Gene cloning and expression of Leifsonia alcohol dehydrogenase (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols

Article abstract of DOI:10.1271/bbb.70.418

The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30-50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed lsadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).

Full text of DOI:10.1271/bbb.70.418

Biosci. Biotechnol. Biochem., 70 (2), 418–426, 2006
Gene Cloning and Expression of Leifsonia Alcohol Dehydrogenase
(LSADH) Involved in Asymmetric Hydrogen-Transfer Bioreduction
to Produce (R)-Form Chiral Alcohols
y
Kousuke INOUE, Yoshihide MAKINO, Tohru DAIRI, and Nobuya ITOH
Biotechnology Research Center, Toyama Prefectural University, Kurokawa 5180, Imizu, Toyama 939-0398, Japan
Received August 1, 2005; Accepted September 22, 2005
The gene encoding Leifsonia alcohol dehydrogenase
LSADH), a useful biocatalyst for producing (R)-chiral
microorganisms.5)
Enzymatic acylations for the resolution of chiral
alcohols were developed in the 1980s, and several kinds
of lipases have been used as biocatalysts, but, in most
cases, for enzymatic resolution, only a 50% yield of
desired intermediate is obtained.
Many examples of the reduction of ketones with
reductases and dehydrogenases have also been describ-
ed, because such processes theoretically produce alcohol
(
alcohols, was cloned from the genomic DNA of Leifsonia
sp. S749. The gene contained an opening reading frame
consisting of 756 nucleotides corresponding to 251
amino acid residues. The subunit molecular weight
was calculated to be 24,999, which was consistent with
that determined by polyacrylamide gel electrophoresis.
The enzyme was expressed in recombinant Escherichia
coli cells and purified to homogeneity by three column
chromatographies. The predicted amino acid sequence
displayed 30–50% homology to known short chain
alcohol dehydrogenase/reductases (SDRs); moreover,
the NADH-binding site and the three catalytic residues
in SDRs were conserved. The recombinant E. coli cells
which overexpressed lsadh produced (R)-form chiral
alcohols from ketones using 2-propanol as a hydrogen
donor with the highest level of productivity ever
reported and enantiomeric excess (e.e.).
6
)
4
,7,8)
with 100% conversion from ketone.
But, in most
cases, they have the disadvantages of a narrow substrate
spectrum and low productivity due to the need for a
cofactor regeneration system such as NADH and
NADPH, although they show rather high enantioselec-
tivity. Therefore, such bioreduction processes are eco-
nomical only when the cofactor can be regenerated in
situ in a second catalytic cycle, for example, formate/
formate dehydrogenase (FDH) or glucose/glucose de-
9
)
hydrogenase (GDH). More recent studies concerning
bioasymmetric reduction have revealed that much
enhanced productivity can be obtained only when a
powerful cofactor regenerating system is coupled with
Key words: alcohol dehydrogenase; Leifsonia sp.; asym-
metric bioreduction; biocatalyst; (R)-chiral
alcohols
1
0,11)
the process.
From the viewpoint of the regeneration of NAD(P)H,
2-propanol is another suitable hydrogen donor for
bioreduction because of its chemical properties and
low cost. Recently, Itoh et al. reported that phenyl-
acetaldehyde reductase (PAR) from the styrene-assim-
ilating Rhodococcus (former Corynebacterium) sp.
strain ST-10 is a unique NADH-dependent alcohol
dehydrogenase (ADH) with a broad substrate range and
high enantioselectivity to give (S)-form alcohols without
an additional coenzyme regeneration system, because
In recent years, enantiometrically pure compounds,
including D- and L-amino acids, organic acids, amines,
alcohols, epoxides, and so on, have been produced with
the use of biocatalysts, because such methods are
superior to general chemical methods in terms of
enantioselectivity. Among them, secondary alcohols
are the most important chiral syntons for pharmaceut-
icals and agrochemicals.
7
)
1
)
Noyori et al. have developed efficient transfer hydro-
genation catalysts: ruthenium complexes of BINAP and
derivatives,2 with which not only hydrogen but also
formic acid or 2-propanol serves as the hydrogen donor
the enzyme itself is able to regenerate NADH in the
12–14)
,3)
presence of 2-propanol.
PAR or its mutated enzyme (Sar268) system
Therefore, a recombinant
1
5)
and
chemotolerant ADH reported in the R. ruber DSM44541
4
)
for ketone reduction. But, in many cases, difficulties
remain in the cost of the catalyst and the operability, and
in attaining sufficient optical purity and productivity. An
alternative to chemical asymmetric reduction processes
is a biocatalytic transformation system using enzymes or
1
6)
strain, are regarded as superior asymmetric hydrogen-
transfer biocatalysts with which to produce (S)-alcohols.
We have also found a novel ADH (LSADH) produc-
ing (R)-alcohols in Leifsonia sp. strain S749, and have
y
To whom correspondence should be addressed. Tel: +81-766-56-7500 ext. 560; Fax: +81-766-56-2498; E-mail: itoh@pu-toyama.ac.jp
Abbreviations: LSADH, Leifsonia alcohol dehydrogenase; PTK, trifluoroacetophenone

Cloning of Leifsonia ADH Gene for Asymmetric Bioreduction
419
OH
O
OH
Reductive reaction
O
CF3
CF3
LSADH
(R)-2-Heptanol
(S)-1-Phenyltrifluoro-
ethanol
((S)-PTE)
2
-Heptanone
Phenyltrifluoro-
acetophenone
+
NADH
NAD
(PTK)
O
OH
LSADH
Acetone
Oxidative reaction 2-propanol
Fig. 1. Principle of the Hydrogen-Transfer Asymmetric Reduction Catalyzed by E. coli Cells Expressing LSADH.
characterized it in detail.17,18) LSADH is a NAD -
dependent ADH, and it produces many chiral alcohols
from ketones, and ꢀ-keto and ꢁ-ketoesters with high
enantioselectivity. This process can function without the
additional coenzyme regeneration system in the pres-
ence of 2-propanol in a similar manner to PAR
þ
easy vector (Promega, Madison, WI), pUC118 and
pGEM5Zf(+) (Promega) were used as cloning vectors.
