Characterization of two carbonyl reductases from Ogataea polymorpha NBRC 0799

Article abstract of DOI:10.1007/s00253-017-8668-8

The enzyme responsible for the enantioselective production of (S)-1,1,1-trifluoro-2-propanol ((S)-TFP) from 1,1,1-trifluoroacetone (TFA) has been identified in Ogataea polymorpha NBRC 0799. We purified two carbonyl reductases, OpCRD-A and OpCRD-B from this strain, and revealed their characteristics. Both enzymes were specific to NADH, but the following characteristics were different: The molecular mass of subunit OpCRD-A was 40?kDa and that of OpCRD-B was 43?kDa. Amino acid sequences of both enzymes were only 21% identical. OpCRD-B contained 4?mol of zinc per mole of enzyme, but OpCRD-A did not. The optimal pH, temperature, pH stability, thermostability, and inhibitor specificity were also remarkably different. With regard to substrate specificity, both enzymes exhibited high reductase activity toward a wide variety of ketones, aldehydes and fluoroketones, and dehydrogenase activity toward 2-propanol and 2-butanol. The reductase activity was much higher than the dehydrogenase activity at acidic pH. OpCRD-A enantioselectively produced (S)-TFP from TFA, but OpCRD-B preferentially produced (R)-TFP. Thus, we concluded that OpCRD-A plays the main role in the production of (S)-TFP by a reaction of O. polymorpha NBRC 0799 cells and that OpCRD-A has great potential for efficient production of (S)-TFP, as it is an S-specific enzyme and does not catalyze the dehydrogenation of (S)-TFP.

Full text of DOI:10.1007/s00253-017-8668-8

Appl Microbiol Biotechnol (2018) 102:1307–1316
DOI 10.1007/s00253-017-8668-8
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Characterization of two carbonyl reductases from Ogataea
polymorpha NBRC 0799
1
,2
3
1,2
1,2
Kimiyasu Isobe & Shinsuke Miki & Ryoko Ueda & Sayaka Shichida
&
1
,2
1,2
1,2
Daisuke Matsui & Yuko Oku & Yasuhisa Asano
Received: 21 June 2017 /Revised: 7 September 2017 /Accepted: 14 September 2017 /Published online: 13 December 2017
Springer-Verlag GmbH Germany, part of Springer Nature 2017
#
Abstract The enzyme responsible for the enantioselective
production of (S)-1,1,1-trifluoro-2-propanol ((S)-TFP) from
cells and that OpCRD-A has great potential for efficient pro-
duction of (S)-TFP, as it is an S-specific enzyme and does not
catalyze the dehydrogenation of (S)-TFP.
1
,1,1-trifluoroacetone (TFA) has been identified in Ogataea
polymorpha NBRC 0799. We purified two carbonyl reduc-
tases, OpCRD-A and OpCRD-B from this strain, and revealed
their characteristics. Both enzymes were specific to NADH,
but the following characteristics were different: The molecular
mass of subunit OpCRD-Awas 40 kDa and that of OpCRD-B
was 43 kDa. Amino acid sequences of both enzymes were
only 21% identical. OpCRD-B contained 4 mol of zinc per
mole of enzyme, but OpCRD-A did not. The optimal pH,
temperature, pH stability, thermostability, and inhibitor spec-
ificity were also remarkably different. With regard to substrate
specificity, both enzymes exhibited high reductase activity
toward a wide variety of ketones, aldehydes and
fluoroketones, and dehydrogenase activity toward 2-
propanol and 2-butanol. The reductase activity was much
higher than the dehydrogenase activity at acidic pH.
OpCRD-A enantioselectively produced (S)-TFP from TFA,
but OpCRD-B preferentially produced (R)-TFP. Thus, we
concluded that OpCRD-A plays the main role in the produc-
tion of (S)-TFP by a reaction of O. polymorpha NBRC 0799
.
Keywords 1,1,1-Trifluoroacetone
.
.
(S)-1,1,1-Trifluoro-2-propanol Carbonyl reductase Alcohol
dehydrogenase Ogataea polymorpha
.
Introduction
Chiral secondary alcohols and their derivatives are valuable
building blocks for synthesis of pharmaceuticals, agrochemi-
cals, or fine chemicals. Stereospecific alcohol dehydrogenases
(ADHs) and carbonyl reductases (CRDs), which catalyze
asymmetric reduction of prochiral ketones, are widely used
for synthesis of chiral alcohols (Nakamura et al. 2003; Ni
and Xu 2012; Yamada-Onodera et al. 2004). Among the chiral
secondary alcohols, (S)-1,1,1-trifluoro-2-propanol ((S)-TFP)
is one of the most useful optically active alcohols, and chem-
ical methods for the production of (S)-TFP have been devel-
oped using a ruthenium phosphine complex (Puentener and
Waldmeier 2008). Biochemical methods for enantioselective
production of (S)-TFP from 1,1,1-trifluoroacetone (TFA) were
also reported, utilizing baker’s yeast (Bucciarelli et al. 1983;
Doswald et al. 2007), and ADH (Rosen et al. 2006) or a crude
enzyme solution of ADH from Rhodococcus erythropolis,
Arthrobacter paraffineus, or Phichia farinose (Hayashi et al.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00253-017-8668-8) contains supplementary
material, which is available to authorized users.
*
Yasuhisa Asano
asano@pu-toyama.ac.jp
2
007). Our group also screened microorganisms capable of
1
2
3
enantioselectively producing (S)-TFP from 1,1,1-
trifluoroacetone and discovered that Ogataea polymorpha
Department of Biotechnology, Toyama Prefectural University, 5180
Kurokawa, Imizu, Toyama 939-0398, Japan
(
formerly Hansenula polymorpha), Candida mycoderma,
and Pichia naganishi are producers of (S)-TFP (Asano et al.
011). Among these strains, O. polymorpha NBRC 0799 ex-
hibited the highest conversion yield of (S)-TFP. However, the
Asano Active Enzyme Molecule Project, ERATO,, JST, 5180
Kurokawa, Imizu, Toyama 939-0398, Japan
2
Chemical Research Center, Central Glass Co. Ltd, 2-17-5 Nakadai,
Kawagoe, Saitama 350-1159, Japan

1
308
Appl Microbiol Biotechnol (2018) 102:1307–1316
enzyme catalyzing this reaction has not yet been identified.
