Metabolic characterization of newly isolated Pseudomonas nitroreducens Jin1 growing on eugenol and isoeugenol

Article abstract of DOI:10.1021/jf0715929

Newly isolated soil bacterium strain Jin1 was able to grow on both eugenol and isoeugenol each as sole source of carbon and energy. Based on bacterial 16S rDNA analysis, Jin1 belongs to Pseudomonas nitroreducens with a similarity of 98.92% (14/1297). P. nitroreducens Jin1 was found to biotransform eugenol and isoeugenol to vanillin by different pathways. Eugenol was biotransformed to vanillin through coniferyl alcohol and ferulic acid similarly to the pathway shown previously by Pseudomonassp. HR199 and vanillin produced from eugenol was rapidly metabolized to vanillic acid. Contrastively, Pseudomonas nitroreducens Jin1 did not appear to produce metabolic intermediates during the biotransformation of isoeugenol to vanillin which was finally biotransformed to vanillic acid with much slower rate. These results indicate that there seems to be different metabolic regulation systems for the biotransformation of eugenol and isoeugenol by this bacterium. Herein, we report on Pseudomonas nitroreducens Jin1, a novel bacterium that produces vanillin from eugenol and isoeugenol by two different metabolic pathways.

Full text of DOI:10.1021/jf0715929

8556 J. Agric. Food Chem. 2007, 55, 8556–8561
Metabolic Characterization of Newly Isolated
Pseudomonas nitroreducens Jin1 Growing on
Eugenol and Isoeugenol
TATSUYA UNNO,† SOO-JIN KIM,† ROBERT A. KANALY,‡ JOONG-HOON AHN,§
SU-IL KANG,| AND HOR-GIL HUR*
Department of Environmental Science and Engineering and International Environmental Research
Center, Gwangju Institute of Science and Technology, Gwangju, Korea, Department of Environmental
Biosciences, Yokohama City University, Yokohama, Japan, Department of Bioscience and
Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Korea, and
International Environmental Research Center, Gwangju Institute of Science and
Technology, Gwangju, Korea
Newly isolated soil bacterium strain Jin1 was able to grow on both eugenol and isoeugenol each as
sole source of carbon and energy. Based on bacterial 16S rDNA analysis, Jin1 belongs to
Pseudomonas nitroreducens with a similarity of 98.92% (14/1297). P. nitroreducens Jin1 was found
to biotransform eugenol and isoeugenol to vanillin by different pathways. Eugenol was biotransformed
to vanillin through coniferyl alcohol and ferulic acid similarly to the pathway shown previously by
Pseudomonassp. HR199 and vanillin produced from eugenol was rapidly metabolized to vanillic acid.
Contrastively, Pseudomonas nitroreducens Jin1 did not appear to produce metabolic intermediates
during the biotransformation of isoeugenol to vanillin which was finally biotransformed to vanillic acid
with much slower rate. These results indicate that there seems to be different metabolic regulation
systems for the biotransformation of eugenol and isoeugenol by this bacterium. Herein, we report on
Pseudomonas nitroreducens Jin1, a novel bacterium that produces vanillin from eugenol and
isoeugenol by two different metabolic pathways.
KEYWORDS: Biotransformation; eugenol; isoeugenol; vanillin; Pseudomonas nitroreducens
INTRODUCTION
possible to engineer plants to produce vanillin from eugenol
and isoeugenol with expression of bacterial genes involved in
the metabolism of eugenol and isoeugenol.
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is widely used
in foods, beverages, perfumes, and pharmaceuticals, and in
various medical industries. Natural vanillin extracted from
botanical sources represents approximately only 0.2 % of the
global market and costs 3000 USD/kg, whereas chemically
synthesized vanillin costs 11 USD/kg (1). The demand for
natural flavors is growing and production of vanillin from natural
raw materials by biotransformation processes is becoming
attractive because the product can be regarded as a natural
aromatic chemical. One of the methods by which to produce
natural vanillin is to biotransform both eugenol and isoeugenol.
