A novel reductase from Candida albicans for the production of ethyl (S)-4-chloro-3-hydroxybutanoate

Article abstract of DOI:10.1271/bbb.120048

A novel NADPH-dependent reductase (CaCR) from Candida albicans was cloned for the first time. It catalyzed asymmetric reduction to produce ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). It contained an open reading frame of 843 bp encoding 281 amino acids. When co-expressed with a glucose dehydrogenase in Escherichia coli, recombinant CaCR exhibited an activity of 5.7 U/mg with ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. In the biocatalysis of COBE to (S)-CHBE, 1320mM (S)-CHBE was obtained without extra NADP⁺/NADPH in a water/butyl acetate system, and the optical purity of the (S)-isomer was higher than 99% enantiomeric excess.

Full text of DOI:10.1271/bbb.120048

Biosci. Biotechnol. Biochem., 76 (6), 1210–1212, 2012
A Novel Reductase from Candida albicans for the Production
of Ethyl (S)-4-Chloro-3-hydroxybutanoate
y
Mingdong AN, Ping CAI, Ming YAN, Ning HAO, Shanshan WANG, Huan LIU, Yan LI, and Lin XU
College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology,
Nanjing 210009, PR China
Received February 7, 2012; Accepted March 12, 2012; Online Publication, June 7, 2012
[doi:10.1271/bbb.120048]
A novel NADPH-dependent reductase (CaCR) from
Materials and Methods
Candida albicans was cloned for the first time. It
catalyzed asymmetric reduction to produce ethyl (S)-4-
chloro-3-hydroxybutanoate ((S)-CHBE). It contained an
open reading frame of 843 bp encoding 281 amino acids.
When co-expressed with a glucose dehydrogenase in
Escherichia coli, recombinant CaCR exhibited an activ-
ity of 5.7 U/mg with ethyl 4-chloro-3-oxobutanoate
Cloning and expression of the CaCR gene in E. coli. The gene of
CaCR from Candida albicans was amplified by PCR with designed
0
0
primers, 5 -GGAGGCTCATGAGCAAAGAAACAT-3 (BspHI site
0
underlined) and 5 -CGCGGATCCTTAAATAACTGTGTAACCACC-
0
ATCAA-3 (BamHI site underlined), based on the nucleotide sequence
of P87218 in GenBank. The amplified DNA fragment was double-
digested with NdeI and BspHI and inserted into expression vector
pET-22b.
(
(
COBE) as substrate. In the biocatalysis of COBE to
S)-CHBE, 1320 mM (S)-CHBE was obtained without
þ
Construction of a polycistronic co-expressing plasmid. Cloning of a
glucose dehydrogenase (GDH) gene from Bacillus megaterium was
finished in our laboratory previously.8,9) To construct an efficient
polycistronic plasmid with a high level of enzyme co-expression, we
changed the order of the genes, altering the Shine–Dalgarno (SD)
regions, and aligned spacing (AS) between the SD sequence and the
translation initiation codon. The optimal SD sequence was 5-
TAAGGAGG-3, and the AS distance was eight nucleotides. A
polycistronic plasmid, pET-22b-GDH-CaCR (22b-GC), encoding the
GDH gene and the CaCR gene, was constructed (Fig. 1) and
transformed into E. coli Rosetta (DE3).
extra NADP /NADPH in a water/butyl acetate system,
and the optical purity of the (S)-isomer was higher than
9
9% enantiomeric excess.
Key words: biocatalysis; reductase; ethyl 4-chloro-3-
hydroxybutanoate; ethyl 4-chloro-3-oxobu-
tanoate; candida albican
The asymmetric reduction of 4-chloro-3-oxobuta-
noate ethyl (COBE) to ethyl (S)-4-chloro-3-hydroxybu-
tanoate [(S)-CHBE], a promising chiral intermediate in
the synthesis of pharmacologically active compounds
such as hydroxymethylglutaryl-CoA reductase inhibi-
tors1 and 4-hydroxypyrrolidone, by reductases has
several positive attributes, including low cost, mild
reaction conditions, high yield, and remarkable enantio-
selectivity, and has received much attention in recent
years. There are two points in the biosynthesis of (S)-
CHBE. One is a reductase with high activity and
remarkable enantioselectivity, and the other is an
efficient coenzyme regeneration system.
Biocatalysis of COBE to (S)-CHBE. A 25-mL reaction mixture of
00 mM potassium phosphate buffer (pH 6.2), COBE (4,800 mM),
1
glucose (4,800 mM), butyl acetate (12.5 mL), Triton X-100 (1%, v/v)
and cells (300 mg dry weight, 100 mL culture broth) was incubated at
)
ꢀ
30 C at 220 rpm for 25 h in a 50-mL vessel. The pH of the reaction
mixture was controlled at 6.2 with 5 M sodium hydroxide. After mixing
and centrifugation when the reaction finished, the upper phase (the
organic layer) was assayed to identify the product (CHBE) and the
substrate (COBE).
Enzyme assay and analysis of COBE and CHBE. The assay mixture
comprised 100 mM potassium phosphate buffer (pH 6.2), 1 mM
ꢀ
Several reductases have been cloned and used in
the asymmetric synthesis of chiral alcohols, including
NADPH, and 10 mM COBE. The reaction was monitored at 30 C by
the absorbance at 340 nm. CaCR and GDH activity were determined
spectrophotometrically, as described previously.10) One unit of CaCR
was defined as the amount catalyzing the reduction of 1 mmol NADPH
per min. The protein concentration was measured by the Bradford
method, using bovine serum albumin (BSA) as standard. The
concentrations of COBE and CHBE were determined by gas
chromatography, and the optical purity of (S)-CHBE was determined
2
)
ScCR from Streptomyces coelicolor, PsCR and PsCRII
–5)
3
from Pichia stipitis,
aestuarii, and S1 from Candida magnolia. Nearly all
of these reductases use glucose/glucose dehydrogen-
KaCR from Kluyveromyces
4)
3)
5–7)
ase as a enzyme-coupled coenzyme system to produce
þ
by HPLC.11) The method of measuring intracellular NADP /NADPH
þ
(S)-CHBE, but all of them require extra NADP /
NADPH in the reaction, which greatly increases costs
was followed ref. 20.
4
,8)
and is an impediment of their application.
In this study, we identified a novel reductase CaCR by
the genome data mining method from Candida albicans
for the first time. This can produce (S)-CHBE at a very
high enantioselectivity. Highly efficient synthesis of
(S)-CHBE was achieved in an organic solvent–water
þ
biphasic system without adding extra NADP /NADPH.
Results and Discussion
Amino acid sequences analysis
According to amino acid sequence analysis with other
COBE-catalyzed reductases (Fig. 2), CaCR appeared to
belong to the SDR superfamily based on three conserved
sequences in the black boxes. The first was a conserved
y
To whom correspondence should be addressed. Tel: +86-25-83172074; Fax: +86-25-83172075; E-mail: yanming@njut.edu.cn