During experiments to purify the enzyme, we found that this
strain produces two enzymes that catalyze the reduction of
TFA. Therefore, we have identified both of the enzymes from
O. polymorpha NBRC 0799 that are responsible for the
enantioselective production of (S)-TFP (Fig. 1a). The present
paper describes the purification and characterization of two
CRDs produced by O. polymorpha NBRC 0799.
harvested by centrifugation, washed with 0.85% saline, and
stored at − 20 °C until use.
Assay of enzyme activity
CRD activity was assayed at 30 °C and pH 6.0 or 6.5 using
TFA and NADH, as follows: the standard reaction mixture
contained 50 mM TFA, 0.1 mM NADH, and 170 mM potas-
sium phosphate buffer (KPB) at pH 6.5 (OpCRD-A) or pH 6.0
(OpCRD-B) and appropriate amounts of enzyme in a final
Materials and methods
Materials
volume of 1.0 mL. The reaction was initiated by the addition
of TFA and followed spectrophotometrically at 30 °C by mea-
suring the absorbance at 340 nm. One unit of enzyme activity
was defined as the amount of enzyme catalyzing the reduction
of 1 μmol of TFA per minute, which coincides with the oxi-
dation velocity of NADH. The molar absorption efficiency of
TFA was purchased from Merck Millipore (MA, USA). (S)-
and (R)-TFP were a gift from Central Glass Co., Ltd. (Tokyo,
Japan). 1,1-Difluoroacetone and acetone were purchased from
INDOFINE Chemical Company Inc. (NJ, USA) and Wako
Pure Chemical Industries, Ltd. (Osaka, Japan), respectively.
Fluoroacetone, 1,3-difluoroacetone, and 4,4,4-trifluoro-2-
butanone were from Sigma-Aldrich Inc. (MO, USA). β-
3
−1
−1
NADH, 6.22 × 10 M cm , was used for the calculation of
the enzyme activity.
ADH activity was assayed at 30 °C and at pH 6.5 and 9.0
(OpCRD-A) or pH 6.0 and 10.0 (OpCRD-B), using 2-
+
propanol and NAD , as follows: the standard reaction mixture
+
+
NADH, β-NADPH, and β-NAD were from Oriental Yeast
contained 50 mM 2-propanol, 1.0 mM NAD , and 170 mM
Co., Ltd. (Osaka, Japan). Trypsin (sequence grade modified
trypsin) and ammonium bicarbonate were from Promega
KPB at pH 6.0 or 6.5, or 170 mM glycine-NaOH buffer at pH
9.0 or 10.0, and appropriate amounts of enzyme in a final
volume of 1.0 mL. The reaction was initiated by the addition
of 2-propanol and followed spectrophotometrically at 30 °C
by measuring the absorbance at 340 nm. One unit of enzyme
activity was defined as the amount of enzyme catalyzing the
dehydrogenation of 1 μmol of 2-propanol per minute, which
coincides with the formation velocity of NADH.
(
Madison, WI, USA) and Nacalai Tesque Inc. (Kyoto,
Japan), respectively. All other chemicals used were of analyt-
ical grade and commercially available.
Cultivation of O. polymorpha NBRC 0799
O. polymorpha NBRC 0799 was first cultivated in a test tube
containing 5 mL of YM medium (1.0% glucose, 0.5% pep-
tone, 0.3% yeast extract, 0.3% malt extract, 0.3% KH PO ,
Purification of enzyme
2
4
0
.2% K HPO , pH 6.5), at 30 °C for 24 h, with shaking at 300
All procedures were performed at 4–10 °C, and the buffer
used was KPB, pH 7.0, unless otherwise stated.
2
4
strokes per minute. The culture (2 mL) was then inoculated
into a 500-mL shaker flask containing 200 mL of YM medi-
um, and the second culture was incubated at 30 °C for 24 h.
Ten milliliters of the second culture was transferred into a 2-L
shaker flask containing 1 L of YM medium. After the third
cultivation was performed at 30 °C for 24 h, the cells were
Preparation of cell-free extract: Cells from 5 L of culture
broth (34.8 g wet weight) were resuspended in 174 mL of
10 mM buffer and disrupted with glass beads (φ0.5 mm) at
2700 rpm for 6 min (time-on 1 min, time-off 1 min, 6 cycles)
using a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). The
Fig. 1 The enantioselective
production of (S)- or (R)-TFP
from TFA. a Whole-cell reaction
of O. polymorpha NBRC0799. b
Enzyme reaction with OpCRD-A
or OpCRD-B

Appl Microbiol Biotechnol (2018) 102:1307–1316
1309
supernatant (230 mL) obtained by centrifugation at 20,000×g
for 10 min was used as a crude enzyme solution.
200 °C for 4 min. The temperature of the vaporizing chamber
and the flame ionization detector was kept at 230 °C. Under
these conditions, the retention time for TFA, (R)-TFP, and (S)-
TFP was 2.5, 7.7, and 8.4 min, respectively (Fig. S1a). The
optical purity of the reaction products was calculated using the
peak area obtained under the above analyzing conditions.
Ammonium sulfate fractionation: Solid ammonium sulfate
(
40.5 g) was added to the crude enzyme solution (final 30%
saturation), and the resulting precipitates were removed by
centrifugation at 20,000×g for 30 min.
Phenyl-Toyopearl column chromatography: The superna-
tant from the 30% ammonium sulfate precipitation was ap-
plied to a Phenyl-Toyopearl column (14 × 2.8 cm diameter),
equilibrated with 10 mM buffer containing 1.33 M ammoni-
um sulfate. After the column was washed with 10 mM buffer
containing 1.33 M ammonium sulfate, the adsorbed enzyme
was eluted by a linear gradient of 10 mM buffer containing
Amino acid sequence of the enzyme proteins
The N-terminal amino acid sequence of each enzyme was
determined using a gas-phase protein sequencer equipped
with an online reverse-phase chromatography system, for
identification of PTH-amino acids.
1
.33 M ammonium sulfate and 10 mM buffer (400 ml each),
followed by 10 mM buffer (200 ml). Since the enzyme activ-
ity was separated in this step, OpCRD-A which eluted at a
higher conductivity and OpCRD-B which eluted at lower con-
ductivity were separately collected and each enzyme solution
was dialyzed against 10 mM buffer.