Eugenol and isoeugenol are thought to be derived from lignin
precursors (2, 3) and are major constituents of essential oil from
clove trees, and they are sold relatively cheaply (4). Recently,
production of eugenol and isoeugenol by sweet basil and petunia
flowers was characterized (2), and considering this, it may be
Many bacteria have been isolated and found to metabolize
eugenol. Among them, Pseudomonassp. HR199 was the most
intensively studied at the genetic and biochemical levels for its
ability to metabolize eugenol to coniferyl alcohol, ferulic acid,
vanillin, vanillic acid, and protocatechuate (5). However, there
has been little specific information available in regard to the
metabolism of isoeugenol by bacteria. To date, it has been
shown that certain strains of Bacillus (Subtilis B2 and HS8 and
fusiformis CGMCC1347) and Pseudomonas (putida I58 and
IE27) metabolize isoeugenol and produce vanillin or vanillic
acid (6–9), but more research is needed.
Pseudomonas nitroreducens Jin1 isolated from soil was found
to metabolize eugenol and isoeugenol each as sole sources of
carbon and energy to produce vanillin as a metabolite, and to
our knowledge this is the first report of a bacterium that shows
this metabolic capability. Vanillin produced from isoeugenol
by P. nitroreducens Jin1 was stably maintained in the medium,
which was not observed in the metabolism of eugenol. We report
herein on strain Jin1, a novel strain in terms of its metabolic
capability for the biotransformation of eugenol and isoeugenol
* Corresponding author phone: +82-62-970-2437; fax: +82-62-970-
2434 e-mail: hghur@gist.ac.kr.
† Department of Environmental Science and Engineering.
‡ Yokohama City University.
§ Konkuk University.
| International Environmental Research Center.
10.1021/jf0715929 CCC: $37.00
2007 American Chemical Society
Published on Web 09/15/2007

Bacterial Growth on Eugenol and Isoeugenol
J. Agric. Food Chem., Vol. 55, No. 21, 2007 8557
Table 1. Time to Reach OD600 ) 0.2 on the Different Growth Substrates for P. nitroreducens Jin1 without or with Induction by Eugenol, Isoeugenol, and
Vanillin, Respectively
time (h) to reach OD600 ) 0.2 with growth substrate
induction conditions
eugenol
isoeugenol
coniferyl alcohol
ferulic acid
vanillin
vanillic acid
no induction
eugenol
isoeugenol
vanillin
10.7 ((0.5)a
4.2 ((0.2)
6.4 ((0.5)
6.1
15.2 ((0.3)
9.8 ((0.2)
9.5 ((0.4)
10.5 ((0.8)
5.6
10.0 ((1.0)
8.9 ((0.3)
10.5 ((0.4)
10.7 ((0.1)
7.5 ((0.3)
4.3 ((0.1)
7.6
5.1 ((0.1)
3.4
3.8 ((0.1)
3.8
3.5 ((0.2)
4.5 ((0.1)
4.6 ((0.4)
5.0 ((0.1)
a Mean value ( standard deviation of duplicates.
Figure 1. Growth curves in 3 mM of eugenol (A), isoeugenol (B), coniferyl alcohol (C), ferulic acid (D), vanillin (E), and vanillic acid (F) for P. nitroreducens
Jin1 without (-3-) or with induction by eugenol (-b-), isoeugenol (-O-), or vanillin (-1-), respectively.
Bacterial Isolation and Identification. Soil from a rice field in
Jangseong, South Korea, was used to isolate bacteria capable of growing
on eugenol and isoeugenol. The soil (5 g) was suspended in 100 mL
of Stainer’s basal minimal salt buffer (MSB) (10) containing 3 mM of
eugenol and isoeugenol, respectively, and incubated with shaking at
200 rpm at 27 °C in the dark. After approximately 10 transfers of the
bacterial culture using the same growth conditions, the culture solution
was spread on nutrient agar plates. Twenty colonies were isolated, and
reinoculated into MSB medium containing 1 mM eugenol and iso-
and discuss the possibility of natural vanillin production by a
single bacterial strain.