Plasmids pET21b (Novagen/Merck Biosciences, San
Diego, CA), pKK223-3 and pTrc99A (Amersham
Pharmacia Biotech, Tokyo) were also used as expression
vectors. For gene cloning and enzyme purification,
4,15,17)
ꢁ
(
Fig. 1).1
E. coli cells were cultivated at 37 C in Luria-Bertani
In this report, we describe the cloning, sequence
(LB) medium (1% tryptone, 0.5% yeast extract, and
0.5% NaCl, pH 7.0) containing 0.1 mg/ml ampicillin,
unless otherwise noted. For induction of the gene under
the control of the lac promoter, 0.4 mM isopropyl ꢁ-D-
thiogalactoside (IPTG) was added to LB medium. To
select recombinant cells harboring pGEM-T easy or
pUC118 with inserted DNA, 0.01% (w/v) 5-bromo-4-
chloro-3-indolyl-ꢁ-D-galactoside (X-Gal) was added to
the 1.5% (w/v) LB agar. For the preparation of genomic
DNA from Leifsonia sp. strain S749, the bacteria were
analysis, and expression in Escherichia coli of the gene
encoding LSADH from Leifsonia sp. S749, and the
purification of the recombinant enzyme. We have also
confirmed that the established expression system func-
tions well as a hydrogen-transferring biocatalyst from
2
-propanol to produce chiral (R)-alcohols.
Materials and Methods
ꢁ
Chemicals. SDS–PAGE molecular weight standards
low range) were purchased from Nippon Bio-Rad
cultivated at 30 C for about 18 h in a liquid medium
containing 0.5% (w/v) peptone and 0.5% (w/v) yeast
extract (pH 7.0).
(
Laboratories (Tokyo). The marker protein kit for HPLC
0
was obtained from Oriental Yeast (Tokyo). 2,3 -Di-
0
chloroacetophenone and (R,S)-2-chloro-1-(3 -chloro-
Partial NH2-terminal and internal amino acid se-
quences of LSADH. The enzyme was electrophoresed on
an SDS–PAGE gel, transferred to a PVDF (polyvinyli-
dene difluoride) membrane (Bio-Rad) using a semidry
electroblotting apparatus (NA-1512, Nippon Eido,
phenyl)ethanol were kindly supplied by Sumitomo
Chemical (Osaka, Japan). All other chemicals used in
this study were of analytical grade and were commer-
cially available.
2
Tokyo) at constant current of 0.8 mA/cm gel for
1
9)
Bacterial strains, vectors, and culture conditions.
7)
90 min by the method of Hirano and Watanabe, and
then stained with Coomassie brilliant blue G-250. The
NH2-terminal amino acid sequence of the enzyme on the
PVDF membrane was determined using a HP G1005A
protein sequencing system (Hewlett Packard, Palo Alto,
CA), and its internal amino-acid was determined by
APRO Life Science Institute (Tokushima, Japan).
1
Leifsonia sp. strain S749, a coryneform Gram positive
bacterium, was used as the source of chromosomal
0
DNA. E. coli XL1-Blue MRF (ꢀ(mcrA)183 ꢀ(mcrCB-
hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96
0
relA1 lac[F proAB laclqZDM15 Tn10 (Tetr)]) was used
as a host strain for the construction of a cosmid genomic
library. E. coli JM109 (recA1, endA1, gyrA96, thi,
ꢀ
ꢀ
þ
ꢀ
hsdR17(rK mK ), e14 (mcrA ), supE44, relA1, ꢀ
General recombinant DNA techniques. The genomic
DNA from Leifsonia sp. strain S749 was isolated as
0
ꢀ
þ
q
(
lac-proAB)/F [traD36, proAB , lac I , lacZꢀM15]),
ꢀ
ꢀ
20)
BL21(DE3) (F , ompT, hsdSB(rB mB ), gal(ꢂcI 857,
ind1, Sam7, nin5, lacUV5-T7gene1), dcm(DE3)), and
described by Hopwood et al.
Plasmid DNA was
purified with a kit from Qiagen (Chatsworth, CA).
Restriction enzymes were purchased from Takara Shuzo
(Kyoto, Japan), Toyobo Biochemicals (Osaka, Japan),
and New England BioLabs (Herts, UK) and used
according to the manufacturer’s directions. T4 DNA
ꢀ
ꢀ
ꢀ
BL21 (F ompT hsdS (r mB ) gal dcm) were used as
B
B
host strains for the expression of LSADH. Cosmid
pWE15 (Toyobo, Osaka) was used as a vector for the
construction of the genomic library. Plasmids pGEM-T

420
K. INOUE et al.
Table 1. Primers Used for PCR Amplification
Primer
Sequencea
Restriction site
ULA-F
ULA-R
KLA-F
TLA-F
ELA-F
5′ -GTCGGATCCCTGAAGGGAGATTTTCATGGCTC-3′
5′ -GGGCTGCAGCCCGGTCACTG-3′
BamHI
PstI
5′ -GAAGGGAGGAATTCATGGCTCAGTACGAC-3′
5′ -GGAGATTTCCATGGCTCAGTACGACG-3′
5′ -GGAGATTCATATGGCTCAGTACGACG-3′
EcoRI
NcoI
NdeI
aUnderlined nucleotides represent the restriction sites.
ligase was purchased from New England BioLabs. DNA
polymerases (Takara Ex Taq and Takara LA Taq) were
purchased from Takara Shuzo. Transformation of E. coli
with plasmid DNA by electroporation was performed
under standard conditions with a Gene Pulser electro-
poration system (Bio-Rad). DNA hybridization was non-
radioactively performed with an AlkPhos direct label-
ling and detection system using a CDP-Star kit (Amer-
sham Biosciences, Tokyo, Japan) following the manu-
facturer’s instructions. Other general DNA manipulation
procedures were carried out as described by Sambrook
et al.2 All the primers were purchased from Espec
Oligo Service (Ibaraki, Japan).