The internal amino acid sequence was analyzed as follows:
After separating the purified enzyme using 12% SDS-PAGE,
the protein was excised and incubated with trypsin (sequence
grade modified trypsin). The trypsin-digested peptides were
separated by a nanoUPLC equipped with a trap column
(nanoACQUITY UPLC Symmetry C18 Trap Column,
Waters, Milford, MA, USA) and a reverse-phase capillary
column (ACQUITY UPLC Peptide CSH C18
nanoACQUITY Column 10 K psi, Waters), using acetonitrile
water containing 0.1% formic acid. The molecular mass of
each peptide was determined using an electrospray
ionization/quadrupole time-of-flight mass spectrometer
(nanoACQUITY UPLC-SYNAPT G2-Si, Waters). The se-
quence of each peptide was analyzed using the Biolynx soft-
ware suite (Waters).
Q-Sepharose column chromatography: The dialyzed
OpCRD-A solution was applied to a Q-Sepharose column
(
7 × 2.8 cm diameter) equilibrated with 10 mM buffer. Since
OpCRD-A did not adsorb to this resin, unadsorbed active
fractions were collected. On the other hand, OpCRD-B
adsorbed to this resin and the enzyme was eluted by a linear
gradient of 10 mM buffer and 10 mM buffer containing
0
.15 M NaCl (150 mL each). The active fractions were com-
bined and dialyzed against 10 mM buffer.
Hydroxyapatite column chromatography: Solutions of
OpCRD-A and OpCRD-B were applied to a hydroxyapatite
column (6 × 1.6 cm diameter) equilibrated with 10 mM buffer.
After the column was washed with 10 mM buffer, the
adsorbed enzyme was eluted by a linear gradient of 10 mM
buffer and 0.3 M buffer (100 mL each). The active fractions
were collected and concentrated by ultrafiltration (Amicon
Ultra-15, Merck Millipore) with 10 mM buffer.
Gel filtration on a Superdex 200 column: The concentrated
OpCRD-A solution was applied to a Superdex 200 10/300GL
column (GE Healthcare UK Ltd., Buckinghamshire, UK)
equilibrated with 10 mM buffer containing 0.3 M NaCl.
Gene cloning of the enzyme
As mentioned in the BResults^ section, the N-terminal and in-
ternal amino acid sequences of OpCRD-A and OpCRD-B were
similar to those of ADH 2, alcohol dehydrogenase, and a puta-
tive threonine dehydrogenase/Zn-dependent dehydrogenase-
like protein from O. parapolymorpha DL-1, respectively.
Therefore, the DNA for OpCRD-A was cloned by PCR with
the following two primers that were synthesized based on the
N-terminal and C-terminal DNA sequences of ADH 2 of
O. parapolymorpha DL-1. The forward and reverse primers
were 5′-gaaggagatatacatATGACTTCCATTCCAAAGAC
TCAAA-3′ and 5′-ttagcagccggatccCTATTTGGAAGTGT
CAAGAACG-3′, respectively. The nucleotides denoted by
lower case letters indicate the 15-bp overlap sequence of
pET11a (Merck KgaA, Darmstadt, Germany), which is neces-
sary for use of the In-Fusion HD Cloning kit (Takara Bio Inc.,
Shiga, Japan). The following two primers, which were prepared
using a sequence 200–300 bases away from the N-terminus and
C-terminus of the DNA sequence from ADH.2 were also used.
T h e f o r w a r d a n d r e v e r s e p r i m e r s w e r e 5 ′ -
gaaggagatatacatTGTGAAATACCCCGCCACGGCCATC-3′
and 5′-ttagcagccggatccACTGGGACAGGTGCCGGAGTCG-
Identification of reaction products
A 56 mM TFA sample was incubated with 10 mM NADH,
5
0 mM d-glucose, 1.0 U of glucose dehydrogenase, and an
appropriate amount of OpCRD at 30 °C for 24 h in 0.2 M
KPB, pH 7.0 (total volume, 1 mL). The reaction products
were identified by gas chromatography using a capillary col-
umn (Rt-βDEXsa, Restek Corporation, PA, USA), in which
helium was used as the carrier gas and the pressure was
1
00 kPa. The temperature of the column was kept at 80 °C
for 25 min, then increased to 200 °C at 20 °C/min, followed by

1
310
Appl Microbiol Biotechnol (2018) 102:1307–1316
3
1
1
′, respectively. PCR was performed for 35 cycles, consisting of
0 s of denaturation at 98 °C, 10 s of annealing at 55 °C, and
5 s of extension at 72 °C, using PrimeSTAR Max DNA
Other analytical methods
The molecular mass of native enzymes was calculated by gel
filtration on a Superdex 200 10/300GL column using the fol-
lowing marker proteins: glutamate dehydrogenase (290 kDa),
lactate dehydrogenase (140 kDa), enolase (67 kDa), myokinase
(32 kDa), and cytochrome c (12.4 kDa). The molecular mass of
denatured enzymes was calculated using SDS-PAGE, with the
markers myosin (200 kDa), β-galactosidase (116 kDa), phos-
phorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ov-
albumin (45 kDa), carbonic anhydrase (31 kDa), and soybean
trypsin inhibitor (21.5 kDa). SDS-PAGE was carried out ac-
cording to the method of Laemmli (Laemmli 1970).
Polymerase (Takara Bio Inc.). Genomic DNA of
O. polymorpha NBRC0799 was extracted by using a Wizard
Genomic DNA Purification Kit (Promega Co., Wisconsin,
USA), according to the manufacturer’s protocol. The PCR
product was inserted into a linearized pET11a vector using
the In-Fusion HD Cloning Kit. The linearized vector was am-
plified by inverse PCR using the following two primers; 5′-
ATGTATATCTCCTTCTTAAAGTTAAAC-3′, and 5′-
GGATCCGGCTGCTAAC-3′. The sequence was analyzed
using an Applied Biosystems 3500 Genetic Analyzer
(
Applied Biosystems, CA, USA).
Protein concentration was measured by the method of
Bradford (Bradford 1976) using Bio-Rad protein assay kit
(Bio-Rad Laboratories, Hercules, CA, USA) and bovine se-
rum albumin as a standard protein.
The carbohydrate content of the enzyme was assayed with
phenol/sulfuric acid, according to the method of Hodge and
Hofreiter (Hodge 1962). The reaction mixture, containing
0.5 mL of the purified enzyme or mannose, 0.5 mL of 5%
phenol, and 2.5 mL of concentrated sulfuric acid, was allowed
to stand at room temperature for 20 min. The absorbance value
was then measured at 490 nm.
The metal species and their content in the enzymes were
analyzed by ICP-MS 7700× (Agilent Technologies, Inc., CA,
USA) using the ICP multi-element standard solution I (Merck
Millipore, MA, USA).