MATERIALS AND METHODS
Chemicals. Eugenol, coniferyl alcohol, vanillin, and vanillic acid
were purchased from Aldrich (Milwaukee, WI). Isoeugenol and
ferulic acid were purchased from Sigma (St. Louis, MO). Organic
solvents (HPLC grade) were obtained from Fisher Scientific (Fair
Lawn, NJ).

8558 J. Agric. Food Chem., Vol. 55, No. 21, 2007
Unno et al.
Figure 3. LC–MS spectrum of metabolite V produced from isoeugenol
by P. nitroreducens Jin1.
multiplier voltage were set at 150 °C, 350 °C, 25 V, 3.0 kV, and 700
V, respectively. Nitrogen gas was used as the nebulizer gas and set at
30 L/h and ultrapure nitrogen gas was used as the desolvation gas and
set at 500 L/h.
Effect of Induction on Bacterial Growth. To investigate the effect
of induction on growth of strain Jin1, the strain was cultured in 50 mL
of nutrient broth overnight with additions of eugenol, isoeugenol, and
vanillin each at final concentrations of 1 mM. The overnight culture
was washed twice with the MSB medium, and adjusted to give an OD600
) 0.05 in 30 mL of MSB medium. Eugenol, isoeugenol, and possible
metabolites such as coniferyl alcohol, ferulic acid, vanillin, and vanillic
acid were then added to the MSB medium at final concentrations of 3
mM. The incubations were performed at 27 °C in the dark with shaking
at 200 rpm. Bacterial growth was determined by OD600 every two hours
until the end of the exponential phase.
Figure 2. Elution profiles of HPLC chromatograms from eugenol and
isoeugenol metabolism analyses. Authentic chemicals (A), and cultures
of P .nitroreducens Jin1 grown with eugenol (B), and isoeugenol (C),
and without eugenol or isoeugenol (D) in MSB medium.
eugenol, respectively. One colony capable of utilizing eugenol and
isoeugenol as a sole source of carbon and energy was isolated and
named Jin1. Chromosomal DNA was extracted from strain Jin1 using
the procedure of Pitcher et al. (11) as modified by Chun and Goodfellow
(12). The 16S rDNA was amplified with universal primers; 27F
(AGAGTTTGA TCMTGGCTCAG) and 1492R (GGY TACCTTGT-
TACGACTT). The PCR program used was as follows: 95 °C for 15
min, followed by 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and
72 °C for 1 min, and a final extension step at 72 °C for 7 min. Amplified
16S rDNA fragments were purified by a PCR fragment purification kit
(Bioneer, Daejeon, Korea), and the sequence was determined at Takara
Korea Biomedical Inc. (Seoul, Korea). Sequence similarity searches
were performed using the web-based program BLAST at http://
www.ncbi.nlm.nih.gov/. The sequence information was then imported
into the PHYDIT program http://plaza.snu.ac.kr/∼jchun/phydit/ (12)
for further assembly and alignment. The sequence of 16S rDNA for
strain Jin1 was then compared to sequences of type strains registered
in the GenBank database. The program was also used to calculate
nucleotide substitution rates.
High-Performance Liquid Chromatography (HPLC). Analytical
HPLC was performed using a Varian ProStar HPLC equipped with a
photodiode array (PDA) detector (Varian, Walnut Creek, CA) and a
reverse phase C18 column (5 µm particle size, 4.6 mm × 25 cm,
Spherisorb, Waters, Milford, MA). The mobile phase consisted of
acetonitrile and water containing 0.1% formic acid as follows: 10%
acetonitrile at 0 min, 20% acetonitrile at 20 min, 40% acetonitrile at
25 min, 90% acetonitrile at 30 min, 10 min washing with 90%
acetonitrile at 40 min. Twenty µL from each sample was injected, the
flow rate was 1 mL/min, and UV detection was performed at 270
nm.
