Asymmetric oxidation of 1,3-propanediols to chiral hydroxyalkanoic acids by Rhodococcus sp. 2N

Article abstract of DOI:10.1080/09168451.2018.1559031

Rhodococcus sp. 2N was found as a 1,3-propanediols-oxidizing strain from soil samples through enrichment culture using 2,2-diethyl-1,3-propanediol (DEPD) as the sole carbon source. The culture condition of the strain 2N was optimized, and the highest activity was observed when 0.3% (w/v) DEPD was added in the culture medium as an inducer. Chiral HPLC analysis of the hydroxyalkanoic acid converted from 2-ethyl-2-methyl-1,3-propanediol (EMPD) revealed that the strain 2N catalyzed the (R)-selective oxidation of EMPD. The reaction products and intermediates from DEPD and EMPD were identified by nuclear magnetic resonance analyses, and the results suggested that only one hydroxymethyl group of the propanediols was converted to carboxy group via two oxidation steps. Under optimized conditions and after a 72-h reaction time, the strain 2N produced 28 mM (4.1 g/L) of 2-(hydroxymethyl)-2-methylbutanoic acid from EMPD with a molar conversion yield of 47% and 65% ee (R).

Full text of DOI:10.1080/09168451.2018.1559031

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Asymmetric oxidation of 1,3-propanediols to chiral
hydroxyalkanoic acids by Rhodococcus sp. 2N
Hiroshi Kikukawa, Rena Koyasu, Yoshihiko Yasohara, Noriyuki Ito, Koichi
Mitsukura & Toyokazu Yoshida
To cite this article: Hiroshi Kikukawa, Rena Koyasu, Yoshihiko Yasohara, Noriyuki Ito, Koichi
Mitsukura & Toyokazu Yoshida (2018): Asymmetric oxidation of 1,3-propanediols to chiral
hydroxyalkanoic acids by Rhodococcus sp. 2N, Bioscience, Biotechnology, and Biochemistry, DOI:
10.1080/09168451.2018.1559031
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2018.1559031
Asymmetric oxidation of 1,3-propanediols to chiral hydroxyalkanoic acids by
Rhodococcus sp. 2N
a
b
c
c
a
Hiroshi Kikukawa , Rena Koyasu , Yoshihiko Yasohara , Noriyuki Ito , Koichi Mitsukura
a
and Toyokazu Yoshida
a
b
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan; Division of Biomolecular
c
Science, Graduate School of Engineering, Gifu University, Gifu, Japan; Biotechnology Research Laboratories, Kaneka Corporation,
Hyogo, Japan
ABSTRACT
ARTICLE HISTORY
Rhodococcus sp. 2N was found as a 1,3-propanediols-oxidizing strain from soil samples through
enrichment culture using 2,2-diethyl-1,3-propanediol (DEPD) as the sole carbon source. The
culture condition of the strain 2N was optimized, and the highest activity was observed when
Received 31 October 2018
Accepted 6 December 2018
KEYWORDS
Asymmetric oxidation;
0.3% (w/v) DEPD was added in the culture medium as an inducer. Chiral HPLC analysis of the
hydroxyalkanoic acid converted from 2-ethyl-2-methyl-1,3-propanediol (EMPD) revealed that
the strain 2N catalyzed the (R)-selective oxidation of EMPD. The reaction products and inter-
mediates from DEPD and EMPD were identified by nuclear magnetic resonance analyses, and
the results suggested that only one hydroxymethyl group of the propanediols was converted to
carboxy group via two oxidation steps. Under optimized conditions and after a 72-h reaction
time, the strain 2N produced 28 mM (4.1 g/L) of 2-(hydroxymethyl)-2-methylbutanoic acid from
EMPD with a molar conversion yield of 47% and 65% ee (R).