Sau3AI. Fragments with a molecular size of about 15–
20 kb were separated and purified by sucrose density
gradient centrifugation. The fragments were inserted
into BamHI-treated pWE15, packaged into bacterio-
phage ꢂ particles with Gigapack III gold packaging
extract (Stratagene, La Jolla, CA), and used to transfect
0
E. coli XL1-Blue MRF cells to construct a genomic
library. The library was screened by colony hybrid-
ization with the alkaline phosphatase-labeled 280-bp
DNA fragment. The colony hybridization was per-
formed following the manufacturer’s directions. A
homology-based search was performed with the FASTA
program.
1)
Preparation of the probe for colony hybridization.
Two degenerate oligonucleotide primers were synthe-
sized for cloning the lsadh gene based on the NH2-
terminal amino acid sequence of native LSADH
Construction of the vector for expressing lsadh in
E. coli. Primers used for the amplification of genes by
PCR are listed in Table 1. Six recombinant plasmid
vectors were constructed as follows: The primers ULA-
F and ULA-R were used to amplify the 0.8-kb lsadh
fragment from the genomic DNA of Leifsonia sp. S749
(pWELA6), and the PCR fragment purified on agarose
gel was inserted into the BamHI-PstI site of vector
pUC118 to give pULA. In a similar manner, primers
KLA-F and ULA-R were used to amplify lsadh, and the
purified fragment was inserted into the EcoRI-PstI site
of vector pKK223-3 to give pKLA. The lsadh gene
amplified using primers TLA-F and ULA-R was purified
and then inserted into the NcoI-PstI site of vector
pTrc99A to give pTLA. Primers ELA-F and ULA-R
were used to amplify lsadh, and the DNA fragment was
inserted into the NdeI-PstI site of vector pGEM5Zf(+)
to give pG5-LA. Next, pG5-LA was double-digested
with NdeI and NotI, and the DNA fragment was isolated
and then inserted into the NdeI-NotI site of pET21b to
construct pELA. pELA was double-digested with XbaI
and PstI to give a DNA fragment, which was inserted
into the XbaI-PstI site of plasmid pUC118 to give
pUELA. pUELA was further digested with EcoRI and
PstI to isolate a DNA fragment, which was ligated into
the EcoRI-PstI site of pKK223-3 to construct pKELA.
Plasmid pULA, pKLA, pTLA, pUELA, or pKELA was
introduced into E. coli JM109. pELA was introduced
into E. coli BL21(DE3). pKELA was also introduced
1
7)
(
trypsin-treated LSADH (IAVNNAGIGGEA), respec-
AQYDVADRSAIVTGG) described previously and
0
0
tively: 5 -primer, 5 -CARTAYGAYGTIGCNGAHMG-
0
0
0
0
3
, and 3 -primer, 5 -CCDATICCNGCRTTRTTNAC-3 .
Degenerate positions are indicated by R for A or G, Y
for T or C, M for A or C, H for A, T, or C, N for all
bases, and I for inosine. PCR amplification was carried
out in a reaction mixture of 20 ml containing 100 pmol of
each of the two primers, 90 ng of Leifsonia sp. strain
S749 chromosomal DNA, 0.25 mM of each deoxynu-
cleotide, and 2.5 units of Takara Ex Taq. The initial
ꢁ
denaturation consisted of 5 min at 94 C. The amplifi-
ꢁ
ꢁ
ꢁ
cation profile (30 s at 94 C, 30 s at 55 C, 30 s at 72 C)
was repeated for 30 cycles. The amplified fragment of
about 280 bp was analyzed by gel electrophoresis,
purified, and cloned into a pGEM-T easy vector. Its
nucleotide sequence was analyzed with the dye termi-
nator cycle sequencing kit and an ABI prism 377 DNA
sequencer (ABI, Perkin Elmer, Chiba, Japan). This
fragment was labelled with alkaline phosphatase using
the AlkPhos direct labelling and detection system with
the CDP-Star kit and used as a probe for colony
hybridization.
Cloning of the lsadh gene. Genomic DNA prepared
from Leifsonia sp. S749 cells was partially digested with
0
into E. coli JM109 XL1Blue MRF and BL21.

Cloning of Leifsonia ADH Gene for Asymmetric Bioreduction
421
ꢁ
Purification of recombinant LSADH. All purification
ꢁ
with shaking (2,500 rpm) for 24 h at 25 C. After the
reaction, the mixture was extracted twice with ethyl
acetate. The combined ethyl acetate extracts were dried
with Na2SO4, and analyzed by gas chromatography
(GC) or high performance liquid chromatography
procedures were performed at 0–4 C in 20 mM KPB
(
pH 7.0) containing 10% (v/v) glycerol, unless other-
wise specified. E. coli BL21 (pKELA) cells were grown
ꢁ
with shaking at 37 C for 18 h in LB medium containing
1
1
7,18)
00 mg/ml of ampicillin. The washed cells (1.17 g
(HPLC), as described previously.