Biocatalysis of (S)-CHBE
1211
sequence region covering variable N-terminal Thr-Gly-
X-X-X-Gly-X-Gly as cofactor binding motif. The
second was a P-/G motif followed by a conserved
Thr, concerned with reaction direction. The last was a
conserved Tyr-X-X-X-Lys segment essential for the
catalytic activity of SDR proteins. All previous studies
confirm that Tyr functions as a catalytic base and is
the most conserved residue within the whole family,
whereas Lys interacts with the nicotinamide ribose and
parapsilosis.19) Amino acid sequence alignments of
CaCR with other SDRs are shown in Fig. 2. Despite low
sequence identity, three conserved segments in CaCR
showed a high degree of similarity to those of other
SDRs.
1
2)
Enzyme activity analysis and cofactor specificity of
CaCR
CaCR is a oxido-reductase in Candida albicans that is
annotated as sorbose reductase in the NCBI, inferred
from homology, but the enzyme has no activity toward
sorbose, and any function remains unknown as to this
novel reductase. Recombined E. coli Rosseta (pET-15b-
CaCR) exhibited an activity of 5.7 U/mg for COBE with
NADPH as electron donor, but exhibited only 0.12 U/
mg with NADH as electron donor. Hence, we consider
CaCR an NADPH-dependent reductase. When CaCR
and GDH coexpressed in plasmid pET-15b-GDH-CaCR,
CaCR exhibited an activity of 4.2 U/mg for COBE and
GDH an activity of 3.7 U/mg for Glucose. E. coli
Rosseta (pET-15b) showed no activity for COBE.
1
3–15)
lowers pKa of the Tyr-OH.
analysis of CaCR indicated that the homology was
Amino acid sequence
3
)
4
5
4.0% with KpCR from Kluyveromyces aestuarii,
1
6,17)
5.1% with PsCRII from Pichia stipiti,
1
55.8% with
8)
S1 from Candida magnolia, 66.7% with PsCR from
Pichia stipiti,1 and 68.2% with CpCR from Candida
1)
Bioreduction of COBE to (S)-CHBE
Most researchers use glucose/glucose dehydrogenase
as the coenzyme regeneration system for the low cost of
glucose and high activities of glucose/glucose towards
both NADH and NADPH, but the extra NAD(P)/
NAD(P)H added in these system is the biggest obstacle
for them to be applied in industry. Our goal was to find
a novel reductase with high activity and remarkable
enantioselectivity and to build a coenzyme regeneration
system for the biosynthesis of (S)-CHBE without adding
extra NAD(P)/NAD(P)H.
We found that 300 mg dry cells in the reaction is
sufficient to produce 4500 mM (S)-CHBE without adding
extra NAD(P)/NAD(P)H. The e.e. value of the product
was excellent (>99%). The transformants gave a molar
yield of 93.8%. The process is shown in the Fig. 3. The
Fig. 1. Construction of Polycistronic Plasmids.
Fig. 2. Amino Acid Sequence Alignments of Short-Chain Alcohol Dehydrogenase/Reductase Using the Clustalx Tool.
Sequences are CaCR from Candida albicans in this study (GenBank accession no. P87218), Pichia. stipitis (PsCR, GenBank accession
no. XP 001387287), Pichia. stipiti (PsCRII, GenBank accession no. XP 001387285), Candida magnoliae (S1, GenBank accession
no. BAB21578), Kluyveromyces aestuarii (KaCR, GenBank accession no. BAD01116), Candida parapsilosis (CpCR, GenBank accession
no. GI: 189096232).

1212
M. AN et al.
Table 1. Comparison of Different Reductases in (S)-CHBE Product
Enzyme
S1
PsCRII
ScCR
KaCR
CmMR
CaCR
Coenzyme specificity
NADPH
200
34
NADH
1,000
30
NADH
1,000
22
NADH
40
NADPH
2,000
36
NADPH
300
25
Biomass (mg, dry weight)
Time of reaction (h)
Production of (S)-CHBE (mm)
6
2,600
13.0
1,398
1.4
3,600
3.6
300
7.5
—
1,550
0.8
1,320
15.0
(S)-CHBE/biomass (mm/mg)
Turnover number
Add coenzyme or not
e.e.%
21,600
YES
>99%
4)
13,980
YES
>99%
17)
12,900
YES
>99%
2)
12,900
NO
38,200
NO
NO
>99%
3)
92.1%
5)
>99%
This study
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4
4
3
3
2
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,000
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,000
,500
,000
,500
,000
,500
,000
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