The isoelectric point (pI) was determined using an isoelec-
tric focusing apparatus (Nippon Eido, Tokyo, Japan) in 1%
Pharmalyte (GE Healthcare UK Ltd., Buckinghamshire, UK),
pH 3.0–10.0, with a sucrose gradient at 400 V for 2 days at
4 °C. The pI was also determined using a Novex pH 3–10 IEF
Gel (Thermo Fisher Scientific, MA, USA), according to the
manufacturer’s instructions.
The DNA for OpCRD-B was also cloned under the same
conditions as that of OpCRD-A, using the N-terminal and C-
terminal DNA sequences of a putative threonine
dehydrogenase/Zn-dependent dehydrogenase-like protein from
O. parapolymorpha DL-1. The forward primer of 5′-
gaaggagatatacatATGATGAAAGCATTGTGTTACTTA-3’and
reverse primer of 5′-ttagcagccggatccCTATTCCGAGGTCA
GAACGACC-3′ were used to amplify the OpCRD-B gene.
The primers 5′-gaaggagatatacatGTGCCTCGCATGTT
CCACGTGAG-3′ and 5′-ttagcagccggatccATCCGCAA
GCACGCGTGCTGCAC-3′ were used as the amplifiers of a
region outside of the OpCRD-B gene.
The recombinant plasmids pET11a-OpCRD-A and
pET11a-OpCRD-B, named pOpCRD-A and pOpCRD-B, re-
spectively, were used to transform Escherichia coli (E. coli)
BL21 (DE3) cells (Agilent Technologies Inc., Santa Clara,
CA, USA).
Expression of the genes for the enzymes
E. coli BL21 (DE3) cells harboring either pOpCRD-A or
pOpCRD-B plasmids were inoculated into 5 mL of Luria-
Bertani (LB) medium (10 g tryptone, 5 g yeast extract, and
Accession numbers
1
0 g NaCl in 1 L of water) containing 100 μg/mL of ampicil-
lin, and then incubated at 37 °C for 18 h with reciprocal shak-
ing at 300 strokes per min. The culture (50 μL) was then
inoculated into 5 mL of the same medium and incubation
continued at 37 °C, with shaking at 300 rpm. When the optical
density of the medium at 600 nm reached 0.5–1.0, isopropyl
β-d-thiogalactopyranoside (IPTG) was added to a final con-
centration of 0.5 mM and the incubation was further continued
at 30 or 20 °C for 24 h. Cells from 4 mL of the culture were
harvested by centrifugation (10,000×g, 5 min, 4 °C) and
washed with 20 mM KPB, pH 7.0. Wet cells (ca. 0.1 g) were
suspended in the same buffer, and then disrupted with glass
beads (φ0.5 mm) at 2700 rpm for 6 min using a Multi-beads
Shocker. The supernatant was obtained after centrifugation
The amino acid sequences of OpCRD-A and OpCRD-B from
O. parapolymorpha NBRC 0799 are deposited at DDBJ as
LC275242 and LC275243, respectively.
Results
Enzyme production
O. polymorpha NBRC 0799 was incubated in a 2-L shaker
flask containing 1 L of YM medium at 30 °C for 48 h, and
CRD activity was assayed every 6 h. The strain grew well in
the YM medium, and enzyme production reached a maximum
at 24 h of cultivation, which did not decrease until 48 h
(
20,000×g, 15 min, 4 °C) and used to assay the CRD activity
and the reaction products from TFA.

Appl Microbiol Biotechnol (2018) 102:1307–1316
1311
(
3
Fig. 2). We therefore incubated the strain with YM medium at
0 °C for 24 h to obtain sufficient enzyme for purification.
For example, enzyme activity of OpCRD-A toward
fluoroacetone, formaldehyde, and acetoaldehyde was more
than 10 times higher than that toward TFA, and that to-
ward 4,4,4-trifluoro-2-butanone was 30% of TFA. On the
other hand, OpCRD-B exhibited more than 20 times
higher activity toward acetone, 2-butanone, fluoroacetone,
Purification and molecular mass
Before purification of the enzymes catalyzing the reduction of
TFA, we discovered that O. polymorpha NBRC 0799 pro-
duced two CDRs, OpCRD-A and OpCRD-B, by isoelectric
focusing analysis (Fig. S2a). Both enzymes were efficiently
separated by Phenyl-Toyopearl column chromatography and
finally purified to a homogeneous state with a specific activity
of 1.36 U/mg of protein and 2.57 U/mg of protein, respective-
ly (Table S1, Fig. S3). The purified enzyme solution of
OpCRD-A exhibited no absorption maxima in the visible re-
gion, but OpCRD-B exhibited absorption maxima at 275 nm
and around 650 nm (data not shown).
4,4,4-trifluoro-2-butanone, acetoaldehyde, propionaldehyde,
and butyraldehyde than that toward TFA (Table 1a).
The substrate specificity in the dehydrogenation reaction
was assayed under standard assay conditions at the optimal
pH of both enzymes (OpCRD-A, pH 6.5 and 9.0; OpCRD-
B, pH 6.0 and 10.0), using 50 mM alcohols and 1.0 mM
Table 1 Substrate specificity of CRDs from O. polymorpha NBRC
0
799
Substrate
OpCRD-A
OpCRD-B
The molecular mass of the native form of OpCRD-A and
OpCRD-B was estimated to be 115 and 69 kDa on a Superdex
2
Specific activity
(U/mg)
Specific activity
(U/mg)
00 10/300GL column and that of the denatured form was 40
and 43 kDa by SDS-PAGE, respectively (Fig. S3). As men-
tioned in the section on amino acid sequences, molecular
masses of 36.6 kDa (OpCRD-A) and 39.3 kDa (OpCRD-B)
were also estimated from the deduced amino acid sequences.
These results indicated that OpCRD-A and OpCRD-B might
be composed of three and two identical subunits, respectively.
(
a) Reduction
Acetone
-Butanone
Fluoroacetone
4.1
2.0
51.4
67.8
109
29.8
26.7
2.57
117
2
21.2
4.5
1
1
,1-Difluoroacetone
,3 -Difluoroacetone
0.9
TFA
1.6
Substrate specificity and kinetic values
4
,4,4-Trifluoro-2-butanone 0.5
Formaldehyde
Acetoaldehyde
Propionaldehyde
Butyraldehyde
67.7
33.0
10.1
6.0
8.2
Substrate specificity in the reduction reaction of both en-
zymes was assayed under standard assay conditions at
each optimal pH (OpCRD-A, pH 6.5; OpCRD-B, pH
170
72.5
99.7
6
.0, as shown below) using 50 mM ketones or aldehydes.