1
-propanediol;
hydroxyalkanoic acid;
Rhodococcus
Optically active hydroxyalkanoic acids are beneficial as
pharmaceutical intermediates, chiral synthons, and
monomers for functional polymers, and various studies
have been performed to develop an efficient synthesis
method of valuable hydroxyalkanoic acids [1–3].
Hydroxyalkanoic acids are enzymatically synthesized
through the following two unique reactions: 1) hydroxy-
lation of the terminal methyl group of alkanoic acids and
2-ethyl-2-methyl-1,3-propanediol (EMPD) oxidation; it
has significant potential as raw material for the synth-
esis of a novel functional polymer. In this study, we
report the screening of microorganisms oxidizing
2,2-diethyl-1,3-propanediols (DEPD) and the synthesis
of hydroxyalkanoic acids, especially chiral HMMBA,
using whole cells of a DEPD-oxidizing strain (Figure 1).
2
) oxidation of the unilateral hydroxymethyl group of
diols. For example, the (R)- and (S)-enantiomers of
-hydroxy-2-methylpropanoic acid can be synthesized
Materials and methods
3
Materials
from 2-methylpropanoic acid by microbial asymmetric
hydroxylation [4–6]. The microbial asymmetric oxida-
tion of 2-methyl-1,3-propanediol by acetic acid bacteria
Gluconobacter and Acetobacter strains produce
The diol compounds, DEPD and EMPD, were pur-
chased from Tokyo Chemical Industry Co., Ltd.
(Japan). Products from the following suppliers were
used: Polypepton (Nippon Pharmaceutical Co., Ltd.,
Japan), meat extract (Kyokuto Pharmaceutical
Industrial Co., Ltd., Japan), and yeast extract (Oriental
Yeast Co., Ltd., Japan). All other chemicals were certi-
fied to be of reagent grade.
(R)-3-hydroxy-2-methylpropanoic acid with high optical
purity [7–9]. (R)- and (S)-2-(Hydroxymethyl)hexanoic
acid, which comprise useful constituents of an anti-
bacterial agent for multidrug-resistant Staphylococcus
aureus [10,11], are produced by Pseudomonas putida
IFO 3738 and A. pasteurianus IAM 12073, respectively
Microorganisms and culture conditions
[12]. However, to our knowledge, the production of
hydroxyalkanoic acids from 2,2-disubstituted-1,3-propa-
nediols has not yet been studied, and the microbial
enzymes accepting various propanediols as substrates
have not been reported.
Various hydroxybutanoic acids are useful as config-
urational molecular glues [13–15]. 2-(Hydroxymethyl)-
The soil samples were added to 80 mL of enrichment
culture medium comprising 0.5% (w/v) DEPD, 0.3%
(
w/v) NH Cl, 0.2% (w/v) K HPO , 0.1% (w/v) NaCl,
4 2 4
0
.01% (w/v) MgSO ·7H O, 0.001% (w/v) FeSO ·7H
4 2 4 2
O, 1% (v/v) vitamin mixture, and 0.1% (v/v) metal
mixture. Microorganisms were isolated on the agar
plate of enrichment culture medium. The vitamin
2
-methylbutanoic acid (HMMBA) can be produced by
CONTACT Koichi Mitsukura
mitukura@gifu-u.ac.jp; Hiroshi Kikukawa
kikukawa@gifu-u.ac.jp
©
2018 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
H. KIKUKAWA ET AL.
a. DEPD oxidation
Alcohol
oxidation
Aldehyde
oxidation
DEPD
2-ethyl-2-(hydroxymethyl)butanal
EHMBA
b. EMPD oxidation
Alcohol
oxidation
Aldehyde
oxidation
EMPD
2-(hydroxymethyl)-2-methylbutanal
HMMBA
Figure 1. Enzymatic pathways for the oxidation of DEPD (a) or EMPD (b) by whole cells of Rhodococcus sp. 2N.
mixture consisted of 0.01% (w/v) biotin, 0.01% (w/v)
inositol, 0.002% (w/v) calcium pantothenate, 0.002%
citrate (pH 5.0–6.0), acetate-sodium acetate (pH
5.0–5.5), KPi (pH 6.0–8.0), Tris-HCl (pH 7.5–9.0),
and glycine-NaOH (pH 9.0–10.0). The effect of tem-
perature was investigated at 20–40 °C.