wet weight), collected from 100 ml of culture broth,
were suspended in 20 ml of the buffer and then disrupted
with an ultrasonic oscillator (Insonator 201 M,
Kubota, Osaka, Japan) for 5 min. After centrifugation
GC was performed using a Shimadzu GC-18A system
(Kyoto, Japan) equipped with a capillary column (J & W
capilary column DB-1, 0:25 mm ꢂ 30 m, Agilent Tech-
nologies, Palo Alto, CA) with an FID (flame ionization
detector) under the following conditions: injection and
(
13;000 ꢂ g, 30 min), the supernatant was applied to a
ꢁ
DEAE-Toyopearl 650 M (Tosoh, Tokyo) column (2.5
by 13 cm) equilibrated with the buffer. The enzyme was
eluted with a linear 0 to 0.6 M NaCl gradient in the same
buffer. The fractions with strong enzyme activity were
collected (total volume, 28 ml). The solution, mixed
with ammonium sulfate up to a concentration of 0.6 M,
was applied to a Butyl-Toyopearl 650 M (Tosoh)
column (2.5 by 13 cm) which had been equilibrated
with the buffer containing 0.6 M ammonium sulfate. The
enzyme was eluted with a linear 0.6 to 0 M ammonium
sulfate gradient in the buffer. The collected fractions
detection temperature of 250 C, column temperature of
ꢁ
ꢁ
ꢁ
40 C for 5 min, raised to 100 C at 5 C/min, the sprit
ratio was 40, and a flow rate of 1 ml/min of He. The
products and substrates showed the following retention
times (min): 2-heptanone, 6.3; 2-heptanol, 7.0; ethyl 3-
methyl-2-oxobutanoate, 12.1; ethyl 3-methyl-2-hydrox-
ybutanoate, 12.9. An injection and detection temperature
ꢁ
ꢁ
was 250 C, and column temperature of 70 C for 5 min
ꢁ
ꢁ
was raised to 250 C at 5 C/min, and a flow rate of
1 ml/min of He. Ethyl 4-chloro-3-oxobutanoate showed
the retention time of 11.1 min and ethyl 4-chloro-3-
hydroxybutanoate of 12.3 min. GC was also performed
using a Shimadzu GC-14 A system equipped with a
coiled column (3 mm ꢂ 2 m) packed with Thermon 1000
(5% on Chromosorb W) with an FID, column temper-
(total volume, 29 ml) with strong enzyme activity were
collected and concentrated using a Centriprep YM-30
(
cutoff MW, 30,000, Millipore, Billerica, MA), and
ꢁ
stored as a purified enzyme preparation at ꢀ20 C.
ꢁ
ature of 70 C, injection and detection temperatures of
ꢁ
Enzyme and protein assays. Activity was assayed
ꢁ
200 C, and a flow rate of 50 ml/min of N2. The product
spectrophotometrically at 25 C by measuring the
and substrates showed the following retention times
(min): ethyl 3-oxobutanoate, 7.6; ethyl 3-hydroxybuta-
noate, 10.1.
decrease in absorption at 340 nm of NADH, as described
in a previous paper using trifluoroacetophenone (PTK)
as a substrate.1 One unit of enzyme was defined as the
amount that converted 1 mmol of NADH and PTK in
7)
To determine the absolute configuration of certain
alcohols, the product was analyzed using GC (HP 6890
GC system, Agilent Technologies) with a Chrompack
CP-cyclodextrin-ꢁ-236-N19 chiral column (0.25 mm by
25 m, 0.25 mm film, Varian, Palo Alto, CA) and a FID.
Helium gas was used as a carrier at 15 psi (0.5 ml/min),
the split ratio was 50, and the injection and detection
1
min. The protein concentration was estimated using a
Bio-Rad protein assay kit with bovine serum as a
standard protein. SDS–PAGE was performed in a 14.0%
polyacrylamide slab gel with the Tris-glycine buffer
system. The molecular mass of the enzyme subunit was
determined from the relative mobility of standard
proteins.
ꢁ
temperatures were 250 C. The column temperature
ꢁ
was maintained isothermally at 80 C. The products and
substrates showed the following retention times (min):
ethyl pyruvate, 3.1; ethyl (R)-lactate, 4.0; ethyl (S)-
lactate, 4.2. When the column temperature was main-
tained at 130 C, PTK, (S)-PTE, and (R)-PTE indicated
the following retention times (min): PTK, 1.8; (S)-PTE,
Nucleotide sequence accession number. The nucleo-
tide sequence reported in this paper is available from the
DDBJ, EMBL, and GenBank databases under accession
no. AB213459.
ꢁ
6
.7; (R)-PTE, 7.3. When the column temperature was
0
ꢁ
Production of chiral alcohols coupled to NADH
regeneration with 2-propanol. To determine the enan-
tiomeric purity of some products and assess the coupling
system of NADH regeneration, we constructed a system
with LSADH and 2-propanol (Fig. 1). The reaction
mixture used for measuring the products from ketones
kept at 160 C, 2,3 -dichloroacetophenone, (S)-2-chloro-
1-(3-chlorophenyl)ethanol, and its (R)-form indicated
the following retention times (min): 2,3 -dichloroaceto-
0
phenone 8.5; (S)-2-chloro-1-(3-chlorophenyl)ethanol,
12.7; (R)-2-chloro-1-(3-chlorophenyl)ethanol 13.1.