(b) Dehydrogenation
Both enzymes exhibited reductase activity toward all sub-
strates tested, and the specific activity of OpCRD-B was
much higher than that of OpCRD-A for most of the sub-
strates. In addition, both enzymes exhibited remarkably
different relative activity toward ketones and aldehydes.
pH 6.5
pH 9.0
pH 6.0
10.8
12.0
0
pH 10.0
2
2
-Propanol
-Butanol
1.15
0.05
0
8.85
0.44
0.15
0.37
0.42
0.40
0
29.5
30.0
0.1
2.6
5.0
1.2
0
Methanol
Ethanol
0.01
0.01
0
0.4
0.7
0.2
0
1
1
-Propanol
-Butanol
(
S)-TFP
R)-TFP
0
(
0
0
0
0
2
2
1
1
-Methyl-2-propanol
-Methyl-2-butanol
,2-Propanediol
0
0
0.1
0.6
1.3
0
0.2
3.7
4.7
0
0
0
0
0.01
0.05
0
,3-Propanediol
0
Glycerol
0
0
0
(a) Reductase activity was assayed under standard assay conditions using
5
0 mM ketones or aldehydes and 0.1 mM NADH at pH 6.5 (CRD-A) or
Fig. 2 Production of CRD from O. polymorpha NBRC 0799. The CRD
activity toward TFAwas assayed every 6 h using cell-free extracts. Closed
triangles, closed quadrangles, and closed circles indicate CRD activity
toward TFA, cell growth, and the pH of the culture broth, respectively
pH 6.0 (CRD-B). (b) Dehydrogenase activity was assayed under standard
assay conditions using 50 mM alcohols and 1 mM NAD at pH 6.5 and
9.0 (CRD-A) or 6.0 and 10.0 (CRD-B). The values are shown as the
average of three-time assay
+

1
312
Appl Microbiol Biotechnol (2018) 102:1307–1316
+
NAD . Both the enzymes exhibited high activity at pH 9.0
or 10.0, and secondary alcohols, such as 2-propanol and 2-
butanol, were good substrates, while the reaction rates of
both enzymes were different from each other. OpCRD-A
exhibited higher activity toward 2-propanol than 2-butanol,
whereas OpCRD-B exhibited similar activity toward 2-
propanol and 2-butanol. Short-chain aliphatic primary alco-
hols were also dehydrogenated by both enzymes, but their
reaction rates were much slower than for 2-propanol. (S)-
TFP and (R)-TFP were not substrates for either enzyme
Stereospecificity
TFA was incubated with OpCRD-A or OpCRD-B, and the
reaction products were analyzed by GC. OpCRD-A
enantioselectively converted TFA into (S)-TFP with 99% ee.
On the other hand, OpCRD-B converted TFA into (R)-TFP
with 40% ee (Fig. S1). In addition, (R)- and (S)-TFP were not
dehydrogenated by either enzyme as mentioned above. These
results indicated that OpCRD-A can enantioselectively pro-
duce (S)-alcohols with high yields. Therefore, we concluded
that OpCRD-A might contribute to the production of (S)-TFP
in O. polymorpha NBRC 0799 cells (Fig. 1).
(
Table 1b). When reaction velocity for the reduction of
ketones and aldehydes was compared with that for dehy-
drogenation of corresponding alcohols, the former reaction
was much faster than the latter reaction at pH 6.0 and 6.5
Cofactor specificity
(
Table S2).
The apparent K values of OpCRD-A for TFA, acetone,
Cofactor specificity of both enzymes was analyzed under stan-
dard assay conditions using 50 mM TFA and 0.1 mM NADH
or NADPH. OpCRD-A exhibited an enzyme activity of
1.02 U/mL by incubation with NADH, but no activity was
detected with NADPH. In the case of OpCRD-B, enzyme
activities of 4.62 and 0.06 U/mL were obtained with NADH
or NADPH, respectively (data not shown). Thus, it was re-
m
and 2-propanol were estimated to be 35.5, 256, and 338
mM, respectively. Those of OpCRD-B for these substrates
were estimated to be 67.8, 0.13, and 0.56 mM, respectively
(
Table 2). Thus, OpCRD-A exhibited high affinity to TFA
compared with acetone, whereas OpCRD-B exhibited high
affinity to acetone compared with TFA. The results indicate
that O. polymorpha NBRC 0799 can properly utilize
OpCRD-A and OpCRD-B in the cells, when the different
concentration of acetone or 2-propanol is contained in a
medium; OpCRD-B reacts with a low concentration of ac-
vealed that both the enzymes were NADH-dependent.
+
The K values for NADH and NAD were analyzed using
m
acetone and 2-propanol, respectively, because both enzymes
did not react with (R/S)-TFP. Both enzymes showed much
+
+
etone or 2-propanol, and NAD or NADH is efficiently
lower apparent Km values for NADH than for NAD
formed, respectively. On the other hand, OpCRD-A reacts
with a high concentration of acetone or 2-propanol.
(Table 2), indicating that they have high affinity for NADH,
and the reduction of ketones or aldehydes proceeds well com-
pared with the dehydrogenation of alcohols under a low con-
centration of cofactor.
Table 2 The K
m
values of CRDs from O. polymorpha NBRC 0799
(mM)
Substrate cofactor
K
m
Effects of pH and temperature on enzyme activity
and stability
OpCRD-A
OpCRD-B
TFA
35.5
256
67.8
0.13
0.56
0.011
0.22
The optimal pH of TFA reduction was assayed under standard
assay conditions, except that the reaction pH was varied be-
tween 5.0 and 11.0. OpCRD-A exhibited reductase activity
over a wide pH range, with highest activity at pH 6.0–6.5.
On the other hand, OpCRD-B exhibited reductase activity at
acidic but not alkaline pH, and the highest activity was obtain-
ed at pH 6.0. (Fig. 3a).
Acetone
2
-Propanol
338
NADH
0.012
0.176
+
NAD
The K
m
values for TFA and acetone were obtained by assay of the en-
zyme activity using 2.5–100 mM TFA or 25–400 mM acetone and
Since neither enzymes exhibited dehydrogenase activity
toward (S)-TFP and (R)-TFP, the optimal pH of the dehydro-
0
0
.1 mM NADH at pH 6.5 (OpCRD-A), and 5.0–200 mM TFA or
.015–2.5 mM acetone and 0.1 mM NADH at pH 6.0 (OpCRD-B).
+
Those for 2-propanol were obtained by assay of the enzyme activity using
genation reaction was analyzed using 2-propanol and NAD .