(w/v) nicotinic acid, 0.002% (w/v) pyridoxine·HCl,
0
.001% (w/v) p-aminobenzoic acid, 0.001% (w/v)
riboflavin, and 1.1 μM folic acid. The metal mixture
consisted of 0.04% (w/v) CaCl ·2H O, 0.03% (w/v)
2
2
H BO , 0.004% (w/v) CuSO ·5H O, 0.01% (w/v) KI,
Analyses and identification of the reaction
3
3
4
2
0
.02% (w/v) FeSO ·7H O, 0.04% (w/v) MnSO ·7H O,
products
4
2
4
2
and 0.02% (w/v) NaMoO ·2H O in 1% (v/v) conc.
4
2
The reaction products were analyzed with thin-layer
chromatography (TLC) using 2,6-dichlorophenolindo-
phenol for carboxy group detection. The products and
intermediates were also analyzed by high-performance
liquid chromatography (HPLC) with a YMC-Triart
C18 column (4.6 × 150 mm; YMC Co., Ltd.) coupled
to an SPD-10AVP UV-Vis detector (Shimadzu, Japan).
The eluent, 50 mM NaH PO /H PO (pH 2.8)/aceto-
nitrile (4:1 v/v), was applied at a flow rate of 1 mL/min
and the eluate was monitored at 210 nm. The reaction
products were extracted with diethyl ether after adjust-
ing the pH of the reaction mixture to pH 3 with 2 M
HCl. The crude products were purified by silica gel
column chromatography (30 × 110 mm, Wakogel
C-300; FUJIFILM Wako Pure Chemical Co., Japan),
and the following eluents (v/v) were run in sequence:
n-hexane only (200 mL), n-hexane/ethyl acetate = 6:1
HCl. The soil isolates were cultivated in 50 mL of
nutrient medium comprising 0.5% (w/v)
a
Polypepton, 0.5% (w/v) meat extract, 0.2% (w/v)
NaCl, and 0.05% (v/v) yeast extract in 500 mL
shake flasks. The isolates were cultured at 28 °C for
2
days with reciprocal shaking (120 strokes/min).
The genome of the soil isolate strain 2N was extracted
2
4
3
4
and purified, and the 16S rRNA gene was amplified and
cloned into the pT7Blue vector. The 16S rRNA gene
sequencing analysis was performed with the primers
27f (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and 1525r
(5ʹ-AAAGGAGGTGATCCAGCC-3ʹ), the GenomeLab
DTCS Quick Start Kit (Sciex, USA), and a Beckman
CEQ8000 DNA sequencer (Beckman Coulter, UK).
Propanediol oxidation reaction using whole cells
(350 mL), n-hexane/ethyl acetate = 4:1 (300 mL), and
The oxidation of propanediols was carried out in 4 mL
of reaction mixtures comprising 10 mM propanediols,
n-hexane/ethyl acetate = 2:1 (600 mL).
The chemical shifts of 2-ethyl-2-(hydroxymethyl)
butanoic acid (EHMBA) were determined: H-NMR
1
1
00 mM potassium phosphate (KPi) buffer (pH 8.0),
and whole cells of the soil isolate Rhodococcus sp. 2N
(600 MHz, D O); δ 0.59 (3H × 2, t, J = 7.2 Hz, -CH -
2
2
(
(
11.9 mg dry cell weight) in 50 mL sample tube
Maruemu Corp., Japan) at 30 °C with reciprocal shak-
CH ), 1.29 (2H × 2, q, J = 7.7 Hz, -CH -CH ), and 3.47
3 2 3
13
(2H, s, -CH -OH), and C-NMR (150 MHz, D O); δ
2
2
ing (120 strokes/min). The strain 2N was precultivated
at 28 °C for a day, then the whole broth was inoculated
into 50 mL of the nutrient medium described above.
