Production of natural fragrance aromatic acids by coexpression of trans-anethole oxygenase and p-anisaldehyde dehydrogenase genes of Pseudomonas putida JYR-1 in Escherichia coli

Article abstract of DOI:10.1021/jf303531u

A gene encoding p-anisaldehyde dehydrogenase (PAADH), which catalyzes the oxidation of p-anisaldehyde to p-anisic acid, was identified to be clustered with the trans-anethole oxygenase (tao) gene in Pseudomonas putida JYR-1. Heterologously expressed PAADH in Escherichia coli catalyzed the oxidation of vanillin, veratraldehyde, and piperonal to the corresponding aromatic acids vanillic acid, veratric acid, and piperonylic acid, respectively. Coexpression of trans-anethole oxygenase (TAO) and PAADH in E. coli also resulted in the successful transformation of trans-anethole, isoeugenol, O-methyl isoeugenol, and isosafrole to p-anisic acid, vanillic acid, veratric acid, and piperonylic acid, respectively, which are compounds found in plants as secondary metabolites. Because of the relaxed substrate specificity and high transformation rates by coexpressed TAO and PAADH in E. coli, the engineered strain has potential to be applied in the fragrance industry.

Full text of DOI:10.1021/jf303531u

Article
pubs.acs.org/JAFC
Production of Natural Fragrance Aromatic Acids by Coexpression of
trans-Anethole Oxygenase and p‑Anisaldehyde Dehydrogenase
Genes of Pseudomonas putida JYR‑1 in Escherichia coli
Dongfei Han,† Somwang Kurusarttra,†,‡ Ji-Young Ryu,† Robert A. Kanaly,§ and Hor-Gil Hur*,†,‡
‡
†School of Environmental Science and Engineering, and International Environmental Research Center,
Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea
§Department of Genome System Science, Yokohama City University, Yokohama 236-0027, Japan
ABSTRACT: A gene encoding p-anisaldehyde dehydrogenase (PAADH), which catalyzes the oxidation of p-anisaldehyde to
p-anisic acid, was identified to be clustered with the trans-anethole oxygenase (tao) gene in Pseudomonas putida JYR-1.
Heterologously expressed PAADH in Escherichia coli catalyzed the oxidation of vanillin, veratraldehyde, and piperonal to the
corresponding aromatic acids vanillic acid, veratric acid, and piperonylic acid, respectively. Coexpression of trans-anethole
oxygenase (TAO) and PAADH in E. coli also resulted in the successful transformation of trans-anethole, isoeugenol, O-methyl
isoeugenol, and isosafrole to p-anisic acid, vanillic acid, veratric acid, and piperonylic acid, respectively, which are compounds
found in plants as secondary metabolites. Because of the relaxed substrate specificity and high transformation rates by coexpressed
TAO and PAADH in E. coli, the engineered strain has potential to be applied in the fragrance industry.
KEYWORDS: p-Anisaldehyde, p-anisaldehyde dehydrogenase gene (paadh), trans-anethole, trans-anethole oxygenase gene (tao),
p-anisic acid, vanillic acid, secondary metabolite
substrates may be further metabolized to the fragrance aromatic
acid compounds12 p-anisic acid, vanillic acid, veratric acid, pip-
eronylic acid, etc., which have also been demonstrated in
diverse industrial applications for producing flavors, spices,
perfumes, cosmetics,13 and pharmaceuticals with antibacte-
rial, antifungal, anti-inflammatory, antispasmodic, and anti-
oxidant activities.14,15
In this study, we tried to biologically produce the diverse
aromatic acid compounds p-anisic acid, vanillic acid, veratric acid,
and piperonylic acid from plant-originated 1-propenylbezene sub-
strates. For this purpose, we cloned the p-anisaldehyde dehydro-
genase (paadh) gene that is located downstream of the trans-
anethole oxygenase gene (tao) in P. putida JYR-1 and coexpressed
both paadh and tao genes in Escherichia coli.
INTRODUCTION
■
Biotechnological production of fragrance and flavor compounds
has been given much attention recently because U.S. and European
legislation has legally designated “natural” flavor substances as
compounds prepared by either physical processes via extraction
from natural sources or enzymatic or microbial processes from
chemical precursors isolated from nature.1 Phenylpropanoid com-
pounds and their metabolic intermediates transformed by
microorganisms have been traditionally used by the flavor and
fragrance industry as starting materials for chemical syntheses.2,3
Compounds with a backbone structure of 1-propenylbenzene,
such as isoeugenol, anethole, isosafrole, etc., are types of aromatic
compounds found widely in essential oils from plants and have
often been attempted as the starting materials to produce aromatic
fragrances and other value-added chemicals.