Whole Cell Reaction for Biotransformation. The whole cell
reaction assay was performed to identify metabolites produced from
eugenol and isoeugenol by strain Jin1. First, overnight cultures were
harvested, washed twice with MSB medium, and adjusted to OD600
)
1.0 in MSB medium. Thirty mL of MSB medium containing 3 mM of
eugenol and isoeugenol each were incubated at 27 °C in the dark with
shaking at 200 rpm. Two mL of the bacterial cultures were extracted
with 5 mL of ethyl acetate and evaporated to dryness with an Automated
Environmental Speed Vac AES1010 (ThermoProd, Riviera Beach, FL).
Dried samples were resuspended in 1 mL of methanol, filtered, and
analyzed by HPLC. Concentrations of the metabolites were calculated
from the peak area of the HPLC chromatogram using standard curves
prepared from each authentic substrate. Thirty mL of MSB medium
containing 3 mM eugenol and isoeugenol each were also prepared
without bacteria as abiotic controls under both neutral (pH 7) and acidic
conditions (pH 4). Escherichia coli strain JM109 was also inoculated
instead of strain Jin1 as a biological control.
RESULTS AND DISCUSSION
Bacterial Identification. Based on the PHYDIT analysis of
1450 bp of the full 16S rDNA sequence obtained from the gram
negative bacterium Jin1, the sequence best matched to Pseudomo-
nas nitroreducens with similarity of 98.92% (14/1297), followed
by Pseudomonas citronellolis (97.41%; 37/1430) and Pseudomo-
nas alcaligenes with (96.50%; 50/1429). The sequence was
registered to GenBank (GenBank accession no. EF534986).
Effect of Induction and Carbon Sources on Growth Of
P. nitroreducens Strain Jin 1. Time to reach OD600 ) 0.2 was
used to compare induction effects of eugenol, isoeugenol, and
vanillin on the growth of P. nitroreducens strain Jin 1 with
eugenol, isoeugenol, and their possible metabolites, coniferyl
alcohol, ferulic acid, vanillin, and vanillic acid (Table 1). The
most conceivable induction effect on the growth of strain Jin1
Liquid Chromatography–Mass Spectrometry (LC–MS). LC–MS
was performed by coupling an Alliance 2695 instrument (Waters,
Milford, MA) with a Quattro LC triple quadrupole tandem mass
spectrometer (Waters, Milford, MA) in negative electrospray ionization
(ESI-) mode. The elution program was the same as employed for
analytical HPLC. The flow rate was set at 0.1 mL/min and UV detection
was performed with photodiode array detector 2996 (Waters, Milford,
MA). Ten µL was set for injection volume. The source temperature,
desolvation temperature, cone voltage, capillary voltage, and electron

Bacterial Growth on Eugenol and Isoeugenol
J. Agric. Food Chem., Vol. 55, No. 21, 2007 8559
Figure 4. Biotransformation kinetics and metabolites of eugenol (A) and isoeugenol (B)by whole cell cultures of P .nitroreducens Jin1. A: eugenol (-b-),
coniferyl alcohol (-O-), ferulic acid (-0-), and vanillic acid (-9-). Ferulic acid and vanillic acid are also shown in inset with the same legend; B: isoeugenol
(-b-), dehydrodiisoeugenol (-O-), vanillin (-9-) and vanillic acid (-0-). Vanillin and vanillic acid are also shown in inset with the same legend.
was found with the MSB medium containing eugenol in which
time to reach OD600 ) 0.2 was less than half of that of the
control experiment. Induction effects of isoeugenol and vanillin
were fairly well observed. As found in Figure 1, isoeugenol
and ferulic acid showed lower growth rate with doubling time
about 6 h and the rest of four substrates showed similar growth
rate with a doubling time of about 3 h. In all the substrates
except for eugenol, exponential growth stopped at approximately
OD600 ) 0.4∼0.5. The highest OD600 observed was 0.70 in the
MSB medium containing eugenol.