The whole cells of the strain 2N were harvested by
centrifugation, washed with 0.85% (w/v) NaCl twice,
and resuspended with 0.85% (w/v) NaCl.
8.23, 26.24, 52.56, 61.44, and 184.34. The chemical
shifts of HMMBA were determined: H-NMR
1
(600 MHz, D O); δ 0.64 (3H, t, J = 7.6 Hz, -CH -
2
2
CH ), 0.89 (3H, s, -CH ), 1.29 (2H, dq, J = 7.6 Hz,
3
3
7.6 Hz, -CH -CH ), 3.33 (1H, d, J = 11 Hz, -CH -OH),
2
3
2
13
and 3.46 (1H, d, J = 11 Hz, -CH -OH), and C-NMR
2
The effect of pH condition was investigated using
(150 MHz, D O); δ 8.43, 19.50, 28.40, 49.08, 67.85, and
2
100 mM of the following buffers: citrate-sodium
185.10.

MICROBIAL OXIDATION OF DIOLS TO CHIRAL HYDROXYALKANOIC ACIDS
3
The intermediates were extracted with diethyl
ether and derivatized with 15 mM 2,4-dinitrophenyl-
hydrazine to form hydrazone derivatives [16]. The
hydrazone derivatives were fractionated and purified
on PLC Silica gel 60 F254, 2 mm (Merck, Germany)
with ethyl acetate/n-hexane = 4:1. The identification
isolated and analyzed by NMR using the same
method applied to DEPD. The product (r.t. = 3.70
on HPLC analysis; Rf = 0.75 on TLC using ethyl
acetate/methanol = 4:1) converted from EMPD was
identified as HMMBA. The product HMMBA was
derivatized with phenacyl bromide and analyzed
with chiral HPLC. The results showed that the whole-
cell reaction exhibited (R)-selectivity (65% ee) in the
oxidation of EMPD.
1
13
was carried out with H-NMR and C-NMR.
Optical purity of HMMBA
The HMMBA produced from EMPD was derivatized
with phenacyl bromide according to the manufacturer’s
protocol (TCI, Japan). The optical purity of the
obtained derivative was evaluated with chiral HPLC
analysis on a ChiralPak AD-H column (4.6 × 150 mm;
Daicel Corp., Japan) coupled to an SPD-10AVP UV-Vis
detector (Shimadzu). The eluent, n-hexane/ethanol
Identification of the intermediates in the
oxidation reaction
In the early stage of the oxidizing reaction of either
DEPD or EMPD, an unknown product was identified
on HPLC analysis. The unknown products from
DEPD and EMPD disappeared in accordance with
the formation of the corresponding hydroxyalkanoic
acids. Thus, it was suggested that the unknown pro-
ducts were the aldehydes formed during the hydro-
xybutanoic acids formation. The unknown products
were extracted with diethyl ether and derivatized with
(
7:3 v/v), was applied at a flow rate of 1 mL/min, and
the eluate was monitored at 254 nm. (S)- and
R)-HMMBAs were eluted separately and analyzed in
(
comparison with the elution of authentic compounds.
The retention times of (S)- and (R)-HMMBAs were
1
1.2 and 13.6 min, respectively.
2,4-dinitrophenylhydrazine to form 2,4-dinitrophe-
nylhydrazone derivatives. The derivatives were
obtained as yellow solids, then washed with pure
water to thoroughly remove the HCl, and analyzed
with NMR. The unknown product from DEPD was
identified as 2-ethyl-2-(hydroxymethyl)butanal and
that from EMPD was identified as 2-(hydroxy-
methyl)-2-methylbutanal, as shown in Figure 1.