Several genes and enzymes have been reported to be involved
in the transformation of 1-propenylbenzenes to oxidized
products, such as benzaldehydes and benzoic acids.4−9 Among
them, metabolism for isoeugenol oxidation is relatively well-
studied. The genes iso and iem encoding the enzymes that cata-
lyze isoeugenol oxidation to vanillin have been isolated from
Pseudomonas putida IE277 and Pseudomonas nitroreducens Jin1,4
respectively. Another well-characterized metabolism for plant-
originated 1-propenylbenzenes is in regard to trans-anethole,
which has been found in the bacteria Arthrobacter sp. TA135 and
P. putida JYR-1.10,11 In a previous report, we isolated the tao gene
from P. putida JYR-1 encoding trans-anethole oxygenase (TAO),
which can catalyze trans-anethole to p-anisaldehyde. Interest-
ingly, TAO was shown to have a relaxed substrate specificity
toward isoeugenol, O-methyl isoeugenol, and isosafrole, all of which
contain the propenyl functional group on the aromatic ring struc-
ture.10 Aldehydes produced from plant-originated 1-propenylbezene
MATERIALS AND METHODS
■
Chemicals. trans-Anethole, isoeugenol, O-methyl isoeugenol,
isosafrole, p-anisaldehyde, vanillin, veratraldehyde, piperonal, p-anisic
acid, veratric acid, and piperonylic acid were purchased from Sigma-
Aldrich (Milwaukee, WI). Organic solvents [high-performance liquid
chromatography (HPLC) grade] were purchased from Fisher Scientific
(Fair Lawn, NJ).
Plasmids, Bacterial Strains, and Growth Conditions. All plas-
mids and bacterial strains used in this study are listed in Table 1. P. putida
JYR-1 was grown in tryptic soy broth (TSB) or Stanier’s minimal salt broth
(MSB)16 containing 10 mM trans-anethole and incubated with rotary shaking
at 200 rpm and 25 °C. E. coli strains DH5α and BL21(DE3) were routinely
grown in Luria-Bertani (LB) medium and incubated by rotary shaking at
Received: August 14, 2012
Revised: November 10, 2012
Accepted: November 10, 2012
Published: November 10, 2012
© 2012 American Chemical Society
11972
dx.doi.org/10.1021/jf303531u | J. Agric. Food Chem. 2012, 60, 11972−11979

Journal of Agricultural and Food Chemistry
Article
Table 1. Bacterial Strains and Plasmids Used in This Study
strain or plasmid
description
Strains
source
P. putida JYR-1
E. coli BL21(DE3)
E. coli EC100
trans-anethole transformation strain
ref 31
host strain for expression vector, F− ompT hsdSB (rB− mB−) gal dcm (DE3)
Novagen
Epicenter
host strain for transposon Tn5 insertion, F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 recA1
endA1 araD139 Δ(ara, leu)7697 galU galK λ− rpsL nupG
E. coli EPI100
E. coli DH5α
host strain for fosmid genomic library, F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 recA1 endA1
araD139 Δ(ara, leu)7697 galU galK λ− rpsL nupG trfA tonA dhfr
Epicenter
ref 32
host strain for cloning vector, F− endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG φ 80dlacZΔM15
Δ(lacZYA-argF)U169, hsdR17(rK− mK+), λ−
Plasmids
Apr; expression vector
pET21-a
Novagen
Promega
Novagen
this study
this study
this study
this study
this study
this study
pGEM-Teasy
pETDuet-1
Apr; TA cloning vector
Apr; expression vector
pTA163
Cmr; 41 kb pEpiFos-5 containing tao gene
Apr; pGEM-Teasy cloning vector containing tao gene
Apr; pGEM-Teasy cloning vector containing tao gene
Apr; pET21-a expression vector containing paadh gene
Apr; pETDuet-1 expression vector containing paadh gene
Apr; pETDuet-1 expression vector containing paadh and tao genes
pG-TAO
pG-PAADH
pET-PAADH
pETD-PAADH
pETD-PAADH-TAO
Figure 1. (A) Phylogenetic tree and (B) amino acid alignment among p-anisaldehyde dehydrogenases (PAADH), p-hydroxybenzaldehyde
dehydrogenases (PchA), benzaldehyde dehydrogenases (BADH), and salicylaldehyde dehydrogenases (SADH). Catalytic residues and NAD(P)
△
binding sites conserved in the ALDH superfamily are indicated by ☆ and , respectively.