The absolute configuration of 2-heptanol was deter-
mined as its benzoyl derivative by HPLC with a chiral
column. HPLC was also performed for certain alcohols
with a Shimadzu LC-10AT system equipped with a
Chiralcel OB-H or OD-H (4:6 ꢂ 250 mm, Daicel Chem-
ical Industries, Tokyo) under the following conditions in
the order (column, mobile phase, flow rate, wavelength
þ
consisted of 1 mmol NAD , 100 mmol KPB (pH 7.0),
1
0% (v/v) 2-propanol, E. coli cells from 1 ml of culture
broth containing approximately 5 units of LSADH, and
one hundred mg of each substrate (10% w/v) in a total
volume of 1 ml, was suspended in a 2-ml polypropylene
tube in a BioShaker MBR-022 (Taitec, Saitama, Japan)

422
K. INOUE et al.
Table 2. Properties of the Constructed Vectors for LSADH Expression
Restriction site of
-terminal regionb
a
Plasmid
Promoter
Sequence
0
5
pULA
pKLA
pTLA
lac
tac
trc
T7
5′ -GGGGATCCCTGAAGGGAGATTTTCATGGC-3′
5′ -AATTTCACACAGGAAACAGAATTCATGGC-3′
5′ -ACAATTTCACACAGGAAACAGACCATGGC-3′
5′ -TAACTTTAAGAAGGAGATATACATATGGC-3′
5′ -TAACTTTAAGAAGGAGATATACATATGGC-3′
5′ -TAACTTTAAGAAGGAGATATACATATGGC-3′
BamHI
EcoRI
NcoI
pELA
NdeI
pUELA
pKELA
lac
tac
NdeI
NdeI
aUnderlined nucleotides represent the restriction sites, and doubly underlined nucleotides the SD sequence.
b
0
The restriction site of the 3 -terminal region of lsadh in each vector was PstI for pULA, pKLA, pTLA, pUELA, and pKELA, and PstI and NotI for pELA.
for detection and column temperature) with the corre-
sponding retention time: (OB-H, hexane/2-propanol
sequence (IAVNNAGIGGEA) were in agreement with
the sequences which were used to prepare the PCR
primers.
ꢁ
(49:1), 0.5 ml/min, 254 nm and 30 C) with the retention
times of 7.2 min for the (R)-derivative and 7.4 min for
(
(
S)-derivative of 2-heptanol; (OB-H, hexane/2-propanol
ꢁ
Comparison of the LSADH amino acid sequence with
other proteins
9:1), 1.0 ml/min, 220 nm and 30 C) with the retention
times of 11.6 min for ethyl (S)-3-hydroxybutanoate and
2.6 min for (R)-form; (OB-H, hexane/2-propanol (9:1),
1.0 ml/min, 220 nm and 30 C) with the retention times
of 7.4 min for ethyl (R)-4-chloro-3-hydroxybutanoate
A search of protein amino acid sequence databases
(DDBJ, GenBank, EMBL and PIR) revealed that
LSADH was similar to the SDR family enzymes
(identity %), viz., cyclohexanonol dehydrogenase from
Acinetobacter sp. (50.6), 2,5-dichloro-2,5-cyclohexa-
diene-1,4-diol dehydrogenase from Sphingomonas pau-
cimobilis (42.8), D-arabinitol 2-dehydrogenase from
Pichia stipitis (27.9), gluconate 5-dehydrogenase from
Gluconobacter suboxydans (31.9), 3-oxoacyl-[acyl-car-
rier-protein] reductase from E. coli (37.7), glucose 1-
dehydrogenase from Bacillus megaterium (32.7), and
acetoacetyl-CoA reductase from Zoogloea ramigera
(35.3). The predicted amino acid sequence of LSADH
displayed 30–50% homology to known SDRs, including
the putative proteins which had been subscribed with
SDR. The amino acid sequence alignment and accession
numbers are shown in Fig. 2.
1
ꢁ
and 7.9 min for (S)-form; (OD-H, hexane/2-propanol
ꢁ
(
9:1), 0.5 ml/min, 220 nm and 30 C) with the retention
times of 7.9 min for ethyl (S)-2-hydroxy-3-methylbuta-
noate and 8.4 min for (R)-form.
Results
Sequence analysis of lsadh
Total DNA isolated from Leifsonia sp. S749 cells was
partially digested with Sau3AI into fragments of 15 to
2
0 kb, which were then inserted into pWE15. To screen
the cosmid genomic library thus obtained, a probe DNA
was synthesized by PCR using primers designed for the
NH -terminal and the internal amino acid sequences of
2
1
7)
purified LSADH. We obtained an amplified DNA
fragment of 280 bp, which was used as a probe for
colony hybridization. One of the positive recombinant
cosmids, pWELA6, was analyzed by Southern hybrid-
ization, subcloned, and sequenced. Nucleotide sequence
analysis of the double-stranded DNA revealed that the
lsadh ORF consists of 756 bp starting with an initiation
codon, ATG, and ending with a termination codon,
TGA. A potential ribosome-binding sequence, AGGGA,
Construction of E. coli transformants overexpressing
LSADH
To overexpress lsadh in E. coli cells, the six ex-
pression vectors in Table 2 were constructed. They had
the following names and properties in the order
(designated name, promoter, origin of the SD sequence
and downstream region, original vector): vector pULA,
lac, AGGGA from Leifsonia sp. S749 chromosomal
DNA, pUC118; vector pKLA, tac, AGGA from
pKK223-3, pKK223-3; vector pTLA, trc, AGGA from
pTrc99A, pTrc99A; vector pELA, T7, AGGA from
pET21b, pET21b; vector pUELA, lac, AGGA from
pET21b, pUC118; vector pKELA, tac, AGGA from
pET21b, pKK223-3. E. coli BL21 carrying pKELA
showed the highest level of enzyme activity (5.2 units/
ml culture broth) without induction by IPTG. It was 170-
fold higher than that for Leifsonia sp. S749 (Table 3).