The 2,4-dinitrophenylhydrazone derivative of
2-ethyl-2-(hydroxymethyl)butanal (r.t. = 9.32 on
Results
Isolation and selection of DEPD-oxidizing
microorganisms
To establish the efficient synthesis of optically active
hydroxyalkanoic acids usingꢀmicrobial enzymes, we
isolated 30 strains of DEPD-consuming microorgan-
isms through enrichment culture with medium com-
prising DEPD as the sole carbon source. Three strains
of these DEPD-consuming microorganisms were
selected for further analysis based on the spot intensity
of carboxylic acids on TLC analysis. After a 24-h
incubation of the whole cells with DEPD, the forma-
tion of monocarboxylic acid and dicarboxylic acid was
evaluated by HPLC analysis. Among the strains, only
one, named as 2N, was able to synthesize monocar-
boxylic acid without forming dicarboxylic acid, indi-
cating the oxidization of the unilateral hydroxymethyl
group of DEPD. The NMR analysis of the isolated
monocarboxylic acid [retention time (r.t.) = 5.57 on
HPLC analysis; Rf = 0.48 on TLC using ethyl acetate]
revealed that the monocarboxylic acid was EHMBA.
The strain 2N exhibited colonial morphology of
smooth surface, moist texture, and pink color. The
HPLC analysis; Rf = 0.47 on TLC using ethyl acetate/
1
n-hexane = 4:5): H-NMR (600 MHz, CDCl ); δ 0.90
3
(3H × 2, t, J = 7.6 Hz, -CH -CH ), 1.63 (2H × 2, q,
2
3
J = 7.6 Hz, -CH -CH ), 1.77 (1H, t, J = 5.9 Hz, -OH),
2
3
3.74 (2H, d, J = 3.4 Hz, -CH -OH), 7.43 (1H, s, -C
2
(= N-)-H), 7.85 (1H, d, J = 9.6 Hz, -NH-C-CH-CH-),
8.31 (1H, dd, J = 2.8, 9.6 Hz, -NH-C-CH-CH-C(NO ),
2
)-
9.12 (1H, d, J = 2.8 Hz, -NH-C-C(NO )-CH-C(NO )-),
2
2
13
and 11.04 (1H, s), and C-NMR (150 MHz, CDCl ); δ
3
7.85, 25.07, 46.58, 65.03, 116.29, 123.55, 128.96, 130.10,
137.94, 144.96, and 157.28. The 2,4-dinitrophenylhy-
drazone derivative of 2-(hydroxymethyl)-2-methylbu-
tanal (r.t. = 5.28 on HPLC analysis; Rf = 0.41 on TLC
1
using ethyl acetate/n-hexane = 1:1.25): H-NMR
(600 MHz, CDCl ); δ 0.95 (3H, t, J = 7.6 Hz, -CH -
3
2
CH ), 1.19 (3H, s), 1.59–1.67 (2H, m, -CH -CH ), 1.89
3
2
3
16S rRNA gene (1,511 bp, accession no. LC434455)
(1H, s, -OH), 3.65 (1H, d, J = 11.0 Hz, -CH -OH), 3.76
2
sequencing analysis of strain 2N showed 100% identity
to Rhodococcus sp. JCM 28273 (accession no.
LC133620) and Rhodococcus sp. S10 (accession no.
MG551531), and the strain 2N was identified as
Rhodococcus sp. 2N.
(1H, d, J = 11.0, -CH -OH), 7.48 (1H, s, -C(= N-)-H),
7.88 (1H, d, J = 9.7 Hz, -NH-C-CH-CH), 8.32 (1H, dd,
2
J = 2.8, 9.6 Hz, -NH-C-CH-CH-C(NO )-), 9.13 (1H, d,
2
J = 2.8 Hz, -NH-C-C(NO )-CH-C(NO )-), and 11.05
2
2
13
(1H, s), and C-NMR (150 MHz, CDCl ); δ 8.18, 19.10,
3
The whole cells of Rhodococcus sp. 2N also cata-
lyzed the oxidation of EMPD. The product was
28.40, 43.90, 68.14, 116.32, 123.51, 128.93, 130.06,
137.91, 144.99, and 157.33.

