Journal of Environmental Biotechnology
Vol. 7, No. 1, 39–44, 2007

Original paper (regular paper)

Bioproduction of 3-Methylcatechol from Toluene
in a Two-Phase (Organic-Aqueous) System by a Genetically Modified
Solvent-Tolerant Pseudomonas putida Strain T-57
IRVAN FAIZAL1, MASAKI OHBA1, AKIO KURODA1, NOBORU TAKIGUCHI1, HISAO OHTAKE2,
KOSUKE HONDA2 and JUNICHI KATO1*
1

Department of Molecular Biotechnology, Hiroshima University, Higashi-Hiroshima, Hiroshima 739–8530, Japan2
Department of Biotechnology, Osaka University, Suita, Osaka 565–0871, Japan* TEL: +81–(0)82–424–7757 FAX: +81–(0)82–424–7047* E-mail: jun@hiroshima-u.ac.jp(Received; 14 December, 2006/Accepted; 20 January, 2007)

Pseudomonas putida strain T-57 is a solvent-tolerant toluene-oxidizing bacterium, which utilizes toluene via toluene dioxygenase (TDO) pathway. In this study, we investigated bioproduction of 3-methylcatechol from toluene by using a 3-methylcatechol accumulating mutant derived from P. putida strain T-57. TDO assays revealed that TDO expression in P. putida
strain T-57 is inducible and toluene is a good inducer. It was shown that TDO induction by toluene was under the control ofcatabolite repression by glucose. Glucose repressed TDO induction by toluene in a concentration-dependent manner. TDO
induction by toluene was also repressed when cells were grown in LB medium. An deletion-insertion mutant of the 3-methylcatechol 2,3-dioxygense gene (todE) was constructed by inserting a kanamycin resistant gene cassette into the chromosomal
todE gene of P. putida strain T-57 and the resulting mutant, designated P. putida strain TODE1, was used for bioconversionof 3-methylcatechol from toluene. To avoid catabolite repression, TODE1 cells were cultivated in a mineral salts basal medium
supplemented with 2 g l–1 of glucose+toluene vapor or n-butanol+toluene vapor. It was found that 3-methylcatechol was
accumulated in culture supernatants at 6 and 12 mM, respectively. Introduction of the second organic phase improved
3-methylcatechol production by TODE1. When TODE1 was grown in a two-liquid-phase system with oleyl alcohol as thesecond liquid extraction phase for 3-methylcatechol, and glucose and n-butanol as additional carbon and energy sources, 48
and 107 mM 3-methylcatehcol was accumulated in the organic solvent phase, respectively. The overall concentrations of
3-methylcatechol in the two-phase culture were twice higher than those in the single-phase culture.
Key words: bioproduction, 3-methylcatechol, solvent-tolerant bacterium, Pseudomonas putida strain T-57

1.

Introduction

Pseudomonas putida strain T-57, which was isolated
from activated sludge at a wastewater treatment facility of afermentation plant in Kumamoto, Japan, is a solvent-tolerant
aerobic bacterium3). It can grow even when organic solvents
such as toluene, ethylbenzene, xylenes, styrene, n-octane,
and n-decane are provided in a two-phase (organic-aqueous)system. This bacterium utilizes toluene, p-xylene, ethylbenzene, and n-butanol as sole sources of carbon and energy.
Genetic and biochemical analysis revealed that P. putida
strain T-57 catabolizes aromatic hydrocarbons via the toluene
dioxygenase (TDO) pathway (Fig. 1). From these characteristics, P. putida strain T-57 seems very promising as a biocatalyst for the bioproduction of value added chemicals
from hydrophobic substrates.
In the previous studies, we investigated the bioproductionof cresol from toluene by using a mutant derived from P.
putida T-573,20). In the initial step of the TDO pathway,

