Bioconversion of aromatic compounds by Escherichia coli that expresses cytochrome P450 CYP153A13a gene isolated from an alkane-assimilating marine bacterium Alcanivorax borkumensis

Article abstract of DOI:10.1016/j.molcatb.2010.05.015

The cytochrome P450 CYP153 family has been isolated from alkane-assimilating bacteria. CYP153 has been shown to mediate terminal hydroxylations of linear alkanes or alkyl aromatics. We here performed the biotransformation of various aromatic compounds by Escherichia coli cells that expressed the CYP153A13a (P450balk) gene, which was isolated from an alkane-degading marine bacterium Alcanivorax borkumensis. Aromatic compounds including a short alkyl moiety or methyl ether moiety, and phenolic compounds were converted to their respective hydroxylated products, whose structures were determined by HRMS and NMR analyses. The present study revealed that the catalytic function of CYP153A13a is multifunctional, i.e., it can hydroxylate not only the terminal of short alkyl groups that attached to aromatic rings but also the p-position of phenolic compounds substituted with a halogen or the acetyl group. CYP153A13a was also shown to demethylate methylether-including aromatic compounds.

Full text of DOI:10.1016/j.molcatb.2010.05.015

Journal of Molecular Catalysis B: Enzymatic 66 (2010) 234–240
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Bioconversion of aromatic compounds by Escherichia coli that expresses
cytochrome P450 CYP153A13a gene isolated from an alkane-assimilating marine
bacterium Alcanivorax borkumensis
Toshihiko Otomatsua,∗, Liming Baia, Naoya Fujitaa, Kazutoshi Shindob,
Keiko Shimizuc, Norihiko Misawac,1
a KNC Bio Research Center, KNC Laboratories Co., Ltd., 1-1-1, Murodani, Nishi-ku, Kobe 651-2241, Japan
b Department of Food and Nutrition, Japan Women’s University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
c Marine Biotechnology Institute, 3-75-1, Heita, Kamaishi-shi 026-0001, Japan2
a r t i c l e i n f o
a b s t r a c t
Article history:
The cytochrome P450 CYP153 family has been isolated from alkane-assimilating bacteria. CYP153 has
been shown to mediate terminal hydroxylations of linear alkanes or alkyl aromatics. We here per-
formed the biotransformation of various aromatic compounds by Escherichia coli cells that expressed the
CYP153A13a (P450balk) gene, which was isolated from an alkane-degading marine bacterium Alcanivorax
borkumensis. Aromatic compounds including a short alkyl moiety or methyl ether moiety, and phenolic
compounds were converted to their respective hydroxylated products, whose structures were deter-
mined by HRMS and NMR analyses. The present study revealed that the catalytic function of CYP153A13a
is multifunctional, i.e., it can hydroxylate not only the terminal of short alkyl groups that attached to aro-
matic rings but also the p-position of phenolic compounds substituted with a halogen or the acetyl group.
CYP153A13a was also shown to demethylate methylether-including aromatic compounds.
© 2010 Elsevier B.V. All rights reserved.
Received 27 December 2009
Received in revised form 13 May 2010
Accepted 28 May 2010
Available online 8 June 2010
Keywords:
Alcanivorax borkumensis
Aromatic compound
Bioconversion
CYP153
Cytochrome P450
Escherichia coli
1. Introduction
genation, oxidative aryl coupling, oxidative aryl rearrangement,
Beayer–Villiger-type oxidation, and NO synthesis [1,2].
Cytochromes P450 (P450s) compose the largest superfamily
of heme-containing enzymes, and are ubiquitously distributed
in organisms from bacteria to plants and mammals. P450s are
involved in the activation of hormones or the metabolism of
pharmaceuticals, agrichemicals and other xenobiotics in ani-
mals and plants, and mediate the biosyntheses of secondary
metabolites such as antibiotics or the degradation of alkanes and
aromatic compounds in microbes, while many of their functions
remain unknown [1,2]. P450s catalyze mono-oxygenation reac-
tions towards a vast variety of hydrophobic low-molecular-weight
compounds such as alkanes, terpenes and aromatic compounds.