4
H. KIKUKAWA ET AL.
Enzyme induction, optimization of the reaction
conditions, and production of hydroxyalkanoic
acids
than for DEPD. The strain 2N cells did not accept
1,3-propanediols comprising a butyl group, hydroxy-
methyl group, methylene group, or amino group in the
2
-position, such as 1,3-propanediol, 2-butyl-1,3-propa-
The alcohol-oxidizing activity of the strain 2N was
inducibly expressed by the addition of various propa-
nediols. No activity was detected without propane-
diols in the culture medium (Table 1). The addition
of 0.3% (w/v) DEPD induced the highest level of
alcohol-oxidizing activity, whereas the addition of
nediol, glycerol, 2-phenyl-1,3-propanediol, 2-methy-
lene-1,3-propanediol, 2-butyl-2-ethyl-1,3-propanediol,
2
1
-hydroxymethyl-2-methyl-1,3-propanediol, 2-amino-
,3-propanediol, 2-amino-2-methyl-1,3-propanediol,
and 2-amino-2-hydroxymethyl-1,3-propanediol.
2
-amino-1,3-propanediols induced no oxidizing
activity.
The 2N cells exhibited the highest oxidation activity
Discussion
We isolated and screened microorganisms through
enrichment culture comprising DEPD as a sole carbon
source. We identified one strain, Rhodococcus sp. 2N,
capable of catalyzing the asymmetric oxidation of
EMPD to (R)-HMMBA. Several diol dehydrogenases
when cultured for 60 h at 28 °C in the nutrient medium
comprising 0.3% (w/v) DEPD, pH 7.0. The highest activ-
ity was observed when the whole-cell reaction was per-
formed at 30 °C in 100 mM KPi buffer, pH 8.0, with
reciprocal shaking at 120 strokes/min. In the 100 mM
KPi (pH 7.0), the oxidation activity was about 70% of
that in 100 mM KPi (pH 8.0). The oxidizing activity of
the strain 2N cells was kept well in the presence of less
than 60 mM EMPD (Figure 2), indicating that excess
addition of EMPD inhibits the oxidation activity.
The conversion of DEPD and EMPD to EHMBA
and HMMBA, respectively, was performed under the
optimal conditions to achieve efficient production of
the hydroxyalkanoic acids (Figure 3). The productivity
of EHMBA reached 28 mM (4.1 g/L) with a molar
conversion yield of 47% and 28 mM (4.1 g/L)
HMMBA was formed from 60 mM EMPD with
a molar conversion yield of 47% and 65% ee (R). The
final pH of the reaction supernatant was around pH 7.0.
+
from Rhodococcus sp. are reported as NAD /NADH-
dependent enzymes [17,18]. With respect to the
Rhodococcus sp. 2N, the cell-free extract exhibits no
oxidation activity upon the addition of coenzymes
+
+
NAD , NADP , NADH, and NADPH (data not
shown). For the characterization of coenzyme depen-
dence, enzyme(s) identification and production of
recombinant enzyme(s) are required to clarify the coen-
zyme requirement. The oxidation activity of the strain
2N cells was highly induced on addition of 0.3% (w/v)
16
14
60
5
0
0
1
2
0
8
6
4
2
0
4
1
30
Substrate specificity of the oxidizing enzyme(s) in
Rhodococcus sp. 2N
2
1
0
0
Substrate specificity of the oxidizing enzyme(s) was
investigated using the whole cells of the strain 2N
and 10 mM various 1,3-propanediols (Table 2). The
strain 2N cells accepted 2-methyl-1,3-propanediol,
0
0
20
40
60
80
EMPD (mM)
2
1
,2-dimethyl-1,3-propanediol, and 2-methyl-2-propyl-
,3-propanediol besides DEPD and EMPD. The activ-
Figure 2. Effect of EMPD concentration on HMMBA produc-
tion (solid circle) and conversion rate (open bar).
ity was 1.66-fold higher for 2-methyl-1,3-propanediol
Table 1. Induction of oxidation activity by propanediol.