toluene is oxidized to toluene cis-glycol, which is further
oxidized to 3-methylcatechol by toluene cis-glycol dehydrogenase (the todD product). It is known that this first intermediate, toluene cis-glycol, is nonenzymatically dehydrated
to cresol12). Therefore, we expected that a todD mutant
strain of T-57 accumulates cresol from toluene. In fact,
when the todD mutant cells were exposed to toluene during
growing with n-butanol, o-cresol was accumulated in themedium3). When the todD mutant was grown in a two-liquidphase system with oleyl alcohol as a second liquid extraction phase for o-cresol, 40 g l–1 of o-cresol was accumulated
in the organic solvent phase and the overall concentrationof o-cresol in the two-phase culture reached 6.6 g l–1 20).
Substituted catechols are important in pharmaceuticalproduction process2,5) and catechol and its derivatives arevaluable precursors in the production of stains and synthetic
flavors15). Multi-step and energy-consuming process is
employed for chemical synthesis of 3-substituted catechols,
resulting in mixtures of diﬀerent catechols and a low overall

FAIZAL et al.

40

Fig. 1. Catabolic pathway for toluene metabolism by P. putida T-57 (A) and restriction map of the tod operon (B). The locations and orientations of the todC1C2BADE genes are indicated by horizontal arrows. The vertical arrow indicates the insertion position of a kanamycin
resistant gene cassette in P. putida TODE1. Restriction sites: E, EcoRI; P, PstI; N, NotI; S, SalI.

product yield7). It is known that various bacteria utilize
aromatic compounds via metabolic pathways with catechols
as their intermediates. If these bacterial cells can be used as
biocatalysts, catechols can be produced at the atmosphericpressure and moderate temperature. There have been several
investigations to attempt bioproduction of catechols byusing a monooxygenase6) and dioxygenases7,13,19). However,toxicity of substrates and products limited these bioproduction levels.
In this study, we used solvent-tolerant P. putida strain
T-57 as the parental strain to construct a biocatalyst forbioproduction of 3-methylcatechol from toluene, and evaluated the resulting biocatalyst for 3-methylcatechol productionin the single- and two-liquid-phase cultivation systems.

2.
2.1.

Strain/plasmid
Strains
E. coli
MV1184

P. putida
T-57
TODE1

Standard procedures were used for plasmid DNA preparations, restriction enzyme digestions, ligations, transformations and agarose gel electrophoresis14). P. putida strain T-57
was transformed by electroporation as described previously3).

aSource orreferenceara∆(lac-proAB) rpsL thi (φ80lacZ∆M15) ∆(srl-recA)306::
Tn10(Tcr) F’[traD36 proAB+lacI q
lacZ∆M15]

17

Solvent-tolerant toluene-oxidizing
bacterium3

T-57 derivative, todE::kan

This
study

pUC4 containing a 1.3-kb kan
gene cassette; Apr, Kmr
pT57Tod01
pUC11817) containing
todC1C2BADE, Apr
pT57Tod01EK pT57Tod01 derivative containing
todE disrupted by insertion of
kan cassette, Apr, Kmr

Bacterial strains and plasmids

DNA manipulation and electroporation

Relevant characteristicsa

Plasmids
pUC4K

Materials and Methods

The bacterial strains and plasmids used in this study arelisted in Table 1. Escherichia coli MV1184 was used forplasmid construction and DNA manipulation. P. putida
strains were grown at 28°C with shaking in mineral salts
basal (MSB) medium3) with appropriate carbon sources.
Luria-Bertani (LB) medium14) was used as complete medium.
For solid media, 2% agar was added to MSB medium and
LB medium. E. coli was grown at 37°C with shaking in
2xYT medium14) supplemented with appropriate antibiotics.
2.2.

Table 1. Bacterial strains and plasmids used in this study.

16
3
This
study

Ap, ampicillin; Km, kanamycin; Tc, tetracycline.