200 rpm and 37 °C. When required, ampicillin (Amp) at 50 μg/mL,
kanamycin (Kan) at 50 μg/mL, and chloramphenicol (Chl) at 12.5 μg/mL
were used for selecting the corresponding recombinant E. coli strains.
Subcloning of the Gene Encoding p-Anisaldehyde Dehydro-
genase (paadh) and Expression of paadh in E. coli. To clone the
open reading frame (ORF) 11 for gene paadh from Contig2 of pTA163,
11973
dx.doi.org/10.1021/jf303531u | J. Agric. Food Chem. 2012, 60, 11972−11979

Journal of Agricultural and Food Chemistry
Article
Figure 2. HPLC elution profile of the metabolites produced from (A) p-anisaldehyde, (B) vanillin, (C) veratraldehyde, and (D) piperonal by (A1, B1,
C1, and D1) resting cells of E. coli BL21(DE3)(pET-PAADH) expressing PAADH after 30 min of incubation, (A2, B2, C2, and D2) resting cells of E. coli
BL21(DE3)(pET21a) without PAADH, and (A3, B3, C3, and D3) authentic aldehyde and acid compounds.
a fosmid, carrying 35 kb of P. putida JYR-1 genomic DNA and containing
the gene cluster encoding enzymes for metabolizing trans-anethole,10
polymerase chain reaction (PCR) was performed by forward primer
attachment to the NdeI recognition sequence, 5′-GGGAATTCCA-
TATGAGCAAGCCAGAGCATTACA-3′, and reverse primer attach-
ment to the BamHI recognition site, 5′-CGCGGATCCTCAGAACG-
GGTAAGGGCGTG-3′. The PCR product digested by NdeI and BamHI
was ligated into an expression vector pET21-a (Novagen, Madison, WI)
under the control of the T7 promoter. The resulting plasmid named
pET-PAADH was transformed into E. coli BL21(DE3) (Novagen,
Madison, WI). A coexpression vector called plasmid pETD-PAADH-
TAO for expressing paadh and tao under the contol of the T7 promoter
was constructed by cloning the genes into plasmid pETDuet-1 (Novagen,
Madison, WI). The gene paadh was amplified from pTA163 in P. putida
JYR-1 by forward primer attachment to the NcoI recognition sequence, 5′-
CATGCCATGGGCAAGCCAGAGCATTACA-3′, and reverse primer
attachment to the BamHI recognition site, 5′-CGCGGATCCTCAGAA-
CGGGTAAGGGCGTG-3′, and the PCR product was ligated into the
pETDuet-1 vector after digestion with NcoI and BamHI, resulting in
vector pETD-PAADH. The coding region of the tao gene and 134
nucleotides of its 5′-untranslated region were amplified from pTA163 in P.
putida JYR-1 by forward primer attachment to the XhoI recognition
sequence, 5′-CCGCTCGAGGAGACCGATGCGAAGGGCAA-3′, and
reverse primer attachment to the XhoI recognition site, 5′-CCGCTCG-
AGTCAGTTAGTCCTCAAGTCGGAATT-3′. The PCR product was
ligated into the pETD-PAADH vector after digestion with XhoI. Finally,
the coexpression plasmid pETD-PAADH-TAO was confirmed by
sequencing the inserted amplicons.
into fresh LB medium and cultured for 2.5 h at 37 °C by rotary shaking at
200 rpm. Expression of TAO was induced for 4 h after the addition of
isopropyl-β-D-thiogalactoside (IPTG) to the medium at a final
concentration of 1 mM. The cells were harvested by centrifugation at
10000g for 10 min and suspended in MSB medium. This process was
repeated twice more for washing, and the cells were resuspended in MSB
medium and adjusted to an optical density (OD) of 2 at 600 nm. The
resuspended cells that were supplemented with 0.5 mM glucose as
an energy source were reacted with 1 mM p-anisaldehyde, vanillin,
veratraldehyde, and piperonal in the case of E. coli BL21(DE3)(pET-
PAADH) and 1 mM trans-anethole, isoeugenol, O-methyl isoeugenol,
and isosafrole in the case of E. coli BL21(DE3)(pETD-PAADH-TAO)
(all from 100 mM stock solutions prepared in methanol). Reactions
were performed by incubation by rotary shaking at 200 rpm and 30 °C
for 10 h. Three volumes of ethyl acetate were used to extract the reaction
solutions. The ethyl acetate extracts were evaporated in a speed vacuum
centrifugal concentrator (Vision Scientific Co., Suwon, South Korea),
and the residue was dissolved in an appropriate volume of methanol and
filtered through polyvinylidene fluoride (PVDF) syringe filters (What-
man, Maidstone, U.K.). The amounts of remaining parent compounds
and aldehyde and acid products in the reaction solutions were deter-
mined by HPLC. Each metabolite was identified by comparison of
ultraviolet (UV) spectral data and liquid chromatography−mass spec-
trometry (LC−MS) data to those from the analyses of authentic com-
pounds. All analyses were performed in triplicate.