Propanediol
Concentration [% (w/v)]
Relative activity (%)
None
-
0
1
2
2
2
2
2
,3-Propanediol
-Methyl-1,3-propanediol
-Butyl-1,3-propanediol
,2-Dimethyl-1,3-propanediol
-Ethyl-2-methyl-1,3-propanediol (EMPD)
,2-Diethyl-1,3-propanediol (DEPD)
0.3
0.3
0.3
0.3
0.3
0.1
10
17
11
36
64
25
93
0.2
0.3
0.4
0.5
a
100
80
52
0
8
20
2
2
-Amino-1,3-propanediol
-Amino-2-methyl-1,3-propanediol
0.3
0.3
0.3
Glycerol
a
7
.8 mM EHMBA was formed.

MICROBIAL OXIDATION OF DIOLS TO CHIRAL HYDROXYALKANOIC ACIDS
5
30
25
20
15
10
5
0
0
10
20
30
40
50
60
70
80
Time (h)
Figure 3. Production of EHMBA (solid squares) from 60 mM DEPD, and HMMBA (open circles) from 60 mM EMPD, by whole cells
of Rhodococcus sp. 2N. All the values are means for triplicate experiments.
Table 2. Substrate specificity of the oxidizing enzyme(s) in
Rhodococcus sp. 2N cells.
In the conversion of EMPD, the oxidation activity
of the strain 2N cells decreased with over 60 mM
EMPD. During the oxidation of DEPD and EMPD
Propanediol
Relative activity (%)
2
2
2
2
2
-Methyl-1,3-propanediol
,2-Dimethyl-1,3-propanediol
-Ethyl-2-methyl-1,3-propanediol (EMPD)
,2-Diethyl-1,3-propanediol (DEPD)
-methyl-2-propyl-1,3-propanediol
166
79
102
100
20
(Figure 3), both DEPD and EMPD were converted
with a molar conversion yield of 47%. The final pH of
the reaction was around pH 7.0, indicating that the
dehydrogenase(s) in the strain 2N was not deacti-
vated by the pH decrease because the enzyme(s) can
work at pH 7.0 as described in the Results section.
These results suggest that the oxidation activities of
the strain 2N cells are inhibited by the produced
EHMBA and HMMBA.
DEPD to the culture medium. Conversely, the addition
of >0.4% (w/v) DEPD decreased the oxidation activity.
The oxidation activity was less induced by the propa-
nediols with lower steric hindrance than DEPD, such as
2
-methyl-1,3-propanediol, 2,2-dimethyl-1,3-propane-
In the present study, we found Rhodococcus sp. 2N
that synthesizes EHMBA and (R)-HMMBA from
DEPD and EMPD, respectively. To isolate this strain,
we used DEPD as a sole carbon source for enrich-
ment culture. Enrichment culture using EMPD
instead of DEPD would lead to acquire a much
higher number of microorganisms oxidizing various
1,3-propanediols because EMPD has less steric hin-
drance than DEPD. To achieve the production of
S-form of HMMBA, a promising molecular glue of
polymers for development of novel functional mate-
rials or a building block for chemical synthesis, we
need to further isolate microorganisms capable of
catalyzing the asymmetric oxidation of 1,3-propane-
diols. Furthermore, to establish a more efficient
asymmetric oxidation system, we would need to iden-
tify the genes encoding the alcohol dehydrogenase(s)
and aldehyde dehydrogenase(s) oxidizing the 1,3-pro-
panediols in the strain 2N.
diol, and EMPD. The aminopropanediols barely
induced the activity.