The lsadh gene was so highly expressed in E. coli cells
2
2)
similar to those utilized in E. coli, was found seven
bases upstream of the ATG translation initiation codon
(
Table 2), but no consensus promoter sequence similar
2
3)
to that of the E. coli ꢀ35 sequence (TTGACA) or the
2
4)
0
was found in the 5 -
ꢀ
10 sequence (TATAAT)
flanking region. The ORF encoded a protein of 251
amino acid residues, the deduced molecular mass of
which was 24,999 Da. The deduced NH -terminal amino
2
acid sequence (AQYDVADRSAIVTGG) and an internal

Cloning of Leifsonia ADH Gene for Asymmetric Bioreduction
423
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LSADH
ACDH
SDCDDH
ARDH
GADH
ACPRD
G1DH
- - - - - - - - M A QY D V A D R S A I V T GGGS G I GR A V A L T L A A S GA A V L V T D L N - E EH A QA V V A E
- - - - - - - - M S N - K F N N K V A L I T GA GS G I GK S T A L L L A QQGV S V V V S D I N - L EA A QK V V D E
- - - - - - - - M S D - - L S G K T I I V T GGGS G I GR A T V E L L V A S GA N V P V A D I N - D EA G E A V V A T
M D Y S Y A N V V P N F R L D G R L A I I T GGS GGL A A V I S R A L L A QGA D V A L I DM N L ER T K S A A K E V
- - - - - M S H P D L F S L S G A R A L V T GA S R G I GL T L A K GL A R Y GA E V V L N GR N - A ES L D S A QS G
- - - - - - - - - - - M N F EG K I A L V T GA S R G I GR A I A E T L A A R GA K V I GT A T S - EN GA QA I S D Y
- - - - - - - - - M Y K D L EG K V V V I T GS S T GL GK SM A I R F A T EK A K V V V N Y R S K ED EA N S V L E E
- - - - - - - - - - - - - - M S R V A L V T GGS R G I GA A I S I A L K A A GY K V A A S Y A G - N D - - D A A K P F
ACRD
7
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LSADH
ACDH
SDCDDH
ARDH
GADH
ACPRD
G1DH
I
I
- - - - - - - - - - - EA A G GK A A A L A GD V T D P A F G E A S V A GA N - A L A P L K I A V N N A G I GG E A A
- - - - - - - - - - - V A L G GK A A A N K A N T A E P E DM K A A V E F A V S T F GA L H L A F N N A G I L G E V N
- - - - - - - - - - - - - - S G GK A A Y F R C D I A Q E E D V K A L V A QT L A A F GGL D GS F N N A A I P QA GL
L GWG E E T L K G E H A S A I GQV S AWS C N I GD A E A V D A T F S S I N E H H GK I A D L L I N T A GY C E N F
F - - - - - - - - - - - EA EG L K A S T A V F D V T D QD A V I D GV A A I ER DM GP I D I L I N N A G I QR R A P
L - - - - - - - - - - - GA N G K GL M L N V T D P A S I E S V L E - - - K I R A E F G E V D I L V N N A G I T - R D N
I
- - - - - - - - - - - K K V G G E A I A V K GD V T V E S D V I N L V QS A I K E F GK L D V M I N N A G L E N P V S
ACRD
K - - - - - - - - - - - A ET G - - I A V Y KWD V S S Y E A C V E G I A K V EA D L GP I D V L V N N A G I T - K D A
1
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80. .|
LSADH
ACDH
T V G D Y S L D S WR T V I EV N L N A V F Y GM QP Q L K AM A A N G - GGA I V NM A S I L GS V G F - - A N S S A
S T E E L S I E GWR R V I D V N L N A V F Y S M H Y E V P A I L A A G - GGA I V N T A S I A GL I G I - - QN I S G
SDCDDH P L A E V S L E R F R QS M D I N V T GT F L CM K Y Q I L AM I E R GT K GS I V N T A S A A GV V GV - - PM H G E
ARDH
GADH
ACPRD
G1DH
ACRD
P A E T Y P A T N A E S I M K V N GL GS F Y V S QS F A R P L I QN N L R GS I I L I GS M S GT I V N D P QP QCM
- L E E F S R K DWD D L M S T N V N A V F F V GQA V A R HM I P R G - R GK I V N I C S V QS E L A R - - P G I A P
L L M RM K D E EWN D I I ET N L S S V F R L S K A V M R AMM K K R - H GR I I T I GS V V GT M GN - - GGQA N
- S H EM S L S DWN K V I D T N L T GA F L GS R EA I K Y F V E N D I K GT V I NM S S V H E K I P W - - P L F V H
M F H KM T P D QWN A V I N T N L T GL F NM T H P V WS GM R D R S - F GR I V N I S S I N GQK GQ - - M GQA N
1
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LSADH
ACDH
Y V T A K H A L L GL T QN A A L E Y A A D K V R V V A V GP G F I R T P L V EA N L S - - A D A L A F L E GK H A L G
Y V A A K H GV T GL T K A A A L E Y A D K G I R I N S V H P GY I K T P L I A E F E E - - A EM V K L - - - - H P I G
SDCDDH Y V G A K H A V V GL T R V A A A D Y GK H G I R V N A L V P GA V R T P M L QR AM D N D A GL E P Y L N S I H P I G
ARDH
GADH
ACPRD
G1DH
ACRD
Y NM S K A GV I H L V R S L A C EWA K Y N I R V N T L S P GY I L T P L T R N V I S GH T EM K EAW E S K I P M K
Y T A T K GA V K N L T K GM A T DWGR H GL Q I N G L A P GY F A T EM T ER L V A D - E E F T DWL C K R T P A G
Y A A A K A GL I G F S K S L A R E V A S R G I T V N V V A P G F I E T DM T R A L S D - - D QR - A G I L A QV P A G
Y A A S K GGM K L M T ET L A L E Y A P K G I R V N N I GP GA I N T P I N A E K F A D - P E QR A D V E SM I P M G
Y S A A K A GD L G F T K A L A Q E GA A K G I T V N A I C P GY I GT EM V R A I P E - - K V L N ER I I P Q I P V G
2
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LSADH
ACDH
R L G E P E E V A S L V A F L A S D A A S - F I T GS Y H L V D GGY T A Q - - - - - - - -
R L G QP E E V A QV V A F L L S D D A S - F V T GS Q Y V V D GA Y T S K - - - - - - - -
SDCDDH R F S E P H E QA QA A V WL L S D A A S - F V T GS C L A A D GG F T A I - - - - - - - -
ARDH
GADH
ACPRD
G1DH
ACRD
RM A E P K E F V GS I L Y L A S E T A S S Y T T GH N L V V D GGY E CW - - - - - - - -
RWG QV E E L V GA A V F L S S R A S S - F V N GQV L M V D GG I T V S L - - - - - - -
R L G GA Q E I A N A V A F L A S D E A A - Y I T G ET L H V N GGM Y M V - - - - - - - -
Y I G E P E E I A A V A AWL A S S E A S - Y V T G I T L F A D GGM T QY P S F QA GR G
R L G E P D E I A R I V V F L A S D E A G - F I T GS T I S A N GGQ F F V - - - - - - - -
Fig. 2. Amino Acid Sequence Alignment of LSADH.