2.3. Construction of todE mutant of P. putida strain T-57
The todE deletion-insertion mutant of P. putida strain
T-57 was constructed by the direct gene replacement technique9). Plasmid pT57Tod01 containing todC1C2BADE waspartially digested with Pst I and ligated with a PstI-flankedkan (conferring kanamycin resistance) cartridge from
pUC4K to disrupt todE encoding methylcatechol 2,3-dioxygenase. P. putida strain T-57 was transformed with theresulting plasmid pT57Tod01EK by electroporation totransfer the mutation into the genome of P. putida strain

Bioproduction of 3-methylcatechol by solvent-tolerant P. putida
Table 2. Eﬀects of growth substrates on TDO expression in P.
putida strain T-57.
Growth substratea

Medium

TDO specific activityb(µg indigo min–1 OD600–1)

Glucosen-Butanol

MSB
MSB
MSB
MSB
MSB
LB

0
0
0
1.39±0.03
4.52±0.39
0Oleyl alcohol
Ethylbenzene
TolueneNAc

Toluene, ethylbenzene, and n-butanol were added in the vapor
phase. Liquid oleyl alcohol (9.1% [vol/vol]) was directly added
to MSB medium.
b
Mean±standard deviation for four experiments.
c
Not applicable.

41

and the organic phase were subjected to measurement of
3-methylcatechol.
2.6.

HPLC

Metabolites in culture supernatants and organic phasewere determined by HPLC on a TSK-GEL ODS-80TM
column (260×4.6 mm; TOSOH, Tokyo) at a flow rate of
0.5 ml min–1. The mobile phase consisted of isocratic composition of water:acetonitrile (30 : 70, vol/vol). The elution
was monitored at 220 nm.

aT-57. The deletion-insertion was confirmed by Southernblot analysis.
2.4.

Toluene dioxygenase assay

P. putida strains were inoculated into 10 ml MSB medium
with appropriate carbon sources in 50-ml screw-cappedvials. Hydrocarbons for carbon sources were provided in thevapor phase by putting small tubes with 150 µl of hydrocarbons inside the screw-capped vials, or directly added to
MSB medium at 9.1% (vol/vol). P. putida was grown withreciprocal shaking (120 rpm) at 28°C to a optical density at
600 nm (OD600) of 1, and cells were harvested by centrifugation (10,000×g, 5 min, 4°C). Pelleted cells were resuspended
in 50 mM phosphate buﬀer (pH 7.2), washed once with thesame buﬀer, and resuspended in an original volume ofphosphate buﬀer. Toluene dioxygenase activity was evaluated by formation of indigo from indole. A cuvette contained
1 ml of cell suspension and 100 µl of 100 mM indole in
N,N-dimethylformamide and maintained at 28°C. In thisenzyme assay, toluene dioxygenase oxidizes indole to indolecis-2,3-dihydrodiol. Indole-cis-dihydrodiol is spontaneously
oxidized to indoxyl and indoxyl is dimerized to the dark
blue pigment, indigo, by air oxidation. Formation of indigo
was recorded at 600 nm.
2.5.

Batch cultivation for 3-methylcatechol production

P. putida cells grown in LB medium overnight at 28°C
with reciprocal shaking (120 rpm) were inoculated into
10 ml MSB medium with 2 g l–1 glucose (a 1% inoculum).
Bacterial cells were grown at 28°C with shaking to OD600 of
1 and harvested by centrifugation. Cells were suspended in
10 ml MSB medium in the 50 ml screw-capped vials.
Growth substrates and toluene were added to the vials.
Glucose was added to MSB medium at a concentration of
2 g l–1, liquid oleylalcohol was directly added to MSB medium
at 9.1% (vol/vol), and toluene and n-butanol were provided
in the vapor phase by putting small tubes with 150 µl of
toluene or n-butanol inside the vials. The screw-cappedvials were then incubated with reciprocal shaking (120 rpm)
at 28°C. After 22 h incubation, the aqueous supernatant

3.
3.1.