Analytical Methods. Analytical HPLC was performed using a
Varian ProStar HPLC equipped with a photodiode array (PDA) detec-
tor (Varian, Walnut Creek, CA) and a reverse-phase C18 column (5 μm
particle size, 4.6 mm × 25 cm, Waters Corporation, Milford, MA). The
mobile phase, which was composed of acetonitrile containing 0.1%
formic acid and water, was programmed as follows: 10% acetonitrile at
0 min, 60% acetonitrile at 10 min, 90% acetonitrile at 20 min, and 90%
Kinetics of trans-Anethole and p-Anisaldehye Biotransfor-
mation. E. coli BL21(DE3)(pET-PAADH) and E. coli BL21(DE3)-
(pETD-PAADH-TAO) were cultured in LB medium overnight at
37 °C, by rotary shaking at 200 rpm. Cells (1%, v/v) were transferred
11974
dx.doi.org/10.1021/jf303531u | J. Agric. Food Chem. 2012, 60, 11972−11979

Journal of Agricultural and Food Chemistry
Article
Figure 3. HPLC elution profile of the metabolites produced from (A) trans-anethole, (B) isoeugenol, (C) O-methyl isoeugenol, and (D) isosafrole by
(A1, B1, C1, and D1) resting cells of E. coli BL21(DE3)(pET-PAADH-TAO) expressing PAADH and TAO together after 30 min of incubation, (A2,
B2, C2, and D2) resting cells of E. coli BL21(DE3)(pET21Duet-1) without PAADH and TAO, and (A3, B3, C3, and D3) authentic compounds.
acetonitrile at 30 min. The flow rate with an injection volume of 10 μL
was 1 mL/min, and UV detection was performed at 270 nm. LC−MS
was performed by coupling an Alliance 2695 LC system (Waters
Corporation, Milford, MA) to a Quattro LC triple quadrupole tandem
mass spectrometer (Waters Corporation, Milford, MA) in positive
electrospray ionization (ESI+) mode. For LC analyses, a SunFire C18
column (3.5 m, 2.1 × 150 mm, Waters Corporation, Milford, MA) was
used, and the mobile phase, elution program, and detection were
identical to the analytical HPLC described above; the flow rate was
0.2 mL/min. For MS analyses, the source temperature, desolvation tem-
perature, and capillary voltage were kept at 150 °C, 350 °C, and 3.2 kV,
respectively. The cone voltage was 20 V. The cone gas and desolvation
gas were ultrapure nitrogen set at 30 and 500 h−1, respectively. The
protein concentration was determined by the Bradford assay method17
with the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA), and
bovine serum albumin was used as the standard.
Therefore, it is assumed that ORF 11 is a putative aldehyde
dehydrogenase gene and was named p-anisaldehyde dehydro-
genase gene (paadh). A phylogenetic tree was constructed using
the distance method with BioNJ algorithm,20 and evolutionary
distances were calculated using the LogDet method.21 The
phylogenetic tree showed that PAADH from P. putida JYR-1 was
clustered with three p-hydroxybenzaldehyde dehydrogenases
(ACS34981.1, AAM92190.1, and AAA75634.2), a benzaldehyde
dehydrogenase (EIM15248.1), and a vanillin dehydrogenase
(BAF62890.1) at a genetic distance of 0.260 (Figure 1A), sug-
gesting that PAADH is evolutionally close to those five dehydro-
genases, with the highest similarity to a vanillin dehydrogenase.