ACDH, Acinetobacter sp., Cyclohexanol dehydrogenase (Accession no. AB006902-5); SDCDDH, Sphingomonas pausimobilis, 2,5-dichloro-
,5-cyclodiene-1,4-diol dehydrogenase (Accession no. P50197); ARDH, Pichia stipitis, D-arabinitol 2-dehydrogenase (Accession no. P50167);
2
GADH, Gluconobacter suboxydans, gluconate 5-dehydrogenase (Accession no. P50199); ACPRD, Escherichia coli, 3-oxoacyl-[acyl-carrier-
protein] reductase (Accession no. P25716); G1DH, Bacillus megaterium, glucose 1-dehydrogenase (Accession no. P40288); ACRD, Zoogloea
ramigera, acetoacetyl-CoA reductase (Accession no. P23238). The alignment was performed using SDRs which had been confirmed as an
enzyme, and constructed using Clustal W software. Identical residues are shown by white letters on a black background. A putative coenzyme-
binding region is underlined. The amino acid residues reported to be important for enzymatic activity and the coenzyme-interacting Asp residue
are marked with an asterisk and closed square respectively.
that more than 50% of the total cellular protein consisted
of LSADH, as shown in lane 2 of Fig. 3.
E. coli cell was estimated to be 58% of the total amount
of soluble protein in the cell-free extract. According to
analytical HPLC on TSK gel G3000SWXL, the molecu-
lar mass of the purified recombinant enzyme was
estimated to be 105 kDa. SDS–PAGE revealed a single
band, and the subunit molecular mass was 26 kDa. These
results show that LSADH is a homotetramer protein, the
same as the native LSADH purified from Leifsonia sp.
Purification of the recombinant enzyme and its
molecular mass
Purification was performed using a buffer containing
1
0% glycerol, which was effective in preventing the
aggregation of LSADH proteins and loss of activity.
From the result in Table 4, the amount of LSADH in
1
7)
S749.

424
K. INOUE et al.
Production of chiral alcohols by E. coli overexpress-
ing LSADH coupled with in situ regeneration of NADH
21 of LSADH) and the amino acid residues reported to
be important for activity among the SDR family
2
5)
The optimal reaction conditions observed for the free
þ
enzymes, Ser 152, Tyr165, and Lys169,
are fully
enzyme reaction (1 mM NAD , 10% v/v 2-propanol,
conserved in the lsadh sequence. The existence of the
coenzyme-interacting Asp residue (position 42), which
regulates specificity for NADH or NADPH, agrees with
the coenzyme specificity of this enzyme for NADH.
Thus the sequence data clearly indicates that LSADH of
Leifsonia sp. belongs to the SDR family. However, in
spite of the homology in primary structure, LSADH is
quite different in substrate specificity from the dehy-
pH 7.0)1 were adopted for the E. coli biocatalyst
system. As exemplified in Table 5, enantioselective
reduction of the ketones and keto esters by this process
using LSADH with 2-propanol as a hydrogen donor
gave chiral alcohols with an enantiomeric purity of
8)
>
99% e.e. Moreover, conversions of five substrates
were almost 100% (100 mg product/ml reaction mix-
ture: 0.53–0.88 M) except for 2-heptanone and ethyl
pyruvate under conditions without optimization of the
reaction for each substrate. The relatively low conver-
sions for (R)-2-heptanol and ethyl (R)-lactate were
probably due to the equilibrium of the reaction, which
have been improved by the addition of a higher
concentration of 2-propanol (data not shown). The
results clearly demonstrate the ability of intact E. coli
cells to express lsadh as an asymmetric bioreduction
biocatalyst for producing (R)-form chiral alcohols.
2
6–29)
drogenases described in Fig. 2
form alcohol-producing ADHs previously reported.
and from other (R)-
7
)
We constructed six vectors in order to express lsadh
sufficiently in E. coli cells. Among them, E. coli BL21
harboring pKELA derived from pKK233-3, showed the
highest level of LSADH activity (about 5 units/ml
culture), although the expression was leaky and not
controlled by IPTG (Table 2). LSADH activity in
E. coli BL21(pKELA) was about 170 times stronger
than that in Leifsonia sp. S749 cells (about 0.03 units/
ml culture), which was enough to reduce ketones into
chiral alcohols. The results also show that selection of a
promoter, the SD sequence, and its downstream nucleo-
tide sequence, and the distance between the SD and
Discussion
In order to establish a suitable expression system of
LSADH and also to study its structural and functional
relationships, we cloned the lsadh gene, determined the
primary structure of the enzyme, and expressed the
lsadh gene in E. coli cells. A homology-based search of
the deduced amino acid sequence of the LSADH showed
that it is similar to the short-chain alcohol dehydrogen-
ase/reductase (SDR) family. A possible consensus
sequence of the coenzyme-binding site of the SDR
family enzyme, GXXXGXG (amino acid positions 15 to
1
2
3
4
9
7,400
6,200
6
45,000
31,000
Table 3. LSADH Activity in Cell-Free Extracts of E. coli Trans-
formants
2
1
1,500
4,400
Activity (U/ml of culture)a
Host
JM109
JM109
Plasmid
With IPTG
Without IPTG
pULA
pKLA
0.03
0.04
0.04
0.81
0.59
1.5
0.02
0.06
0.02
0.89
0.06
1.9
JM109
pTLA
pELA
Fig. 3. Purification of Recombinant LSADH from E. coli (pKELA).