Identification of Metabolites Found in the Metabolism of
Eugenol and Isoeugenol by P. nitroreducens Jin1. Metabolites
of eugenol and isoeugenol, which were found in the bacterial
cultures, were identified using retention time on the HPLC
chromatograms (Figure 2), UV–visible spectroscopy (data not
shown) and molecular weight detected by LC–MS as compared
to those of the corresponding authentic compounds. Metabolites
I, II, and III having [M–H]- ion at m/z 167, 179, and 193,
respectively, were accumulated in the bacterial culture with
eugenol and found to be vanillic acid, coniferyl alchol, and
ferulic acid, respectively. Bacterial culture with isoeugenol
produced metabolites IV having [M–H]- ion at m/z 150 as well
as metabolite I. Metabolite IV was considered to be vanillin.
Another metabolite V having [M–H]- ion at m/z 325 was
accumulated in the bacterial culture with isoeugenol and
appeared to be dehydrodiisoeugenol as suggested in the earlier
study (Figure 3) (9)
Biotransformation Kinetics of Eugenol and Isoeugenol by
Whole Cell Culture of P. nitroreducens Jin1. Biotransforma-
tion kinetics of eugenol, isoeugenol, and metabolites by whole
cell culture of strain Jin1 are shown in Figure 4. To calculate
the concentration of dehydrodiisoeugenol (not commercially
available) found during isoeugenol metabolism, we assumed that
the molar absorptivity (ε) of dehydrodiisoeugenol is two times
that of isoeugenol at 270 nm.
As detected by HPLC, the initial concentration of 2.5 mM
eugenol decreased rapidly for the first 2 h while coniferyl alcohol
concomitantly increased up to 0.52 mM. Ferulic acid and vanillic
acid were then detected in the medium, and the concentrations
increased up to 0.035 and 0.083 mM, respectively, by 3 h of
incubation. All of the compounds were mostly depleted by 5 h
of incubation (Figure 4A). However in the case of isoeugenol,

8560 J. Agric. Food Chem., Vol. 55, No. 21, 2007
Unno et al.
Scheme 1. Proposed Metabolic Pathways of Eugenol (A) and Isoeugenol
(B) by P. nitroreducens Jin1.
this study, optimization to produce higher amount of vanillin
was not particularly developed; however, it may be possible
by addition of organic solvents such as DMSO to dissolve
sparingly soluble isoeugenol or to supply additional nutrition
to enhance metabolism. For example, Yamada et al. (8)
optimized culture conditions for P. putida IE27 by adding 0.1
% (V/V) glycerol and 1% (w/V) yeast extract and reported that
vanillin-producing activity from isoeugenol was increased from
2.01 nmol min-1 ml-1 to 210 nmol min-1 ml-1. They have
also reported that the use of 10% (V/V) DMSO in culture
promoted vanillin-producing activity from isoeugenol to 114%.
Other studies have also shown high conversions of isoeugenol.
For example, 20 mg of resting cells (as dry matter) of
Pseudomonas putida I58 converted 10 mM of isoeugenol into
vanillic acid through vanillin with 98% yield in 40 min (6) and
cell free extracts of Bacillus subtilis B2 biotransformed 12.8
gL-1 (approximately 78 mM) isoeugenol into vanillin with 14%
yield in 96 h without optimization (7). It was suggested that
vanillin production occurs by a pathway from isoeugenol
through isoeugenol diol (9). Much research has shown the
capability of biotransforming eugenol and isoeugenol separately,
yet there has not been discussion in regard to the possibility of
producing vanillin from both eugenol and isoeugenol simulta-
neously. In the future, genes involved in the two metabolic
pathways need to be identified and characterized for further
understanding of the mechanism. The biosynthesis mechanisms
of eugenol and isoeugenol by sweet basil and petunia flowers
were well characterized (2) which implies the possibilities of
production of vanillin in plants from eugenol and isoeugenol
with expression of genes of strain Jin1 described in this study.