These reactions contain not only hydroxylation reactions but also
other redox reactions, e.g., epoxidation of alkenes, dealkylation
of compounds including an O- or N-alkyl group, sulfoxylation,
N-hydroxylation, oxidative cleavage of C–C bond, oxidative dehalo-
Cytochromes P450 belonging to CYP153 family have been iso-
lated from bacteria such as Acinetobacter calcoaceticus EB104 and
Alcanivorax borkumensis SK2, which utilize alkanes as the sole
carbon source [3,4], amino acid sequence of which are found in
a database (http://drnelson.utmem.edu/biblioE.html). CYP153A13a
(called P450balk) that was isolated from marine bacterium A.
borkumensis SK2 [4] is thought to mediate the first terminal hydrox-
ylation reaction for the assimilation of an incorporated carbon
source [5]. In order to act as monooxygenases, P450s must be
coupled with one or two additional protein(s) to transfer two elec-
trons from NAD(P)H to the heme domain of a P450 protein [6].
P450RhF (CYP116B2) derived from Rhodococcus sp. NCIMB 9784
was found as a novel self-sufficient P450 protein, in which the P450
domain is C-terminally fused to a reductase domain [7]. Nodate
et al. [4] succeeded for the first time in functionally expressing a
CYP153 gene in E. coli, i.e., they expressed in E. coli the CYP153A13a
gene with the functional expression vector pRED, which contained
the DNA fragment encoding the P450RhF ferredoxin reductase
(FMN) and ferredoxin (FeS) domains preceded with the linker
sequence derived from P450RhF (called P450 RhF reductase
domain), and showed that resultant recombinant E. coli cells
hydroxylated the terminal regions of alkanes with carbon number
∗
Corresponding author. Tel.: +81-78-990-3211; fax: +81-78-990-3210.
E-mail address: t otomatsu@kncweb.co.jp (T. Otomatsu).
Present address: Research Institute for Bioresources and Biotechnology,
1
Ishikawa Prefectural University, 1-308, Nonoichi-machi, Ishikawa 921-8836, Japan.
2
Closed on June 30, 2008.
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.05.015

T. Otomatsu et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 234–240
235
of 6–8. The E. coli cells were further revealed to convert cyclohex-
ane, 4-phenyl-1-butene, n-butylbenzene, and 2-n-butylbenzofuran
into cyclohexanol, 2-phenyl-oxirane, 4-phenyl-1-butanol, and 4-
benzofuran-2-yl-butan-1-ol, respectively [8,9]. It was also shown
that a gene cluster including another CYP153A gene and genes for
a ferredoxin reductase (FAD) and a ferredoxin was isolated from
soil bacterium Acinetobacter sp. OC4, and E. coli cells expressing
this gene cluster converted n-ankanes with 6–12 carbons to cor-
responding 1-alkanols or ␣,␻-diols [10]. Pseudomonas putida that
harbored CYP153A6 gene from Mycobacterium sp. was reported to
convert limonene to perillyl alcohol [11]. In this paper, we con-
structed a vector pUCRED to have higher expression levels than
pRED, and showed that E. coli cells expressing CYP153A13a on this
vector can achieve biotransformation towards a variety of aromatic
compounds.
of 5-aminolevulinic acid hydrochloride (5-ALA), and solutions of
the Overnight Express Autoinduction system 1 (1 ml of solution 1,
2.5 ml of solution 2 and 50 ␮l of solution 3; Novagen) in an Erlen-
meyer flask, and cultivation was continued with rotary shaking
(200 rpm) for 24 h at 28 ◦C. The cells were collected, and suspended
in 5 ml of 50 mM phosphate buffer (pH 7.2) containing 5% glycerol.
This cell suspension solution was called Mixture for Bioconversion
(MFB).
2.4. CO difference spectral analysis
Two mM of dithiothreitol (DTT), 200 ␮l of 10× BugBuster
(Novagen) and 1 ␮l of Benzonase Nuclease (Novagen) were added
to 2 ml of each cell suspension (MFB). After 20 min of shaking at
room temperature, cell debris and large part of the membranes
were removed by centrifugation at 8000 rpm for 10 min. The solu-
ble fraction was then used to measure CO-reduced CYP absorption
with a spectrophotometer DU 640 (Beckman; Fullerton, CA, USA).
The CO difference spectra were measured as described previously
[13]. In order to calculate the amount of CYP functional form, an
extinction coefficient of 91 mM−1 cM−1 was used [amount of CYP
functional form (nM) = CO difference a peak at OD 450 nm (OD
450 nm–OD 490 nm)/91(mM−1 cM−1) × 1000,000].