During the conversion of DEPD and EMPD into their
corresponding hydroxymethyl carboxylic acids using the
strain 2N cells, the formation of the aldehyde intermedi-
ates 2-ethyl-2-(hydroxymethyl)butanal and 2-(hydroxy-
methyl)-2-methylbutanal was observed, respectively. In
previous studies [7–12], no aldehyde intermediate was
detected. Presumably, the dehydrogenase(s) involving in
the asymmetric oxidation of the present strain 2N have
characteristics different from the membrane-bound
dehydrogenases of acetic acid bacteria [19]. In the oxida-
tion reaction of their membrane-bound dehydrogenases,
no aldehyde intermediate was detected because the alco-
hol substrate can be immediately transferred to following
enzyme aldehyde dehydrogenase. The dehydrogenase(s)
were soluble in cell-free extract, so the aldehyde inter-
mediate might be discharged from the conversion flow.
The chemical synthesis of the abovementioned aldehydes
has been reported [20]; however, their enzymatic or
microbial synthesis remains to be reported. By inhibiting
the aldehyde oxidation involved in the subsequent oxida-
tion reaction of Rhodococcus sp. 2N by gene modification
or with inhibitor for the dehydrogenase(s), the aldehydes
might be exclusively produced.
Author contributions
K. Mitsukura and H. Kikukawa designed the research.
H. Kikukawa, R. Koyasu, and K. Mitsukura performed the
experiments. H. Kikukawa wrote the paper. T. Yoshida
supervised all the experiments and data analyses.

6
H. KIKUKAWA ET AL.
Acknowledgments
Acetobacter pasteurianus. Tetrahedron-Asymmetry.
003;14:2041–2043.
9] Ohta H, Tetsukawa H, Noto N. Enantiotopically selec-
tive oxidation of α,ω-diols with the enzyme systems of
microorganisms. J Org Chem. 1982;47:2400–2404.
2
We would like to thank Enago (www.enago.jp) for English
language editing.
[
[
10] Fujita M, Sakamoto M, Horiuchi N, et al. inven-
tors; Dainippon Pharmaceutical Co., Ltd.,
assignee. Proline derivatives. PCT Int Appl.
WO2004018453A1. 2004.
Disclosure statement
No potential conflict of interest was reported by the
authors.
[11] Kapa PK, Jiang XL, Loeser EM, et al., inventors;
Novartis Ag and Novartis Pharma Gmbh, asignee.
Process for preparing intermediates. PCT Int Appl.
WO2004026824A1. 2004.
Funding
[
12] Mitsukura K, Uno T, Yoshida T, et al. Microbial
asymmetric oxidation of 2-butyl-1,3-propanediol.
Appl Microbiol Biotechnol. 2007;76(1):61–65.
13] Tsuji H, Noda S, Kimura T, et al. Configurational
molecular glue: one optically active polymer attracts
two oppositely configured optically active polymers.
Sci Rep. 2017;7:45170.
This work was supported by the Japan Society for the
Promotion of Science (JSPS) Grant-in-Aid for Research
Activity Start-up (No. 16H06843), The Koshiyama Research
Grant, and The OGAWA Science and Technology Grant.
[
[
[
[
[
14] Tsuji H, Sobue T. Cocrystallization of monomer
units in lactic acid-based biodegradable copolymers,
poly(l-lactic acid-co-l-2-hydroxybutanoic acid)s.
Polymer. 2015;72:202–211.
15] Tsuji H, Sobue T. Stereocomplex crystallization and
homo-crystallization of enantiomeric substituted
poly(lactic acid)s, poly(2-hydroxy-3-methylbutanoic
acid)s. Polymer. 2015;69:186–192.
16] Yamada M, Okada Y, Yoshida T, et al.
Purification, characterization and gene cloning of
isoeugenol-degrading enzyme from Pseudomonas
putida IE27. Arch Microbiol. 2007;187(6):511–517.
17] Baer K, Krausser M, Burda E, et al. Sequential and
modular synthesis of chiral 1,3-diols with two stereo-
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Rhodococcus erythropolis DCL14 contains a novel
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