This result, together with the biotransformation results, suggested
that PAADH is a benzaldehyde dehydrogenase. PAADH contains
not only NAD(P) binding sites but also active catalytic residues,
whichwere also found in the ALDH family (Figure 1B).22 PAADH
activity was confirmed by cloning the gene paadh into a pET
vector and expressing it in E. coli BL21(DE3)(pET-PAADH).
Resting cells of E. coli BL21(DE3)(pET-PAADH) transformed
1 mM p-anisaldehyde in 4 h to p-anisic acid, which was identified
by HPLC (Figure 2) and LC−MS (Figure 4).
Nucleotide Sequence Accession Numbers. The DNA and
deduced protein sequences described in this study have been deposited
in the GenBank database under accession number JX104157.
RESULTS
■
Identification of the Gene paadh Encoding p-Anisaldehyde
Dehydrogenase. ORF 11 of Contig2 of plasmid pTA163 in P.
putida JYR-1, which is located downstream of the trans-anethole
oxygenase gene, showed an amino acid identity of 66% with
p-hydroxybenzaldehyde dehydrogenase (PchA) from P. putida
NCIMB 9866,18 64% with vanillin dehydrogenase (VDH) from
P. putida IE27,7 62% with benzaldehyde dehydrogenase (BADH)
from Pseudomonas chlororaphis O6, and 35% with salicylaldehyde
dehydrogenase (SADH) from Pseudomonas stutzeri AN10.19
Biotransformation of p-Anisaldehyde and Other Similar
Aromatic Aldehyde Compounds by Resting Cells of E. coli
BL21(DE3)(pET-PAADH) Expressing paadh. A total of 2 mL
of resting cells (OD at 600 nm = 2) of E. coli BL21(DE3)(pET-
PAADH) expressing paadh was equivalent to a total protein
concentration of 0.32 mg/mL and was able to completely con-
vert 1 mM p-anisaldehyde to p-anisic acid in 4 h of incubation
11975
dx.doi.org/10.1021/jf303531u | J. Agric. Food Chem. 2012, 60, 11972−11979

Journal of Agricultural and Food Chemistry
Article
Figure 4. LC−MS spectra of aromatic acidic metabolites M1, M2, M3, and M4 produced from trans-anethole, isoeugenol, O-methyl isoeugenol, and
isosafrole, respectively, by resting cells of E. coli BL21(DE3)(pETD-PAADH-TAO) expressing PAADH and TAO together and authentic chemicals
p-anisic acid, vanillic acid, veratric acid, and piperonylic acid.
with a reaction rate of 6.51 nmol min−1 (mg of total protein)−1
(Figure 5A). Metabolites of p-anisaldehyde produced by E. coli
BL21(DE3)(pET-PAADH) were identified by retention time on
HPLC chromatograms (Figure 2) and compared molecular mass
detected by LC−MS spectroscopy (Figure 4) to those of the
corresponding authentic compounds. HPLC elution profiles
from the sample extracted at a reaction time of 30 min showed
two peaks at 9.20 and 11.05 min, and these retention times
were identical to the authentic compounds p-anisic acid and
p-anisaldehyde, respectively (Figure 2A1). Electron impact−mass
11976
dx.doi.org/10.1021/jf303531u | J. Agric. Food Chem. 2012, 60, 11972−11979

Journal of Agricultural and Food Chemistry
Article
Figure 5. Biotransformation kinetics of (A) p-anisaldehyde, (B) vanillin, (C) veratraldehyde, and (D) piperonal by resting cells of E. coli
■
●
BL21(DE3)(pET-PAADH) expressing PAADH. Substrates and products are represented by and , respectively.
Figure 6. Biotransformation kinetics of (A) trans-anethole, (B) isoeugenol, (C) O-methyl isoeugenol, and (D) isosafrole by resting cells of E. coli
■
,
●
, and
▲
,
BL21(DE3)(pETD-PAADH-TAO) expressing PAADH and TAO together. Substrates, intermediates, and products are represented by
respectively.
spectrometry (EI−MS) analyses in negative ionization mode
(Figure 4) showed a molecular weight of 152 giving a [M − H]−
molecular ion peak at 151, confirming p-anisic acid as a
metabolite of p-anisaldehyde. In addition, resting cells of E. coli
BL21(DE3)(pET-PAADH) were incubated for 4 h with 1 mM
vanillin, veratraldehyde, and piperonal, which are structurally
similar to p-anisaldehyde. HPLC and LC−MS analyses identified
that acid products from vanillin, veratraldehyde, and piperonal
were vanillic acid, veratric acid, and piperonylic acid, respectively
(Figures 2 and 4). From the incubations, 0.55 mM p-anisic acid,
11977
dx.doi.org/10.1021/jf303531u | J. Agric. Food Chem. 2012, 60, 11972−11979

Journal of Agricultural and Food Chemistry
Article
0.32 mM vanillin, 0.4 mM veratric acid, and 0.63 mM piperonylic
acid as final products were obtained from 1 mM of the each start-
ing compound.