2. Experimental
2.1. Bacterial strains and genetic manipulations
E. coli DH5␣ (ECOS Competent E. coli DH5␣; Nippon Gene; Tokyo,
Japan) was used for DNA manipulations. BL21 (DE3) (Novagen;
Madison, WI, USA) or E. coli BL21 (Takara Bio; Ohtsu, Japan) was
used as the host for the functional expression of the CYP153a13a
(P450balk) gene. The PCR amplifications were performed using
KOD plus DNA polymerase version 2 (Toyobo; Osaka, Japan) and a
thermal cycler (Applied Biosystems; Foster city, CA, USA). Restric-
tion enzymes and DNA-modifying enzymes were purchased from
New England BioLabs (Beverly, CA, USA) or Takara Bio. Ligation-
Convenience Kit (Nippon Gene) was also used. Plasmid DNA was
prepared with a QIAprep Miniprep Kit (Qiagen; Hilden, Germany).
Nucleotide sequences were confirmed with Bigdye terminator
cycle sequencing ready reaction kit version 3.1 (Applied Biosys-
tems) and a model 3730 DNA analyzer (Applied Biosystems). All
recombinant DNA experiments were basically conducted according
to Sambrook and Russell [12].
2.5. Bioconversion of 4-methylbiphenyl by the individual
recombinant E. coli cells
Five mM of proline, 1 mM of disodium dihydrogen ethylenedi-
amine tetraacetate (EDTA), and 0.2 mM of DTT were added to 1.5 ml
of MFB in a test tube. Then, 4-methylbiphenyl as substrate dissolved
in DMSO was added to final concentration of 1 mM. The reaction
was performed at 28 ◦C for 24 h with rotary shaking (170 rpm).
2.6. Conversion experiments
E. coli BL21 harboring pUCRed-Balk was grown in an LB medium
containing Ap (100 ␮g/ml) at 30 ◦C with rotary shaking for 16 h. E.
coli BL21 harboring vector pUC18 was also used as a control to mon-
itor any side reactions that may occur with endogenous enzymes in
the E. coli BL21 cells. 2.5 ml of these cultures were inoculated into
250 ml of an LB medium including Ap (100 ␮g/ml) in an 1 l Erlen-
meyer flask with baffle, and cultured at 30 ◦C for 5–7 h with rotary
shaking (100 rpm), until the absorbance at OD 600 nm reached
approximately 1. Then, 5-ALA (80 ␮g/ml), ammonium iron (II)
sulfate (0.1 mM), and isopropyl ␤-d-thiogalactopyranoside (IPTG;
0.1 mM) were added to the culture, and cultivation was contin-
ued at 20 ◦C for further 19 h with rotary shaking (100 rpm). The
cells were collected by centrifugation and resuspended in 25 ml of
sodium phosphate buffer (50 mM, pH 7.2) containing glycerol (5%),
proline (5 mM), EDTA (1 mM), and DTT (0.2 mM). The cell suspen-
sion was divided into 96-deep well plate with 0.5 ml each, and each
substrate dissolved in DMSO was added at the final concentration
of 1 mM to the cell suspension, and bioconversion was performed
with cultivation at 28 ◦C for 24 h with vortex shaking.
2.2. Construction of a plasmid
pRED vector [4] was digested with EcoRI and HindIII (Takara
Bio), a 1.0-kb fragment containing the P450 RhF reductase domain
was recovered from agarose gel, and inserted into the EcoRI and
HindIII-double digested pUC18 (Takara Bio), yielding a desirable
vector pUCRED. The P450balk (CYP153A13a) gene was isolated from
a marine bacterium A. borkumensis SK2 (DSM 11573T) that was
purchased from Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH [4]. This gene was amplified by PCR using a
primer set, Balk-MfeIF (5ꢀ-acatcaattggATGTCAACGAGTTCAAGTA-
3ꢀ) and Balk-MfeIR (5ꢀ-cagaccaattgTTTTTTAGCCGTCAACTTA-3ꢀ) as
follows: 12.5 ␮l of 2× PrimeSTAR MaxPremix, 1.0 ␮l of Balk-MfeIF
(10 pmol/␮l), 1.0 ␮l of Balk-MfeIR (10 pmol/␮l), 0.5 ␮l (20 ng) of
plasmid pBalk-Red including the P450balk gene [4,9], and 10.0 ␮l
of water were mixed, and PCR reaction was carried out with 10 s at
98 ◦C, 5 s at 60 ◦C, 7 s at 72 ◦C (32 cycles). The amplified P450balk
gene (1410 bp) was digested with MfeI, and inserted into the EcoRI-
digested pUCRED, yielding pUCRED-Balk.
The substrates used in this study were purchased from
Sigma–Aldrich Co., Wako Pure Chemical Industries Co. (Osaka,
Japan) or Tokyo Chemical Industry Co. (Tokyo, Japan), and ibuprofen
methyl ester was prepared by esterification according to literature
[14].