Results and Discussion

Expression of toluene dioxygenase is inducible

P. putida strain T-57 oxidizes toluene to 3-methylcatechol
through 2-step oxidation in the TDO pathway (Fig. 1A).
When 3-methylcatechol is produced from toluene by using
strains derived from P. putida strain T-57 as biocatalysts,
high expression of TDO, which initially oxidizes toluene totoluene cis-glycol, is important. Therefore, we first investigated eﬀects of growth substrates on TDO expression in
T-57. Strain T-57 cells grown in MSB medium containing
each of growth substrates were examined for TDO activity.
When strain T-57 was grown on glucose as a sole carbon
source, no TDO activity was detected (Table 2). The highest
TDO activity (4.52 µg indigo min–1 OD600–1) was detected incells grown on toluene followed by cells grown ethylbenzene(1.39 µg indigo min–1 OD600–1). But cells grown in MSB
with n-butanol and oleyl alcohol and in LB medium showed
no TDO activity. Thus, these results suggest that TDO expression in P. putida T-57 is inducible and that toluene andethylbenzene are good inducers.
3.2.

TDO expression is under the control of catabolite
repression

In the TDO pathway, toluene is initially oxidized to toluene
cis-glycol, which is further oxidized to 3-methylcatechol(Fig. 1A). This product undergoes meta-cleavage fission
catalyzed by methylcatechol 2,3-dioxygenase encoded by
todE. For production of 3-methylcatechol, the todE geneshould be inactivated to prevent further oxidation of
3-methylcatechol. It is expected that the todE mutant ofstrain T-57 is not able to utilize toluene as a growth substrate. Therefore, additional carbon and energy sources are
required for production of 3-methylcatechol by the todE
mutant in the growing conditions. We then investigatedeﬀects of additional growth substrates on TDO induction by
toluene in T-57. Strain T-57 was grown in MSB medium
with additional growth substrates or in LB medium supplemented with toluene in the vapor phase and TDO activity
in cells was determined. LB medium supported the highest
growth rate with a cell doubling time of 1.5 h, while cell
doubling times were approximately 2.1–2.4 h when grown in
MSB medium with carbon sources. TDO activity decreased
with increase of glucose concentrations (Table 3). Strain
T-57 cells grown in LB medium showed only a trace level ofFAIZAL et al.

42

Table 3. Eﬀects of additional carbon and energy sources on TDO
induction by toluene in P. putida strain T-57a.
Co-substrateb

Medium

TDO specific activityc(µg indigo min–1 OD600–1)

None
Glucose (2 g l–1)
Glucose (5 g l–1)
Glucose (10 g l–1)
Glucose (15 g l–1)
Glucose (20 g l–1)
n-Butanoln-Octanol

MSB
MSB
MSB
MSB
MSB
MSB
MSB
MSB
MSB

4.52±0.39
2.73±0.15
2.37±0.16
1.25±0.21
0.16±0.12
0.07±0.02
2.64±0.30
1.83±0.49
3.71±0.54MSB

2.77±0.67

LB

0.06±0.05

Oleyl alcohol
n-Butanol+
oleyl alcohol—

P. putida strain T-57 was grown in MSB with additional carbon
sources or in LB supplemented with toluene in the vapor
phase.
b
Toluene, n-butanol, and n-octanol were added in the vapor
phase. Liquid oleyl alcohol (9.1% [vol/vol]) was directly added
to MSB medium.
c
Mean±standard deviation for four experiments.
a

TDO activity even in the presence of toluene. These results
suggest that TDO expression in strain T-57 is subject tocatabolite repression by glucose and a component(s) in LB
medium. When cells were cultivated in MSB medium containing liquid oleyl alcohol (9.1% [vol/vol]) or supplemented
with n-butanol in the vapor phase, TDO levels were morethan 60% of the fully-induced level. Thus, oleyl alcohol andn-butanol were indicated to be suitable for additional carbon
and energy sources for 3-methylcatechol production by thetodE mutant.
3.3.