Taken together, although various chemical methods have been
applied for the synthesis of fragrance compounds,26−28 there are
usually limitations because of low yields, requirements for high
temperatures and pressures, and issues with regard to low purity.
Biological production of flavor aromatic acid compounds by
genetically modified bacterial strains is considered an alternative
way to the conventional chemical syntheses and physical extrac-
tion methods from their natural sources to effectively produce
the legally designated “natural” flavor substances from the
biomass containing the precursor compounds.3,29,30 Indeed, the
recombinant E. coli strain expressing TAO and PAADH, which
show the relatively relaxed substrate specificity, has the potential
to produce various fragrance aromatic acids with their high
transformation rates toward plant-originated phenylpropanoid
substrates trans-anethole, isoeugenol, O-methyl isoeugenol, and
isosafrole.
Production of Aromatic Acid Compounds from Plant-
Originated Propenylbenzene by Resting Cells of E. coli
BL21(DE3)(pETD-PAADH-TAO) Coexpressing tao and
paadh. Engineered E. coli BL21(DE3) expressing both genes
tao and paadh in similar amounts (data now shown) transformed
trans-anethole to p-anisic acid (Figure 3A). In addition, the recom-
binant strain also produced vanillic acid, veratric acid, and pip-
eronylic acid from isoeugenol, O-methyl isoeugenol, and isosafrole,
respectively. These aromatic acidic products were also confirmed
by HPLC and LC−MS analyses, as compared to the authentic
compounds (Figures 2 and 4). Figure 6 shows the biotransforma-
tion kinetics of trans-anethole, isoeugenol, O-methyl isoeugenol,
and isosafrole. The initial concentration of 0.7 mM trans-anethole
rapidly decreased with the formation of p-anisic acid (Figure 6A).
Within 2 h of incubation, trans-anethole was almost completely
transformed. Similarly, the initial concentrations of 0.91 mM
isoeugenol and 0.65 mM isosafrole decreased rapidly and were
totally transformed to vanillic acid and piperonylic acid, respec-
tively, after 2 h (panels B and D of Figure 6). However, the initial
concentration of 0.86 mM O-methyl isoeugenol decreased to
0.6 mM with the production of veratric acid in 0.15 mM after 2 h
(Figure 6C).
AUTHOR INFORMATION
Corresponding Author
*Telephone: +82-62-970-2437. Fax: +82-62-970-2434. E-mail:
hghur@gist.ac.kr.
■
Funding
This work was supported by National Research Foundation of
Korea Grant NRF 2010-0029224 funded by the Korean govern-
ment [Ministry of Education, Science and Technology (MEST)].
Notes
The authors declare no competing financial interest.
DISCUSSION
■
Aromatic aldehyde dehydrogenase was known to catalyze oxida-
tion of benzaldehyde to benzoic acid, vanillin to vanillic acid, and
veratraldehyde to veratric acid23 and has been suggested to be
applied in the aromatic fragrance industry.24 The paadh gene
encoding PAADH, which catalyzes the oxidation of p-anisaldehyde
to p-anisic acid, is located downstream of the tao gene of P. putida
JYR-1.10 Similar activity of aromatic aldehyde dehydrogenase
toward p-anisaldehyde has been characterized from maize (Zea
mays).25 However, p-anisaldehyde dehydrogenase activity has not
been identified in microorganisms. Furthermore, there are also no
reports on the biocatalytic oxidation of veratraldehyde and
piperonal to veratric acid and piperonylic acid, respectively.