2.3. Cultivation of bacteria
Individual plasmids, pBalk-Red and pUCRed-Balk, were intro-
duced in E. coli BL21 (DE3) and E. coli BL21, respectively. Each
recombinant E. coli was pre-cultured at 30 ◦C with shaking in an
LB medium (L-broth; 1% tryptone, 0.5% yeast extract, 0.5% NaCl)
containing 100 ␮g/ml of ampicillin (Ap). After 16 h of cultivation,
500 ␮l of the pre-culture was inoculated into 50 ml of an LB medium
containing 100 ␮g/ml of Ap, 100 ␮g/l of FeSO4–7H2O, 80 ␮g/ml
2.7. Extraction and HPLC analysis of the products
Five hundred ␮l of ethyl acetate was added to reaction mix-
ture, and shaken to extract the converted compounds in the organic
layer. After centrifugation, the organic phase was analyzed by high
pressure liquid chromatography (HPLC). The organic phase (10 ␮l)

236
T. Otomatsu et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 234–240
was applied to HPLC in an Xterra MS C18 column (4.6 mm × 100 mm,
Waters), with a photodiode array detector: (model SPD-M20A, Shi-
madzu). Development was at a flow rate: 1 ml/min with solvent A
(5% acetonitrile (CH3CN) and 20 mM phosphoric acid) for 3 min,
then by a linear gradient from solvent A to solvent B (95% acetoni-
trile (CH3CN) and 20 mM phosphoric acid) for 25 min, and finally
with solvent B for 15 min, the maximum absorbance being moni-
tored in the range of 200–500 nm.
in E. coli cells harboring pBalk-Red. Accordingly, the conversion
ratio of 4-methylbiphenyl to 4-hydroxymethybiphenyl with E. coli
(pUCRED-Balk) was higher than E. coli (pBalk-Red).
3.2. Bioconversion of various aromatic compounds
Bioconversion experiments of various aromatic compounds
were performed with the E. coli BL21 harboring pUCRED-Balk. The
conversion ratio was analyzed by HPLC, the results being shown in
Table 2 . Aromatic compounds including short alkyl moieties such
a 4-methylbiphenyl, phenolic compounds such a 2-chlorophenol,
and aromatic compounds including methyl ether moieties such
as 4-methoxybiphenyl were selected as substrates as shown in
Table 2. Compounds converted from 2,6-dimethyl naphthalene,
ibuprofen methyl ether, and 3-bromopehnol generated a small
peak by HPLC analyses, which is likely to be a minor product, in
addition to each major peak, while compounds converted from the
others generated one peak with HPLC. Products (only major prod-
ucts in the case of 2,6-dimethyl naphthalene, ibuprofen methyl
ether and 3-bromopehnol) were purified by large-scale biotrans-
formation, and identified by spectroscopic methods, the results also
being shown in Table 2.
2.8. Preparation experiments
E. coli BL21 harboring pUCRED-Balk was grown in an LB medium
containing Ap (100 ␮g/ml) at 30 ◦C with rotary shaking for 16 h.
Five ml of this culture was inoculated into 500 ml of an LB medium
including Ap (100 ␮g/ml) in 2 l Erlenmeyer flasks with baffle
(×4 times), and cultured at 30 ◦C for 5–7 h with rotary shaking
(100 rpm), until the absorbance at OD 600 nm reached approx-
imately 1. Then, 5-ALA (80 ␮g/ml), ammonium iron (II) sulfate
(0.1 mM), and IPTG (0.1 mM) were added to the culture, and cul-
tivation was continued at 20 ◦C for further 19 h with rotary shaking
(100 rpm). The cells were collected by centrifugation, and resus-
pended in 400 ml of sodium phosphate buffer (50 mM, pH 7.2)
containing glycerol (5%), proline (5 mM), EDTA (1 mM), and DTT
(0.2 mM) in an 2 l Erlenmeyer flask with baffle. Each substrate dis-
solved in DMSO was added at the final concentration of 1 mM to the
cell suspension, and bioconversion was performed with cultivation
at 28 ◦C for 24 h with rotary shaking (160 rpm).
3.2.1. Bioconversion of aromatic compounds including short alkyl
moieties
3.2.1.1. Product converted from 4-methylbiphenyl. The molecular
formula of product 1 converted from 4-methylbiphenyl was deter-
mined to be C13H12O by HRMS (EI). The 1H and 13C NMR spectra of
1 showed that the methyl group in 4-methylbiphenyl (substrate)
was oxidized to the corresponding primary alcohol. Thus, 1 was
determined to be 4-hydroxymethybiphenyl (Table 2).