+
2
5–600 mM 2-propanol and 1.0 mM NAD at pH 9.0 (OpCRD-A) and
Both enzymes exhibited dehydrogenase activity over a wide
pH range. OpCRD-A exhibited the highest activity at pH 9.0,
and 85% activity at pH 10.5. On the other hand, OpCRD-B
exhibited the highest activity at pH 10.0, which was drastical-
ly decreased at pH 11.0 (Fig. 3b).
The pH stability of both enzymes was analyzed by incu-
bating them at 30 °C for 60 min in a pH ranging from 5.0 to
11.5, without substrate. OpCRD-Awas stable in the pH range
+
using 0.05–12.5 mM 2-propanol and 1.0 mM NAD at pH 10.0
OpCRD-B). The K values for NADH was obtained by assay the en-
zyme activity using 300 mM acetone and 0.0075–0.25 mM NADH at pH
.5 (OpCRD-A) and using 0.5 mM acetone and 0.001–0.05 mM NADH
(
m
6
+
at pH 6.0 (OpCRD-B). Those for NAD were obtained by assay the
enzyme activity using 200 mM 2-propanol and 0.01–1.0 mM NAD at
pH 9.0 (OpCRD-A) and using 5.0 mM 2-propanol and 0.025–2.0 mM
NAD at pH 10.0 (OpCRD-B). Those values are shown as the average of
+
+
three-time assay

Appl Microbiol Biotechnol (2018) 102:1307–1316
1313
Fig. 3 Effects of pH and temperature on enzyme activity and stability. a
Optimal pH of the reductase reaction: enzyme activity was assayed under
described above (a), and the remaining activities were assayed under the
standard assay conditions for CRD activity measurement. d Optimum
temperature: CRD activity was assayed under standard assay conditions,
except that the reaction temperature was varied between 20 and 70 °C. e
Thermostability: enzyme activity was assayed after incubation at 30–
70 °C for 60 min at pH 6.5. The remaining activity was calculated by
the ratio to the enzyme activity without heating. In a–d, solid lines are
OpCRD-A and dotted lines are OpCRD-B
standard assay conditions using 170 mM citric acid-Na
2
HPO
.5), KPB (pH 5.8–8.0), Tris-HCl (pH 7.2–8.5), glycine-NaOH (pH 9.0–
0.5), and NaH PO -NaOH (pH 11.0–11.9). b Optimal pH of the dehy-
4
(pH 5.0–
6
1
2
4
drogenase reaction: the enzyme activity was assayed under standard assay
conditions using 2-propanol and the same buffers as (a). c pH stability:
the enzyme was incubated at 30 °C for 60 min with 40 mM buffers as
from 6.5 to 10.5. OpCRD-B was stable in the pH range from
sulfhydryl reagents, or metals. The enzyme activity of
2
+
6
.0 to 8.4 (Fig. 3c).
OpCRD-A was strongly inhibited by Cu , but not by other
metals, carbonyl reagents, chelating reagents, sulfhydryl re-
agents, or chelating reagents. On the other hand, the enzyme
activity of OpCRD-B was strongly inhibited by chelating re-
agents, such as o-phenanthroline, α,α-dipyridyl and 8-
The effect of temperature on TFA reduction was assayed
under standard assay conditions at the optimal pH of both
enzymes, except that the reaction temperature was varied be-
tween 20 and 70 °C. OpCRD-A exhibited the highest activity
at 50–55 °C, and enzyme activity at 20 and 70 °C was less than
2
+
3+
hydroxyquinoline, and Cu and Fe . Both enzymes were
2
+
20 and 10% of the highest activity, respectively. On the other
slightly activated by Mg (Table 3).
hand, OpCRD-B exhibited the highest activity at 30 °C and
more than 70% activity was obtained at 20–70 °C (Fig. 3d).
Thermostability was analyzed after the enzymes were in-
cubated at 20–70 °C for 60 min at pH 6.5, without substrate.
The enzyme activity of OpCRD-A remained intact even after
incubation at 50 °C, but dramatically decreased at 55 °C. For
OpCRD-B, more than 70% of the activity remained after in-
cubation at 50 °C and 30% at 60 °C (Fig. 3e).
Metal content and isoelectric point
OpCRD-B contained 2 mol of zinc per mole of subunit, but
OpCRD-A did not (data not shown). This result is in accor-
dance with the inhibitor specificity, whereby the enzyme ac-
tivity of OpCRD-B was inhibited by chelating reagents, but
that of OpCRD-A was not.
OpCRD-A and OpCRD-B showed a pI of 6.7 and 5.6,
respectively, using a Novex pH 3–10 IEF Gel (Fig. S2b).
These results are similar to the values obtained by an isoelec-
tric focusing apparatus using Pharmalyte, pH 3.0–10.0, as
shown in Fig. S2a.
Effects of compounds on enzyme activity
Effects of compounds on the CRD activity were analyzed by
adding 1 mM carbonyl reagents, chelating reagents,

1
314
Appl Microbiol Biotechnol (2018) 102:1307–1316
Table 3 Effects of chemicals and metals on the CRD activity
by the phenol-sulfuric acid method (data not shown), although
carbohydrate-binding motifs were present in the deduced ami-
no acid sequence of both CRDs as described below.
Chemical
Relative activity (%)
OpCRD-A
OpCRD-B
Amino acid sequence
None
100
92
92
84
90
88
74
77
70
85
90
88
77
111
108
101
103
96
0
100
93
77
94
42
86
16
37
15
76
95
75
80
115
74
64
67
89
0
Phenylhydrazine
Hydrazine
The N-terminal amino acid sequences of OpCRD-A and
OpCRD-B were found to be PSIPKTQKAIVFET and
MKALCYLGQNKGIRW, respectively, in which Cys was
identified using the nanoACQUITY UPLC-SYNAPTsystem.
The internal amino acid sequences of six trypsin-digested pep-
tides from OpCRD-A and five peptides from OpCRD-B were
determined by mass spectrometry (Table S3). Homologous
sequence analysis by BLAST, using above sequences, showed
that OpCRD-A is homologous to ADH 2 (Genbank accession
No. ESW99796.1) and that OpCRD-B is homologous to a
putative threonine dehydrogenase/Zn-dependent
dehydrogenase-like protein (Genbank accession No.
ESW96437.1) from O. parapolymorpha DL-1. Cloning of
OpCRD-A and OpCRD-B genes was performed as described
in the BMaterials and methods^ section, and the whole de-
duced amino acid sequence of both enzymes was determined
as shown in Fig. 4 (sequence data of OpCRD-A and OpCRD-
B were submitted to the DDBJ databases under the accession
numbers LC275242 and LC275243, respectively).