In the current research, by combining both tao and paadh
genes under the control of a T7 promoter in E. coli, we suc-
cessfully convertednotonlyitsphysiologicalsubstratetrans-anethole
to p-anisic acid but also isoeugenol, O-methyl isoeugenol, and iso-
safrole to the aromatic acid compounds vanillic acid, piperonylic acid,
and veratric acid, respectively. In comparison to the other three
substrates, the relatively low biotransformation rate of O-methyl
isoeugenol to the veratric acid through veratraldehyde could be
caused by the low catalytic activity of TAO to O-methyl iso-
eugenol10 and not by PAADH to veratraldehyde. It was pre-
viously suggested that TAO has the least affinity to the aromatic
compound with two methoxyl side chains.10 As shown in Figure
5, PAADH catalyzed veratraldehyde significantly to veratric acid,
as compared to other tested aldehyde compounds, suggesting
that biotransformation of veratraldehyde to veratric acid by
PAADH is not the rate-limiting step. In addition, the inter-
mediate veratraldehyde and the end-product veratric acid pro-
duced from O-methyl isoeugenol could exert the antimicrobial
activities onto E. coli.15 During the isoeugenol biotransformation
by the recombinant E. coli, stoichiometry was not observed,
although isoeugenol fairly well decreased with the time of incuba-
tion. This is probably due to absorption to the cells and volatility
of the compounds isoeugenol, vanillin, and vanillic acid.
REFERENCES
■
(1) Serra, S.; Fuganti, C.; Brenna, E. Biocatalytic preparation of natural
flavours and fragrances. Trends Biotechnol. 2005, 23, 193−198.
(2) Heldt, H.-W. Plant Biochenmistry and Molecular Biology; Oxford
University Press: New York, 1999.
(3) Xu, P.; Hua, D.; Ma, C. Microbial transformation of
propenylbenzenes for natural flavour production. Trends Biotechnol.
2007, 25, 571−576.
(4) Ryu, J. Y.; Seo, J.; Unno, T.; Ahn, J. H.; Yan, T.; Sadowsky, M. J.;
Hur, H. G. Isoeugenol monooxygenase and its putative regulatory gene
are located in the eugenol metabolic gene cluster in Pseudomonas
nitroreducens Jin1. Arch. Microbiol. 2010, 192, 201−209.
(5) Shimoni, E.; Baasov, T.; Ravid, U.; Shoham, Y. The trans-anethole
degradation pathway in an Arthrobacter sp. J. Biol. Chem. 2002, 277,
11866−11872.
(6) Shimoni, E.; Baasov, T.; Ravid, U.; Shoham, Y. Biotransformations
of propenylbenzenes by an Arthrobacter sp. and its t-anethole blocked
mutants. J. Biotechnol. 2003, 105, 61−70.
(7) Yamada, M.; Okada, Y.; Yoshida, T.; Nagasawa, T. Purification,
characterization and gene cloning of isoeugenol-degrading enzyme from
Pseudomonas putida IE27. Arch. Microbiol. 2007, 187, 511−517.
(8) Kurlemann, N.; Lara, M.; Pohl, M.; Kroutil, W.; Liese, A.
Asymmetric synthesis of chiral 2-hydroxy ketones by coupled
biocatalytic alkene oxidation and C−C bond formation. J. Mol. Catal.
B: Enzym. 2009, 61, 111−116.
(9) Seshadri, R.; Lamm, A. S.; Khare, A.; Rosazza, J. P. N. Oxidation of
isoeugenol by Nocardia iowensis. Enzyme Microb. Technol. 2008, 43,
486−494.
(10) Han, D.; Ryu, J. Y.; Kanaly, R. A.; Hur, H. G. Isolation of a gene
responsible for the oxidation of trans-anethole to para-anisaldehyde by
Pseudomonas putida JYR-1 and its expression in Escherichia coli. Appl.
Environ. Microbiol. 2012, 78, 5238−5246.
(11) Ryu, J.; Seo, J.; Lee, Y.; Lim, Y.; Ahn, J. H.; Hur, H. G.
Identification of syn- and anti-anethole-2,3-epoxides in the metabolism
of trans-anethole by the newly isolated bacterium Pseudomonas putida
JYR-1. J. Agric. Food Chem. 2005, 53, 5954−5958.
(12) Priefert, H.; Rabenhorst, J.; Steinbuchel, A. Molecular character-
ization of genes of Pseudomonas sp. strain HR199 involved in
11978
dx.doi.org/10.1021/jf303531u | J. Agric. Food Chem. 2012, 60, 11972−11979

Journal of Agricultural and Food Chemistry
Article
bioconversion of vanillin to protocatechuate. J. Bacteriol. 1997, 179,
2595−2607.
(13) Embong, M. B.; Hadziyev, D.; Molnar, S. Essential oils from spices
grown in Alberta. Anise oil (Pimpinella anisum). Can. J. Plant Sci. 1977,
57, 681−688.