2.9. Purification and identification of the products
The reaction mixture (400 ml) was extracted with ethyl acetate
(EtOAc; 400 ml × 2 times). The organic layer was concentrated in
vacuo, and analyzed by thin-layer chromatography (TLC) on sil-
ica gel [0.25 mm E. Merk silicagel plates (60F-254)]. The converted
compounds were purified by silica gel column chromatography
[12 mm × 250 mm, Silica Gel 60 (Merk)]. Their structures were
analyzed by mass spectrometry (MS) [HRMS (EI), Jeol AX-505W
instrument or HRMS (ESI or APCI), Jeol JMS-T100LP] and nuclear
magnetic resonance (NMR) (400 MHz, Bruker AMX400 instrument
or 500 MHz, Varian INOVA-500AS instrument).
3.2.1.2. Product converted from 3-methylbiphenyl. The molecular
formula of product 2 converted from 3-methylbiphenyl was deter-
mined to be C13H12O by HRMS (EI). The 1H and 13C NMR spectra of
2 showed that the methyl group in 3-methylbiphenyl (substrate)
was oxidized to the corresponding primary alcohol. Thus, 2 was
determined to be 3-hydroxymethybiphenyl (Table 2).
3.2.1.3. Product converted from 4-ethylbiphenyl. The molecular for-
mula of product 3 converted from 4-ethylbiphenyl was determined
to be C14H14O by HRMS (EI). The 1H and 13C NMR spectra of 3
showed that the methyl group in 4-ethylbiphenyl (substrate) was
oxidized to the corresponding primary alcohol. Thus, 3 was deter-
mined to be 4-(2-Hydroxyethyl)biphenyl (Table 2).
2.10. Chromatographic and spectroscopic data of the products
Chromatographic and spectroscopic data of the individual prod-
ucts (1–18) are described in Supplemental Material.
3.2.1.4. Product converted from 4,4ꢀ-diethylbiphenyl. The molecu-
lar formula of product 4 converted from 4,4ꢀ-diethylbiphenyl
was determined to be C16H18O by HRMS (EI). The 1H and 13C
NMR spectra of 4 showed that one of the methyl groups in 4,4ꢀ-
diethylbiphenyl (substrate) was oxidized to the corresponding
primary alcohol. Thus, 4 was determined to be 2-(4ꢀ-ethylbiphenyl-
4-yl)ethanol (Table 2).
3. Results
3.1. Measurement of the functional forms of CYP153A13a
proteins synthesized and conversion ratio of 4-methylbiphenyl in
the individual recombinant E. coli cells
In order to measure amounts of the active form of CYP153A13a
proteins synthesized in E. coli strains carrying the respective plas-
mids, pBalk-Red and pUCRED-Balk, we performed reduced CO
difference spectral analysis on each cultivated cell suspension, the
results being shown in Table 1. The functional proteins in E. coli
cells harboring pUCRED-Balk were 1.6 times larger than those
3.2.1.5. Product converted from 4-isopropylbiphenyl. The molecu-
lar formula of product 5 converted from 4-isopropylbiphenyl was
determined to be C15H16O by HRMS (EI). The 1H and 13C NMR spec-
tra of 5 showed that one of methyl groups in 4-isopropylbiphenyl
(substrate) was oxidized to the corresponding primary alcohol.
Thus, 5 was determined to be 4-(1-Hydroxy-2-propyl)biphenyl
(Table 2).
Table 1
Expression levels of CYP153A13a by the CO difference spectrum method and con-
version ratio of 4-methylbiphenyl to 4-hydroxymethylbiphenyl.
3.2.1.6. Product converted from 2-(p-tolyl)-pyridine. The molecu-
lar formula of product 6 converted from 2-(p-tolyl)-pyridine was
determined to be C12H11NO by HRMS (ESI). The 1H and 13C NMR
spectra of 6 showed that the methyl group in 2-(p-tolyl)-pyridine
Expression level of CYP153A13a
Conversion ratio
pRed-Balk/BL21
pUCRed-Balk/BL21
71 nM
116 nM
28.8%
62.3%

T. Otomatsu et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 234–240
237
Table 2
Bioconversion of various aromatic compounds by Escherichia coli expressing cytochrome P450 CYP153A13a genes.