Hydroxylamine
Semicarbazide
EDTA
o-Phenanthroline
α,α’-Dipyridyl
8
-Hydroxyquinoline
Sodium azide
Potassium cyanide
N-Ethylmaleimide
Iodoacetic acid
MgCl
MnCl
CoCl
NiCl
ZnCl
CuCl
FeCl
2
2
2
2
2
2
3
96
29
The deduced amino acid sequence of OpCRD-A was only
2% identical to that of OpCRD-B (DNA sequence similarity
Enzyme activity was assayed under standard assay conditions using
2
0
1
.27 U of OpCRD-A and 0.62 U of OpCRD-B by the addition of
mM carbonyl reagents, chelating reagents, sulfhydryl reagents, or
was 46%). The deduced amino acid sequences of OpCRD-A
and OpCRD-B were approximately 99% identical to those of
ADH 2 and a putative threonine dehydrogenase/Zn-dependent
dehydrogenase-like protein from O. parapolymorpha DL-1,
respectively (DNA sequence similarity was 96 and 95%, re-
spectively). Recently, completely identical DNA sequences of
OpCRD-A and OpCRD-B were reported in the genome se-
quence of O. parapolymorpha NCYC 495 leu1.1 (Riley et al.
metals. Relative activity was calculated as percentage to the enzyme
activity without chemicals
Carbohydrate analysis
The carbohydrates were not detected in OpCRD-A, but
2
0 mol of mannose were estimated in 1 mol of OpCRD-B
Fig. 4 Deduced amino acid sequence of OpCRD-A and OpCRD-
B. Identical amino acid residues are indicated by asterisks. The
sequences of peptides obtained by trypsin digestion are framed in
boxes. Motifs for carbohydrate binding at asparagine residues (N-
X-T/S) are underlined. The NAD -binding motif (G-X-G-X-X-G)
is double lined
+

Appl Microbiol Biotechnol (2018) 102:1307–1316
1315
2
016). The alcohol dehydrogenase of that strain (Genbank ac-
developed by enantioselective reduction of TFA using baker’s
yeast (Bucciarelli et al. 1983; Doswald et al. 2007) or a crude
enzyme solution of ADH from R. erythropolis, A.paraffineus,
or P. farinose (Hayashi et al. 2007). These methods were use-
ful for enantioselective production of (S)-TFP, but not suffi-
cient for production of enantiomerically pure (S)-TFP (> 99%
ee). Therefore, they were utilized under the limited condition
to obtain the pure (S)-TFP (Bucciarelli et al. 1983). When the
optical purity of (S)-TFP in the reaction mixture is low, some
chemical methods such as a simulated moving bed chroma-
tography are also performed to obtain the pure (S)-TFP. Thus,
the enzyme with higher enantioselectivity is important to ob-
tain enantiomerically pure (S)-TFP. Recently, Asano et al. re-
ported that O. polymorpha NBRC 0799 is useful for the pro-
duction of enantiomerically pure (S)-TFP from TFA, with
high yields (Asano et al. 2011). However, the enzyme respon-
sible for this reaction had not yet been identified. Therefore,
we have now purified the enzymes catalyzing this reaction in
this strain and elucidated their characteristics.
The strain produced two enzymes catalyzing the reduction
of TFA, OpCRD-Awith pI 6.7 and OpCRD-B with pI 5.6, by
isoelectric focusing analysis. Since the pI values were rather
high, separation of the enzymes was difficult by ion exchange
chromatography, but they were efficiently separated by a
Phenyl-Toyopearl column chromatography.
Both OpCRD-A and OpCRD-B exhibited reductase activ-
ity toward not only TFA but also other fluoroacetone deriva-
tives, acetone, 2-butanone, and short-chain primary aldehydes
(Table 1a). In addition, high CRD activity was obtained by
incubation with NADH, but no activity with NADPH. Both
enzymes also exhibited dehydrogenase activity toward certain
alcohols, although these were much lower than toward the
corresponding ketones and aldehydes at pH 6.0 or 6.5
cession No. OBA15472.1) was identical to OpCRD-A, and a
hypothetical protein, OGAPODRAFT_22591 (Genbank acces-
sion No. OBA18267), was identical to OpCRD-B (Fig. S4).
+
In the sequence of OpCRD-A, the typical NAD -binding
motif (G-X-G-X-X-G) was not recognized, but the motif was
present in the OpCRD-B sequence at position 188–193 (G-L-
G-P-I-G). Motifs for carbohydrate binding at an asparagine
residue (N-X-T or S) were confirmed at positions 80–82 (N-
V-T), 96–98 (N-G-S), and 246–249 (N-V-S) in OpCRD-A
and at position 301–303 (N-V-T) in OpCRD-B, indicating that
carbohydrates can bind at these positions in both CRDs.
Overexpression of OpCRD-A and OpCRD-B genes
Each gene of OpCRD-A and OpCRD-B was cloned into the
pET11a vector and transformed into E. coli BL21 (DE3) cells.
Both transformants grew well in LB medium at 20 and 30 °C
and produced the CRD in a soluble fraction. However, the
transformants showed different optimal cultivation tempera-
tures for enzyme production. The transformant harboring the
pOpCRD-A gene produced 0.22 U/mL of broth (specific ac-
tivity of the cell-free extract was 0.40 U/mg of protein) at
3
0 °C, and 0.12 U/mL of broth (specific activity of the cell-
free extract was 0.18 U/mg of protein) at 20 °C. On the other
hand, the transformant harboring the pOpCRD-B gene
showed high enzyme productivity at 20 °C, compared with
3
0 °C. The transformant produced 0.26 U/mL of broth (spe-
cific activity of the cell-free extract was 0.48 U/mg of protein)
at 20 °C and 0.02 U/mL of broth (specific activity of the cell-
free extract was 0.05 U/mg of protein) at 30 °C. Thus, it was
revealed that recombinant OpCRD-A was efficiently pro-
duced by incubation at 30 °C, and OpCRD-B, at 20 °C.
(Table 1, Table S2). Thus, OpCRD-A and OpCRD-B are clas-
Production of 1,1,1-trifluoro-2-propanol
sified into the same group of NADH-dependent CRDs.
While both CRDs are classified into the same enzyme
group and exhibit broad substrate specificity, their enzymatic
properties are significantly different from each other. OpCRD-
A exhibited high CRD activity and stability over a wide pH
range compared with OpCRD-B (Figs. 3a, c). The optimal
temperature, thermostability, and inhibitor specificity were also
remarkably different from each other (Figs. 3d, e; Table 3).