Lane 1, molecular weight standards, including (from top to
bottom) phosphorylase B (Mr, 97,400), serum albumin (66,200),
ovalbumin (45,000), carbonic anhydrase (31,000), trypsin inhibitor
BL21(DE3)
JM109
JM109
pUELA
pKELA
pKELA
pKELA
0
XL1BlueMRF
BL21
2.3
3.1
2.7
5.2
(
21,500), and lysozyme (14,400); lane 2, cell extract (5 mg); lane 3,
DEAE-Toyopearl (5 mg); lane 4, Butyl-Toyopearl (5 mg). The gel
was stained with Quick-CBB (Wako Pure Chemicals, Osaka, Japan).
aActivity was measured using PTK as a substrate.
Table 4. Summary of the Purification of LSADH from E. coli BL21 (pKELA)
Total protein
mg)
Total activity
(U)
Specific activity
(U/mg)
Yield
(%)
Purification factor
(fold)
Step
(
Cell extract
DEAE-Toyopearl
Butyl-Toyopearl
84.5
55.0
31.9
518
461
332
6.1
8.4
100
1
89.0
64.1
1.37
1.70
10.4

Cloning of Leifsonia ADH Gene for Asymmetric Bioreduction
425
Table 5. Conversion and Enantioselectivity of the E. coli (pKELA) Biocatalyst for the Reduction of Ketones
Relative
activity (%)a
Enantiomeric
excess (%)
Conversion
(%)
Substrate
Product
O
OH
(
R)-form
9
2
4
29
88
57
58
9
O
OH
(
R)-form
99
O
O
>
O
O
O
OH
(
R)-form
99
O
O
O
3
3
100
>
O
O
O
O
O
OH
(
R)-form
>
3
8
09
09
100
100
99
O
O
O
O
OH
(
S)-form
99
Cl
Cl
>
O
O
O
OH
(
(
S)-form
99
1
00
100
98
CF3
CF3
>
O
OH
Cl
Cl
S)-form
99
6
7
>
Cl
Cl
aActivity was measured by NADH consumption and is displayed when that for PTK is 100%.
ATG initial codon, greatly influenced the expression
level of LSADH.
to the equilibrium of the reaction. For example, in
the case of 2-heptanol, the K value of [2-heptanol]-
eq
þ
þ
ꢀ8
13)
As shown in Table 5, we found that an E. coli
biocatalyst expressing LSADH can regenerate NADH in
the presence of 2-propanol to produce chiral alcohols
with a high enantioselectivity and yield. The conver-
sions of PTK, ethyl 3-methyl-2-oxobutanoate, ethyl 3-
[NAD ]/[2-heptanone][NADH][H ] is 4:0 ꢂ 10 M,
þ
and that of [2-propanol][NAD ]/[acetone][NADH]-
þ
ꢀ7
[H ] is 8:0 ꢂ 10 M. Therefore, the theoretical con-
version of the product was calculated to be 81%
under the tested conditions from the calculation. A
quite similar conversion (78%) was in fact observed
when the same reaction was carried out using free
0
oxobutanoate, ethyl 4-chloro-3-oxobutanoate and 2,3 -
dichloroacetophenone were almost 100% (100 mg/ml
reaction mixture) without optimization for each sub-
strate after 24 h of reaction, suggesting further produc-
tivity. In fact, we have confirmed that production of
ethyl (S)-4-chloro-3-hydroxybutanoate, a useful building
block of HMG-CoA reductase inhibitor, reached a level
of more than 350 g/l reaction mixture (data will be
reported elsewhere), which is comparable with the
productivity reported by Shimizu et al. using recombi-
nant E. coli biocatalyst expressing NADPH-dependent
carbonyl reductase from Candida magnoliae and glu-
1
8)
LSADH, suggesting that 2-propanol in the E. coli
cells was lowered by the barrier of the cell membrane of
E. coli. This drawback can be overcome by optimization
of the reaction and process engineering, such as the
addition of a higher concentration of 2-propanol,
elimination of the acetone produced from the reaction
mixture, and an organic solvent and water two-phase
9
,10)
reaction system.
system of LSADH in E. coli should provide a powerful
Thus the established expression
means to obtain optically pure alcohols for industry.
9
)
cose dehydrogenase. Our bioprocess is superior to the
previous ones in the following points: (1) a simple
reaction system because there is no need for additional
NADH-regenerating system, (2) easy handling in down-
stream processing and low cost due to using 2-propanol
as a hydrogen donor and coexisting solvent, (3)
application to versatile ketones because of LSADH’s
broad substrate range and high enantioselectivity.
The rather low conversions observed for (R)-2-
heptanol and ethyl (R)-lactate are considered to be due
Acknowledgments
We wish to thank Sumitomo Chemical Co., Ltd., of
Osaka, Japan for the supply of several ketones, and
racemic and chiral alcohol standards. The study was
supported in part by Grant-in-Aids for Scientific
Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan, and Japan
Foundation of Applied Enzymology, Osaka, Japan.
1
7,18)

426
K. INOUE et al.
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