Substrate
Product
Conversion ratio
4-Methylbiphenyl
3-Methylbiphenyl
1
2
90.2%
73.2%
2-Methylbiphenyl
4-Ethylbiphenyl
–
Not converted
90.8%
3
4
4,4ꢀ-Diethylbiphenyl
4-Isopropylbiphenyl
28.8%
40.1%
5
6
2-(p-tolyl)-Pyridine
90.1%
61.6%
2-Methylnaphthalene
7
8
1,6-Dimethylnaphthalene
34.5%
2,6-Dimethylnaphthalene
Ibuprofen methyl ester
9
16.3%
74.3%
10
2-Chlorophenol
2-Bromophenol
3-Bromophenol
11
12
13
56.1%
37.4%
36.5%
2-Iodophenol
14
15
48.8%
28.6%
2-Acetylphenol

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T. Otomatsu et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 234–240
Table 2 (Continued )
Substrate
Product
Conversion ratio
15.4%
1-Phenylnaphtharene
16
4-Methoxybiphenyl
17
18
34.0%
14.0%
1-Methoxynaphthalene
(substrate) was oxidized to the corresponding primary alcohol.
Thus, 6 was determined to be (4-(pyridine-2-yl)phenyl)methanol
(Table 2).
molecular formula, the replacement of one phenolic OH group in
the aromatic ring is proposed. An analysis of the gDQF COSY and
gHMBC spectra of product 11 suggested the attachment of one phe-
nolic group at C-4. The position of this phenolic OH group was
confirmed by the observation of 1H–13C long-range coupling from
H-6 (ı 6.89) to C-4 (ı 149.4), and a vicinal spin network from
H-5 to H-6. These findings enabled product 11 to determine as
2-chlorobenzene-1,4-diol (Table 2).
3.2.1.7. Product converted from 2-methylnaphthalene. The molecu-
lar formula of product 7 converted from 2-methylnaphthalene was
determined to be C11H10O by HRMS (EI). The 1H and 13C NMR spec-
tra of 7 showed that the methyl group in 2-methylnaphthalene
(substrate) was oxidized to the corresponding primary alco-
hol. Thus, 7 was determined to be 2-hyrdoxymetylnaphthalene
(Table 2).
3.2.2.2. Product converted from 2-bromophenol. The molecular
formula of product 12 converted from 2-bromophenol was deter-
mined to be C6H5BrO2 by HRMS (APCI). Consistent with its
molecular formula, the replacement of one phenolic OH group in
the aromatic ring is proposed. An analysis of the DQF COSY and
HMBC spectra of product 12 suggested the attachment of one phe-
nolic group at C-4. The position of this phenolic OH group was
confirmed by the observation of 1H–13C long-range coupling from
H-6 (ı 6.73) to C-4 (ı 150.9), and a vicinal spin network from
H-5 to H-6. These findings enabled product 12 to determine as
2-bromobenzene-1,4-diol (Table 2).
3.2.1.8. Product
The molecular formula of product
converted
from
1,6-dimethylnaphthalene.
converted from 1,6-
8
dimethylnaphthalene was determined to be C12H12O by HRMS
(EI). The 1H and 13C NMR spectra of 8 showed that one of methyl
groups in 1,6-dimethylnaphthalene (substrate) was oxidized to
its primary alcohol. An analysis of the gDQF COSY and gHMBC
spectra of 8 suggested the attachment of one OH group at C-2ꢀ.
The position of this OH group was confirmed by the observation
of 1H–13C long-range coupling from H-5 (ı 7.82) to C-2ꢀ (ı 65.5)
and from H-7 (ı 7.53) to C-2ꢀ (ı 65.5), and a vicinal spin network
from H-7 to H-8. These findings enabled product 8 to determine as
(1-methylnaphthalene-6-yl)methanol (Table 2).
3.2.2.3. Product converted from 3-bromophenol. The molecular
formula of product 13 converted from 3-bromophenol was deter-
mined to be C6H5BrO2 by HRMS (APCI). Consistent with its
molecular formula, the replacement of one phenolic OH group in
the aromatic ring is proposed. An analysis of the gDQF COSY and
gHMBC spectra of product 13 suggested the attachment of one phe-
nolic group at C-4. The position of this phenolic OH group was
confirmed by the observation of 1H–13C long-range coupling from
H-6 (ı 6.89) to C-4 (ı 149.5), and a vicinal spin network from
H-5 to H-6. These findings enabled product 13 to determine as
2-bromobenzene-1,4-diol (Table 2).