OpCRD-A did not contain zinc, but OpCRD-B contained
4 mol of zinc per mole of enzyme. Furthermore, the amino
acid sequence of OpCRD-A was only 21% identical to that
of OpCRD-B. The amino acid sequences of OpCRD-A and
OpCRD-B were approximately 99% identical to those of ADH
The conversion of TFA to TFP was performed using a crude
recombinant enzyme solution, in which recombinant
OpCRD-A and OpCRD-B were prepared from the cells cul-
tivated at 30 and 20 °C, respectively. When 50 mU of recom-
binant OpCRD-A was incubated with 56 mM TFA under the
same conditions as described in the section of BIdentification
of reaction products^ in BMaterials and methods,^ 39.1 mM
(S)-TFP was produced with 99% ee. In the case of recombi-
nant OpCRD-B, 16 mM (R)-TFP was produced with 40% ee.
These results agree with the results of the purified OpCRD-A
and OpCRD-B enzymes (shown in Fig. S1).
2
and a putative threonine dehydrogenase/Zn-dependent
dehydrogenase-like protein from O. parapolymorpha DL-1,
respectively. We confirmed that OpCRD-B did not exhibit
threonine dehydrogenase activity. Thus, we demonstrated that
O. polymorpha NBRC 0799 produces two CRDs. Recently,
completely identical DNA sequences of OpCRD-A and
Discussion
(S)-TFP is one of the most useful optically active alcohols, and
biochemical methods for production of (S)-TFP have been

1
316
Appl Microbiol Biotechnol (2018) 102:1307–1316
OpCRD-B were reported by genome sequencing of
O. parapolymorpha NCYC 495 leu1.1 (Riley et al. 2016).
These results indicate that strains belonging to
O. polymorpha can produce different types of ADHs or CRDs.
The two CRDs from O. polymorpha NBRC 0799 catalyze
the reduction of ketones and dehydrogenation of alcohols, and
the former reaction is much faster than the latter reaction at
acidic pH. However, stereoselectivity of both enzymes was
different. OpCRD-A can produce (S)-TFP from TFA with
high enantioselectivity, whereas OpCRD-B exhibits low
enantioselectivity in the reduction of TFA. Therefore, we con-
cluded that OpCRD-A greatly contributes to the production of
References
Asano Y, Fuhshuku K, Nishii T, Ishii A (2011) Method for industrially
producing (S)-1, 1, 1-trifluoro-2-propanol. PAT-WO2011099595
Bradford MM (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 72:248–254
Bucciarelli M, Forni A, Moretti I, Torre G (1983) Asymmetric reduction
of trifluoromethyl and methyl ketones by yeast: an improved meth-
od. Synthesis 1983(11):897–899
Doswald S, Hanlon SP, Kupfer E (2007) Asymmetric reduction of 1, 1, 1-
trifluoroacetone. PAT-US2007/0009999 A1
Hayashi M, Nikaido T, Yamamoto H (2007) Process for production of
optically active alcohol. PAT-WO2007142210
(
S)-TFP from TFA in O. polymorpha NBRC 0799 cells.
Hodge J (1962) Determination of reducing sugars and carbohydrates.
Methods in carbohydrate chemistry 1:380–394
Rosen et al. isolated two ADHs, ADH RS1 and ADH RS2,
from Rhodococcus sp. and applied them to produce chiral
alcohols. The ADH RS1 exhibited reductase activity toward
TFA and enantioselectively produced (S)-TFP. However, the
activity toward TFAwas much lower than that toward acetone,
and production of (S)-TFP was 93% ee (Rosen et al. 2006).
On the other hand, our OpCRD-A exhibited similar activity
toward TFA and acetone and efficiently produced (S)-TFP
with 99% ee by E. coli. Thus, OpCRD-A from
O. polymorpha NBRC 0799 is useful for the efficient produc-
tion of enantiomerically pure (S)-TFP from TFA.
Laemmli UK (1970) Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 227(5259):680–685
Nakamura K, Yamanaka R, Matsuda T, Harada T (2003) Recent devel-
opments in asymmetric reduction of ketones with biocatalysts.
Tetrahedron Asymmetry 14(18):2659–2681
Ni Y, Xu J-H (2012) Biocatalytic ketone reduction: a green and efficient
access to enantiopure alcohols. Biotechnol Adv 30(6):1279–1288
Puentener K, Waldmeier P (2008) Asymmetric hydrogenation of 1, 1, 1-
trifluoroacetone. PAT-WO200812240
Riley R, Haridas S, Wolfe KH, Lopes MR, Hittinger CT, Goker M,
Salamov AA, Wisecaver JH, Long TM, Calvey CH, Aerts AL,
Barry KW, Choi C, Clum A, Coughlan AY, Deshpande S,
Douglass AP, Hanson SJ, Klenk HP, LaButti KM, Lapidus A,
Lindquist EA, Lipzen AM, Meier-Kolthoff JP, Ohm RA, Otillar
RP, Pangilinan JL, Peng Y, Rokas A, Rosa CA, Scheuner C,
Sibirny AA, Slot JC, Stielow JB, Sun H, Kurtzman CP, Blackwell
M, Grigoriev IV, Jeffries TW (2016) Comparative genomics of bio-
technologically important yeasts. Proc Natl Acad Sci U S A 113(35):
Acknowledgements The authors wish to express their thanks to Prof.
Tomonori Kawakami, Toyama Prefectural University, for his work on
metal content analysis. Thanks are due to Dr. Atsutoshi Ina for the work
on protein sequence determination and to Dr. Ken-ich Fuhshuku, Ms.
Nana Takada and Mr. Shinji Kitagawa for their preliminary experiments.
This work was supported by JST ERATO Asano Active Enzyme
Molecule Project (Grant Number JPMJER1102), Japan.
9
882–9887. https://doi.org/10.1073/pnas.1603941113
Rosen TC, Feldmann R, Dünkelmann P, Daußmann T (2006)
Bioreductive synthesis of perfluorinated chiral alcohols.
Tetrahedron Lett 47(28):4803–4806
Yamada-Onodera K, Nariai H, Tani Y, Yamamoto H (2004) Gene cloning
of dihydroxyacetone reductase from a methylotrophic yeast,
Hansenula ofunaensis, and its expression in Escherichia coli
HB101 for production of optically active 2-pentanol. Engineering
in life sciences 4(5):418–425
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.