Therefore, we conclude that the use of strain Jin1 could provide
a biologically economical tool in related industries.
starting concentration of 2.5 mM, bacterial metabolism to
approximately 0.1 mM occurred after 12 h of incubation (Figure
4B). As strain Jin1 consumed isoeugenol, vanillin and vanillic
acid were detected in the medium with vanillin reaching a
maximum concentration of 0.1 mM by 3 h of incubation and
vanillic acid reaching a maximum concentration of 0.02 mM
by 6 h of incubation. Vanillin and vanillic acid were fully
metabolized in the medium by strain Jin1 after 12 h of
incubation (Figure 4).
Meanwhile, dehydrodiisoeugenol produced by the metabolism
of isoeugenol by P. nitroreducens Jin1 appeared not to be further
metabolized as reported previously (9). The increase of dehy-
drodiisoeugenol was inversely proportional to the concentration
of isoeugenol, which consequently caused low yields of vanillin.
Production of dehydrodiisoeugenol may have been due to
consequences of unknown metabolic activity of strain Jin1
because it was not produced from isoeugenol in abiotic controls
under different pH conditions nor in E. coli controls.
LITERATURE CITED
(1) Priefert, H.; Rabenhorst, J.; Steinbuchel, A. Biotechnological
production of vanillin. Appl. Microbiol. Biotechnol. 2001, 56 (3–
4), 296–314.
(2) Koeduka, T.; Fridman, E.; Gang, D. R.; Vassao, D. G.; Jackson,
B. L.; Kish, C. M.; Orlova, I.; Spassova, S. M.; Lewis, N. G.;
Noel, J. P.; Baiga, T. J.; Dudareva, N.; Pichersky, E. Eugenol
and isoeugenol, characteristic aromatic constituents of spices, are
biosynthesized via reduction of a coniferyl alcohol ester. Proc.
Natl. Acad. Sci. U. S. A. 2006, 103 (26), 10128–33.
(3) Wang, J.; Dudareva, N.; Bhakta, S.; Raguso, R. A.; Pichersky,
E. Floral scent production in Clarkia breweri (Onagraceae). II.
Localization and developmental modulation of the enzyme S-
adenosyl-L-methionine:(iso)eugenol O-methyltransferase and phe-
nylpropanoid emission. Plant Physiol. 1997, 114 (1), 213–21.
(4) Walton, N. J.; Narbad, A.; Faulds, C.; Williamson, G. Novel
approaches to the biosynthesis of vanillin. Curr. Opin. Biotechnol.
2000, 11 (5), 490–6.
(5) Rabenhorst, J. Production of methoxyphenol-type natural aroma
chemicals by biotransformation of eugenol with a new Pseudomo-
nassp. Appl. Microbiol. Biotechnol. 1996, 46, 470–474.
(6) Furukawa, H.; Morita, H.; Yoshida, T.; Nagasawa, T. Conversion
of isoeugenol into vanillic acid by Pseudomonas putida I58 cells
exhibiting high isoeugenol-degrading activity. J. Biosci. Bioeng.
2003, 96 (4), 401–3.