3.2.1.9. Product
The molecular formula of product
converted
from
2,6-dimethylnaphthalene.
converted from 2,6-
9
dimethylnaphthalene was determined to be C12H12O by HRMS
(EI). The 1H and 13C NMR spectra of 9 showed that one of methyl
groups in 2,6-dimethylnaphthalene (substrate) was oxidized to
the corresponding primary alcohol. Thus, 9 was determined to be
(6-methylnaphthalene-2-yl)methanol (Table 2).
3.2.2.4. Product converted from 2-iodophenol. The molecular for-
mula of product 14 converted from 2-iodephenol was determined
to be C6H5IO2 by HRMS (APCI). Consistent with its molecular for-
mula, the replacement of one phenolic OH group in the aromatic
ring is proposed. An analysis of the gDQF COSY and gHMBC spectra
of product 14 suggested the attachment of one phenolic group at
C-4. The position of this phenolic OH group was confirmed by the
observation of 1H–13C long-range coupling from H-6 (ı 6.67) to C-4
(ı 150.9), and a vicinal spin network from H-5 to H-6. These find-
ings enabled product 14 to determine as 2-iodobenzene-1,4-diol
(Table 2).
3.2.1.10. Product converted from ibuprofen methyl ester. The molec-
ular formula of product 10 converted from ibuprofen methyl ester
was determined to be C14H20O3 by HRMS (EI). The 1H and 13C
NMR spectra of 10 showed that one of methyl groups in ibupro-
fen methyl ester (substrate) was oxidized to its primary alcohol.
An analysis of the gDQF COSY and gHMBC spectra of product 10
suggested the attachment of one OH group at C-3ꢀ. The positions of
this OH group was confirmed by the observation of 1H–13C long-
range coupling from H-1ꢀ (ı 2.40, 2.59) to C-3ꢀ (ı 67.6) and from
H-4ꢀ (ı 0.92) to C-3ꢀ (ı 67.6), and a vicinal spin network from H-1ꢀ
to H-4ꢀ. These findings enabled product 10 to determine as methyl
2-(4-(3-hydroxy-2-methylpropyl)phenyl)propanoate (Table 2).
3.2.2.5. Product converted from 2-acetylphenol. The molecular for-
mula of product 15 converted from 2-acetylphenol was determined
to be C8H8O3 by HRMS (APCI). An analysis of the gDQF COSY and
gHMBC spectra of product 15 suggested the attachment of one phe-
nolic group at C-5. The position of this phenolic OH group was
3.2.2. Bioconversion of phenolic compounds
3.2.2.1. Product converted from 2-chlorophenol. The molecular
formula of product 11 converted from 2-chlorophenol was deter-
mined to be C6H5ClO2 by HRMS (APCI). Consistent with its

T. Otomatsu et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 234–240
239
4. Discussion
We here constructed a new vector pUCRED for the functional
expression of a bacterial P450 gene using an E. coli vector pUC18.
In pUCRED, an inserted P450 gene is designed to utilize the lac pro-
moter and lacZ translational signal that includes the lacZ leader
sequence, i.e., ATG ACC ATG ATT ACG AAT TCG ATG (the last ATG is
the start codon of an inserted gene, and the EcoRI site is under-
lined). The CYP153A13a (P450balk) gene was shown to exhibit
better expression level with this vector pUCRED (plasmid PUCRED-
Balk) rather than with vector pRED that utilizes the T7 promoter
derived from pET21a (plasmid pBalk-Red) [4]. Such trials to utilize
the transcriptional and translational signals derived from pUC vec-
tors such as pUC18 were found to be successful for the functional
expression of carotenoid biosynthesis genes such as crtW and crtZ
in E. coli [15–17]. The pUC series may be one of promising vec-
tors for the functional expression of genes involved in chemical
conversions.
Fig. 1. Time course of bioconversions on 3-methylbipehnyl, ibuprofen methyl ether,
and 2-chlorophenol. Each value is shown as mean SE (n = 3).
Cytochrome P450 CYP153 family, particularly CYP153A subfam-
ily, is known as bacterial alkane terminal hydroxylase [4,8,10].
