and no NADP+ or NADPH was added in the mix-
ture. After 5 h of bioconversion, the coexisting system
produced 1050 mM of (S)-CHBE from 1500 mM
COBE. The enantiomeric excess was over 99%.
Enzyme activity assay.
Cells were disrupted ultra-
sonically in 100 mM potassium phosphate buffer (pH
6.2). The debris was removed by centrifugation at
13,000 g for 10 min. The supernatant was used as the
cell-free extract. SOU1 activity was determined using
10 mM COBE with 1 mM NADPH, following changes in
absorbance at 340 nm. The extinction coefficient of
NADPH was 6.22 mM−1 cm−1. One unit of SOU1 was
defined as the disappearance of 1 μmol NADPH per min.
Protein concentration was measured by the Bradford
method using bovine serum albumin as the standard. The
repeatability was determined by three time measurements.
Effect of cell mass ratio on (S)-CHBE yield
Given the differences between SOU1 activity toward
COBE in E. coli Rosseta (pET-22b-SOU1) and the
ability of NADPH regeneration catalyzed by E. coli
Rosseta (pET-22b-GDH), the two-strain mass ratio was
adjusted to establish favorable conditions for two
enzymes functioning corporately in the reaction.
The bioconversion protocol was applied under the
same amount of total biomass, except that different cell
mass ratios of two strains were used. As shown in
Fig. 2, the maximum yield of (S)-CHBE (1240 mM)
Bioconversion of COBE to (S)-CHBE. Cells were
harvested by centrifugation (10,000 g, 20 min, 4 °C)

Biosynthesis of (S)-CHBE with Reductase
3
Fig. 2. Effects of different biomass ratios between E. coli Rosseta (pET-22b-SOU1) and E. coli Rosseta (pET-22b-GDH) on the yield of
(S)-CHBE.
Table 1. Comparison of several biocatalytic processes for the synthesis of (S)-CHBE.
Catalyst
S1
MER
PsCRII
SCR2
CaCR
CAR
SOU1
Coupling strategy
Coexpression
Coexpression
Coexpression
Crude
Coexpression
Two-strain
Two-strain
enzymes
coexisting
coexisting
Reaction time (h)
Biomass (g L−1 DCW)
Space-time yield
(mM L−1h−1g−1 DCW)
Optical purity(%)
Add coenzyme or not
TTN
34
–
–
8
–
–
30
50
1
6
6
26.5
25
12
15
10
10
0.6
5
8
31
99
Yes
92
Yes
99
Yes
99
Yes
99
No
99
No
–
12)
99
No
53,900
This work
21,600
12,900
13,980
40,000
38,200
10)
16)
17)
15)
3)
Reference
was achieved when E. coli Rosseta (pET-22b-SOU1)
and E. coli Rosseta (pET-22b-GDH) were added at a
ratio of 1:2 after 5 h of biotransformation. The enantio-
meric excess was more than 99%. The system per-
formed poorly because of the low regeneration of
NADPH without adding E. coli Rosseta (pET-22b-
GDH).
the e.e. value of (S)-CHBE was higher than 99%. The
best production of (S)-CHBE was achieved under the
mass ratio of 1:2 between SOU1 and GDH. A large
amount of E. coli Rosseta (pET-22b-GDH) was
required to maintain sufficient NADPH for the whole
system, suggesting that the efficiency of NADPH
regeneration is the major limiting factor for the
production of (S)-CHBE.
Several reported biocatalytic processes for the synthe-
sis of (S)-CHBE using GDH as cofactor regenerator were
compared with this system (Table 1). One of the advanta-
ges of our system is that NADP+/NADPH addition can
be omitted. The internal cofactor of E.coli host cells is
sufficient to start and maintain the reaction recycle. The
turnover number of intracellular coenzyme to (S)-CHBE
formed was 53,900 (mol/mol), which is higher than those
known (S)-CHBE production systems. The space-time
yield (31 mmol L−1h−1 g−1DCW) is the highest one and
can compete against the one with crude enzyme
reaction system (26.5 mmol L−1h−1 g−1DCW).15) More-
over, the two strains would be cultivated individually at
optimal culture conditions and mixed in an appropriate
ratio to maximize the yield of (S)-CHBE production,
making the process easy and flexible for practical
operations.
Discussion
An NADPH-dependent SOU1 in C. albicans was
identified to catalyze the reduction of COBE. SOU1
shared a high degree of similarity in three conserved
segments with other COBE-catalytic reductases from
SDR family. The recombinant SOU1 can catalyze the
asymmetric reduction of COBE to (S)-CHBE with high
activity and enantioselectivity.
The present work adopted a two-strain coexisting
system for the production of (S)-CHBE. The SOU1
expressed in E. coli Rosseta(pET-22b-SOU1) catalyzed
the biosynthesis of (S)-CHBE, whereas the GDH
expressed in E. coli Rosseta(pET-22b-GDH) provided
NADPH recycling. The system produced 1240 mM
(S)-CHBE without adding extra NADP+/NADPH, and

4
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Funding
This work was supported by the National Basic Research
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butanoate. Bioresour. Technol. 2010;101:6761–6767.
[10] Kizaki N, Yasohara Y, Hasegawa J, Wada M, Kataoka M,
Shimizu S. Synthesis of optically pure ethyl (S)-4-chloro-3-hy-
droxybutanoate by Escherichia coli transformant cells coexpress-
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Appl. Microbiol. Biotechnol. 2001;55:590–595.
Program of China [grant number 2011CBA0807]; National
High Technology Research and Development Program of
China [grant number 2012AA022101]; Priority Academic
Program Development of Jiangsu Higher Education Institu-
tions [grant number 1001020B].
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