Metabolic Pathway of Eugenol and Isoeugenol in P.
nitroreducens Strain Jin1. Pathways of eugenol and isoeugenol
biotransformation by strain Jin1 are proposed in Scheme 1. The
pathway was also confirmed by feeding each postulated
metabolite to cultures of strain Jin1. Vanillin was not observed
in the bacterial culture with 3 mM of eugenol. However, vanillin
was detected when 3 mM of the slow-growth substrate ferulic
acid was fed as a single source of carbon and energy (data not
shown). On the contrary, vanillin was observed at least for 6 h
in the culture of strain Jin1 containing isoeugenol. Moreover,
isoeugenol induction effects on vanillin metabolism by strain
Jin1 did not result in a shortening of the time to reach OD600
)
0.2 as compared to the control experiments (Table 1). Therefore,
different gene induction systems may be involved in the
eugenol-vanilln and isoeugenol–vanillin metabolisms for strain
Jin1. The pathways shown in Scheme 1 are the same pathways
as found in Pseudomonassp. HR199 (5) for eugenol metabolism
and also similar to that of Bacillus subtilis HS8 (9) for
isoeugenol metabolism. Although some bacteria have been
reported to transform isoeugenol into vanillin, the genes involved
in this metabolism have not been identified yet. However, the
eugenol metabolic pathway of Pseudomonassp. HR199 has been
well studied on the molecular level and was engineered to
produce natural vanillin (13–15). Contrastively, isoeugenol has
not received much attention for the microbial production of
natural vanillin due to isoeugenol toxicity and insolubility. In
(7) Shimoni, E.; Ravid, U.; Shoham, Y. Isolation of a Bacillussp.
capable of transforming isoeugenol to vanillin. J Biotechnol 2000,
78 (1), 1–9.
(8) Yamada, M.; Okada, Y.; Yoshida, T.; Nagasawa, T. Biotransfor-
mation of isoeugenol to vanillin by Pseudomonas putida IE27
cells. Appl. Microbiol. Biotechnol. 2007, 73 (5), 1025–30.
(9) Zhang, Y.; Xu, P.; Han, S.; Yan, H.; Ma, C., Metabolism of
isoeugenol via isoeugenol-diol by a newly isolated strain of
Bacillus subtilis HS8. Appl. Microbiol. Biotechnol. 2006.

Bacterial Growth on Eugenol and Isoeugenol
J. Agric. Food Chem., Vol. 55, No. 21, 2007 8561
(10) Stainer, R. Y.; Palleroni, N. J.; Duodoroff, M. The aerobic
pseudomonads: a taxonomic study. J. Gen. Microbiol. 1966, 43,
159–271.
(11) Pitcher, D. G.; Saunder, N. A.; Owen, R. J. Rapid extraction of
bacterial genomic DNA with guanidium thiocyanate. Lett. Appl.
Microbiol. 1989, 8, 151–156.
(14) Overhage, J.; Priefert, H.; Steinbuchel, A. Biochemical and genetic
analyses of ferulic acid catabolism in Pseudomonassp. Strain
HR199. Appl. EnViron. Microbiol. 1999, 65 (11), 4837–47.
(15) Overhage, J.; Steinbuchel, A.; Priefert, H. Biotransformation of
eugenol to ferulic acid by a recombinant strain of Ralstonia
eutropha H16. Appl. EnViron. Microbiol. 2002, 68 (9), 4315–21.
(12) Chun, J.; Goodfellow, M. A phylogenetic analysis of the genus
Nocardia with 16S rRNA gene sequences. Int. J. Syst. Bacteriol
1995, 45 (2), 240–5.
(13) Overhage, J.; Priefert, H.; Rabenhorst, J.; Steinbuchel, A. Biotrans-
formation of eugenol to vanillin by a mutant of Pseudomonassp.
strain HR199 constructed by disruption of the vanillin dehydro-
genase (Vdh) gene. Appl. Microbiol. Biotechnol 1999, 52 (6), 820–
8.
Received for review May 30, 2007. Revised manuscript received August
1, 2007. Accepted August 2, 2007. This study was carried out with the
support of ”On-Site Cooperative Agriculture Research Project (project
no. 20070101080021)”, RDA, Republic of Korea and the Korea Research
Foundation Grant funded by the Korean Government (MOEHRD)
(project no. 420035) to Tatsuya Unno in GIST, Korea.
JF0715929