Recently, we revealed that CYP153A can achieve the terminal
hydroxylation on aromatic compounds including butyl moieties
such as n-butylbenzene and 2-n-butylbenzofuran [9]. A. borku-
mensis, which the CYP153A13a gene was isolated from [4], was
found as a predominant bacterium among bacterial populations on
oil-contaminated marine environments [18]. Thus in the present
study, we checked bioconversion ability towards a variety of aro-
matic compounds using recombinant E. coli cells that expressed
CYP153A13a, and found that this P450 fused to the P450RhF
reductase domain performed such bioconversions as a multifunc-
tional enzyme. CYP153A13a hydroxylated not only the terminals
of short alkyl groups that attached to aromatic rings but also the
p-positions of phenolic compounds substituted with a halogen
or the acetyl group, as shown in Table 2. The terminal hydrox-
ylation reactions were performed towards a series of biphenyl
derivatives that include methyl group, ethyl group(s) or isopropyl
group with the exception of 2-methylbiphenyl. CYP153A13a also
hydroxylated the methyl group of naphthalene derivatives includ-
ing methyl or dimethyl group (Table 2), while it was not able to
convert 1-methylnaphthalene (data not shown). A drug ibuprofen
methylether was also shown to have the terminal hydroxylation
reaction with the P450 with high conversion efficiency.
Cytochrome P450BM-3 that originated from Bacillus mega-
terium has the structure that a P450 portion is C- terminally fused
to a reductase domain composed of a mammalian-like diflavin
NADPH-P450 reductase [19]. P450BM-3 catalyzed the hydroxy-
lation and epoxidation reactions of long-chain unsaturated fatty
acids [19–21]. Its Phe87Val mutant P450BM-3 (F87V) was shown
to hydroxylate the p-position of various phenolic compounds such
as 2-bromophenol to yield hydroquinone [22]. In this study, we
revealed that CYP153A13a can also hydroxylates the p-position
of such phenolic compounds. Moreover, CYP153A13a was able to
convert a phenolic compound with a bulky substituent, 1-phenyl
naphthalene, into the product hydroxylated at the p-position. We
also showed that it catalyzed demethylation reactions of aromatic
compounds with a methoxy group (Table 2). The broad sub-
strate affinity (specificity) of CYP153A13a (P450balk) should carry
over to the synthesis of various low-molecular-weight compounds
including drug-like compounds, which are difficult to synthesize
chemically.
confirmed by the observation of 1H–13C long-range coupling from
H-3 (ı 6.78) to C-5 (ı 149.5), and a vicinal spin network from H-3
to H-4. These findings enabled product 15 to determine as 1-(2,5-
dihydroxyphenyl)ethanone (Table 2).
3.2.2.6. Product converted from 1-phenylnaphthalene. The molecu-
lar formula of product 16 converted from 1-phenylnaphthalene
was determined to be C16H12O by HRMS (APCI). An analysis of the
gDQF COSY and gHMBC spectra of product 16 suggested the attach-
ment of one phenolic group at C-4. The position of this phenolic OH
group was confirmed by the observation of 1H–13C long-range cou-
pling from H-2 (ı 7.38) to C-4 (ı 154.9), and a vicinal spin network
from H-2 to H-3. These findings enabled product 16 to determine
as 4-(naphthalene-1-yl)phenol (Table 2).
3.2.3. Bioconversion of aromatic compounds including methyl
ether moieties
3.2.3.1. Product converted from 4-methoxybiphenyl. The molecular
formula of product 17 converted from 4-methoxybiphenyl was
determined to be C12H10O by HRMS (APCI). The 1H and 13C NMR
spectra of 17 showed that the methyl group in 4-methoxybiphenyl
(substrate) was cleaved to the corresponding alcohol. Thus, 17 was
determined to be 4-hydroxybiphenyl (Table 2).
3.2.3.2. Product converted from 1-methoxynaphthalene. The molec-
ular formula of product 18 converted from 1-methoxynaphthalene
was determined to be C10H8O by HRMS (ESI). The 1H and 13C NMR
spectra of 18 showed that the methyl group in 1-methoxynaphtol
(substrate) was cleaved to the corresponding alcohol. Thus, 18 was
determined to be 1-naphthol (Table 2).
3.3. Time course of bioconversions
Time course experiments of bioconversions on 3-
methylbipehnyl, ibuprofen methyl ether, and 2-chlorophenol
were further performed with the E. coli BL21 harboring pUCRED-
Balk, the results being shown in Fig. 1. In the log phase (2–8 h),
the order of bioconversion efficiency on these substrates was as
follows: 3-methylbipehnyl > 2-chlorophenol > ibuprofen methyl
ether. However, in 24-h cultivation, their conversion ratios reached
similar levels (70–74%). This result suggests that 24 h is enough for
bioconversions with the recombinant E. coli.
Acknowledgement
This work was in part supported by New Energy and Industrial
Technology Development Organization (NEDO).

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T. Otomatsu et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 234–240
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