(14) Saravanakumar, M.; Raja, B. Veratric acid, a phenolic acid
attenuates blood pressure and oxidative stress in L-NAME induced
hypertensive rats. Eur. J. Pharmacol. 2011, 671, 87−94.
(15) Narasimhan, B.; Ohlan, S.; Ohlan, R.; Judge, V.; Narang, R.
Hansch analysis of veratric acid derivatives as antimicrobial agents. Eur. J.
Med. Chem. 2009, 44, 689−700.
(16) Stanier, R. Y.; Palleroni, N. J.; Doudoroff, M. The aerobic
pseudomonads: A taxonomic study. J. Gen. Microbiol. 1966, 43, 159−
271.
(17) Bradford, M. M. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of protein−dye
binding. Anal. Biochem. 1976, 72, 248−254.
(18) Cronin, C. N.; Kim, J.; Fuller, J. H.; Zhang, X.; McIntire, W. S.
Organization and sequences of p-hydroxybenzaldehyde dehydrogenase
and other plasmid-encoded genes for early enzymes of the p-cresol
degradative pathway in Pseudomonas putida NCIMB 9866 and 9869.
DNA Seq. 1999, 10, 7−17.
(19) Bosch, R.; Garcia-Valdes, E.; Moore, E. R. Genetic character-
ization and evolutionary implications of a chromosomally encoded
naphthalene-degradation upper pathway from Pseudomonas stutzeri
AN10. Gene 1999, 236, 149−157.
(20) Gascuel, O. BIONJ: An improved version of the NJ algorithm
based on a simple model of sequence data. Mol. Biol. Evol. 1997, 14,
685−695.
(21) Lake, J. A. Reconstructing evolutionary trees from DNA and
protein sequences: Paralinear distances. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 1455−1459.
(22) Kotchoni, S. O.; Jimenez-Lopez, J. C.; Kayode, A. P.; Gachomo, E.
W.; Baba-Moussa, L. The soybean aldehyde dehydrogenase (ALDH)
protein superfamily. Gene 2012, 495, 128−133.
(23) Creawford, D. L.; Sutherland, J. B.; Pometto, A. L.; Miller, J. M.
Production of an aromatic aldehyde oxidase by Streptomyces viridosporus.
Arch. Microbiol. 1982, 131, 351−355.
(24) Priefert, H.; Rabenhorst, J.; Steinbuchel, A. Biotechnological
production of vanillin. Appl. Microbiol. Biotechnol. 2001, 56, 296−314.
(25) Liu, F.; Schnable, P. S. Functional specialization of maize
mitochondrial aldehyde dehydrogenases. Plant Physiol. 2002, 130,
1657−1674.
(26) Gilman, H.; Langham, W.; Willis, H. B. Dibenzofuran. XVI. The
two-stage metalation of 2-bromodibenzofuran. J. Am. Chem. Soc. 1940,
62, 346−348.
(27) Jihad, D.; Amikam, Z.; Sasson, Y. Process for the manufacture of
derivatives of benzoic acid. U.S. Patent 4,990,659, 1991.
(28) Gandhi, P. J.; Talia, Y. H.; Murthy, Z. V. P. Production of p-anisic
acid by modified Williamson etherification reaction using design of
experiments. Chem. Prod. Process Model. 2010, 5, 1−27.
(29) Barghini, P.; Di Gioia, D.; Fava, F.; Ruzzi, M. Vanillin production
using metabolically engineered Escherichia coli under non-growing
conditions. Microb. Cell Fact. 2007, 6, 13.
(30) Yoon, S. H.; Li, C.; Kim, J. E.; Lee, S. H.; Yoon, J. Y.; Choi, M. S.;
Seo, W. T.; Yang, J. K.; Kim, J. Y.; Kim, S. W. Production of vanillin by
metabolically engineered Escherichia coli. Biotechnol. Lett. 2005, 27,
1829−1832.
(31) Ryu, J.; Seo, J.; Lee, Y.; Lim, Y.; Ahn, J. H.; Hur, H. G.
Identification of syn- and anti-anethole-2,3-epoxides in the metabolism
of trans-anethole by the newly isolated bacterium Pseudomonas putida
JYR-1. J. Agric. Food Chem. 2005, 53, 5954−5958.
(32) Boyer, H. W.; Roulland-Dussoix, D. A complementation analysis
of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol.
1969, 41, 459−472.
11979
dx.doi.org/10.1021/jf303531u | J. Agric. Food Chem. 2012, 60, 11972−11979