Construction of a todE mutant of T-57

We constructed a todE (encoding catechol 2,3-dioxygenase)mutant of T-57 to produce 3-methylcatechol from toluene.
The todE gene was inactivated by inserting a kan cassette
into the chromosomal todE gene of T-57 and the resulting
mutant was designated P. putida strain TODE1. The rapid
spot test described by Pankhurst11) was carried out to assessmethylcatechol 2,3-dioxygenase activity in TODE1. Methylcatechol dissolved in acetone was sprayed onto TODE1 andT-57 colonies grown on MSB agar plates supplemented with
n-butanol and toluene vapor. Colonies of T-57 turned yellow,
but not those of TODE1 (data not shown), indicating thatmethylcatechol 2,3-dioxygenase was not produced in
TODE1. TODE1 was not able to grow in MSB medium
with toluene as a sole source of carbon and energy. Since
TODE1 showed the expected mutant phenotype, it was
further used for bioproduction of 3-methylcatechol fromtoluene.
3.4.

Bioproduction of 3-methylcaetchol from toluene

We conducted bioproduction of 3-methylcatechol fromtoluene by using P. putida strain TODE1. Since TDO assays

Table 4. Bioproduction of 3-methylcatechol from toluene by P.
putida strain TODE1 grown in MSB medium with toluene
and co-substratesa.
Co-substrate

3-Methylcatechol (mM)cAqueous
Organic
Overalld
phase
phase

None
Oleyl alcohol
GlucoseGlucose+oleyl alcohol
n-Butanolb
n-Butanolb+oleyl alcohol

1.1±0.3
8.9±1.0
5.7±0.6
7.2±2.3
12.1±1.3
16.1±2.3—71.8±4.8—47.6±4.3—
107.3±7.9

1.1±0.3
14.6±1.4
5.7±0.6
10.9±2.4
12.1±1.3
24.4±2.7

Toluene was supplemented in the vapor phase.
n-Butanol was supplemented in the vapor phase.
c
Mean±standard deviation for four experiments.
dOverall concentration=(concentration in aqueous phase×volume
of aqueous phase+concentration in organic phase×volume oforganic phase)/(volume of aqueous phase+volume of organic
phase).
ab

in T-57 revealed that n-butanol and 2 g l–1 of glucose did
not greatly aﬀect TDO induction by toluene (Table 3),
glucose and n-butanol were used as carbon and energy
sources. We first examined bioproduction of 3-methylcatechol
in a single-liquid-phase culture. Strains T-57 and TODE1
were grown in MSB medium supplemented with 2 g l–1 ofglucose+toluene vapor or n-butanol+toluene vapor for
22 h and 3-methylcatechol in culture supernatants wasmeasured. In these culture conditions, both strains showedsimilar growth with cell doubling times of approximately 2.1–2.5 h. In supernatant of the T-57 culture, 3-methylcatechol
was not detected (data not shown), while 5.7 and 12.1 mM
3-methylcatechol was accumulated in culture supernatants
when TODE1 was cultivated in MSB medium with 2 g l–1 ofglucose+toluene and n-butanol+toluene, respectively (Table 4).
In the previous study, we demonstrated that productionlevels of o-cresol by the todD (encoding toluene cis-glycol
dehydrogenase [Fig. 1]) mutant of P. putida strain T-57 increased by addition of liquid oleyl alcohol to medium as thesecond, organic phase 20). Therefore, we examined eﬀects ofthe second, organic phase on 3-mthylcatechol production
by TODE1. Addition of 9.1% (vol/vol) oleyl alcohol significantly increased 3-methylcatechol production by TODE1(Table 4). When n-butanol or glucose were added to medium
as an additional carbon and energy source, overall concentrations of 3-methyl catechol in the two-liquid-phase culture
were twice as high as those in the single-liquid-phase culture.
In the two-liquid-phase culture with n-butanol and oleyl
alcohol (n-butanol+oleyl alcohol) as the additional carbon
and energy sources, the highest overall product concentration of 24.4 mM was obtained, consisting of 107.3 mM in
the oleyl alcohol phase and 16.1 mM in the aqueous phase.
Increased volume of oleyl alcohol in the two-liquid-phaseculture did not improve 3-methylcatehcol production (datanot shown). Since oleyl alcohol can be used as a source ofcarbon and energy by P. putida strain T-57 and it did notBioproduction of 3-methylcatechol by solvent-tolerant P. putida

greatly aﬀect TDO induction by toluene (Table 3), bioproduction of 3-methylcatechol was conducted in MSB medium
with 9.1% (vol/vol) oleyl alcohol+toluene vapor. An overall concentration of 3-methycatechol reached 14.6 mM,
which was about 60% of that in the two-phase-liquid culture
with n-butanol as the additional carbon and energy source.
The result suggests that n-butanol has a positive eﬀect on
3-methylcatechol production by TODE1 in the two-liquidphase culture. In consistent with it, we previously found thatn-butanol increased the oxygen consumption rate of P.
putida strain T-57 cells in the two-liquid-phase culture 20).
Although P. putida strain T-57 and its derivative P. putida
strain TODE1 are tolerant to organic solvents, 3-methylcatechol may still have a detrimental eﬀect on these bacterial
cells. The second organic phase scavenges the toxic productfrom the aqueous phase. Probably, it is the reason why introduction of oleyl alcohol improves 3-methylcatechol production from toluene by P. putida strain TODE1. The colorof medium was white in the start of the single-liquid-phasecultivation and it changed to dark reddish brown after 22 hcultivation. It is due to o-benzoquinone and polymerizedcompounds produced by the oxidation of 3-methylcatecholwith dissolved oxygen10,15). Enzymatic reactions may be also
involved in this coloration of culture broth. Wang et al. proposed that oxidative enzymes such as peroxidase catalyze aone-electron oxidation reaction, generating a free radical
from catechols and initializing the formation of quinones
and their subsequent polymerization, which is detected as a
black precipitate18). Consistent with their proposition, cells
harvested by centrifugation were also dark reddish brown,suggesting that the color compounds deposited on cells. No
TDO activity was detected with these deep-colored cells(data not shown). Deposition of color compounds aroundthe cell may interfere with access of a substrate (toluene) to
TDO inside the cell, resulting in loss of TDO activity. In the
two-liquid-phase culture, the color of the organic andaqueous phases turned pale brown after 22 h incubationeven though higher concentrations of 3-methylcatechol
accumulated in the culture medium. This result suggeststhat removal of 3-methylcatechol from the aqueous phaseby oleyl alcohol decreased further oxidation of 3-methylcatechol by bacterial cells, resulting in decreased polymerization. Thus, it is likely that the second organic phase in the
two-liquid-phase cultivation contributes to improvement of
3-methylcatechol production by protecting TDO activity inbacterial cells from interference by deposition of polymerizedcompounds on cells.
P. putida F1 is a toluene-oxidizing bacterium, which
degrades toluene via the TDO pathway21), and P. putida
F107 is a 3-methylcatechol accumulating mutant (probably
todE mutant) of P. putida F14). Hüsken et al.7) constructed3-methylcatechol hyperaccumulating P. putida MC2 fromP. putida F107 by integrating the extra copy of the
todC1C2BAD genes into the P. putida F107 genome, andinvestigated bioproduction of 3-methylcatechol from toluene by using MC2 cells as a biocatalyst8). When MC2 cells

43

were grown in the medium containing 50% (vol/vol) of
1-octanol as a second liquid extraction phase, the overall
concentration of 3-methylcatechol reached 25 mM. In thiscultivation, it took more than 50 h to reach the maximum
overall concentrations of 3-methylcatechol. In this study,
production levels of 3-methylcatechol by TODE1 were
almost same to that by P. putida MC2, however, the time
to attain the maximum product concentration (22 h) was
shorter than that of P. putida MC2, suggesting that P.
putida strain TODE1 showed the higher production rate of
3-methylcatechol than that of P. putida MC2. Thus, P.
putida strain TODE1 is thought to be a good candidate asa biocatalyst for bioproduction of 3-substituted catecholsfrom aromatic compounds.

Acknowledgments
This work was carried out as part of the Project for Development of a Technological Infrastructure for Industrial Bioprocess on R&D of New Industrial Science and Technology
Frontiers by the Ministry of Economy, Trade, and Industry(METI), which was entrusted by New Energy and Industrial
Technology Development Organization (NEDO).

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