Microbial synthesis of plant oxylipins from γ-linolenic acid through designed biotransformation pathways

Article abstract of DOI:10.1021/jf5058843

Secondary metabolites of plants are often difficult to synthesize in high yields because of the large complexity of the biosynthetic pathways and challenges encountered in the functional expression of the required biosynthetic enzymes in microbial cells. In this study, the biosynthesis of plant oxylipins - a family of oxygenated unsaturated carboxylic acids - was explored to enable a high-yield production through a designed microbial synthetic system harboring a set of microbial enzymes (i.e., fatty acid double-bond hydratases, alcohol dehydrogenases, Baeyer-Villiger monooxygenases, and esterases) to produce a variety of unsaturated carboxylic acids from γ-linolenic acid. The whole cell system of the recombinant Escherichia coli efficiently produced (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid (7), (Z)-9-hydroxynon-6-enoic acid (15), (Z)-dec-4-enedioic acid (17), and (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid (2). This study demonstrated that various secondary metabolites of plants can be produced by implementing artificial biosynthetic pathways into whole-cell biocatalysis.

Full text of DOI:10.1021/jf5058843

Article
pubs.acs.org/JAFC
Microbial Synthesis of Plant Oxylipins from γ‑Linolenic Acid through
Designed Biotransformation Pathways
Sae-Um Kim,† Kyoung-Rok Kim,§ Ji-Won Kim,† Soomin Kim,‡ Yong-Uk Kwon,‡ Deok-Kun Oh,§
and Jin-Byung Park*,†
‡
†Department of Food Science and Engineering and Department of Chemistry and Nano Science, Ewha Womans University,
Seoul 120-750, Republic of Korea
§Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea
S
* Supporting Information
ABSTRACT: Secondary metabolites of plants are often difficult to synthesize in high yields because of the large complexity of
the biosynthetic pathways and challenges encountered in the functional expression of the required biosynthetic enzymes in
microbial cells. In this study, the biosynthesis of plant oxylipinsa family of oxygenated unsaturated carboxylic acidswas
explored to enable a high-yield production through a designed microbial synthetic system harboring a set of microbial enzymes
(i.e., fatty acid double-bond hydratases, alcohol dehydrogenases, Baeyer−Villiger monooxygenases, and esterases) to produce a
variety of unsaturated carboxylic acids from γ-linolenic acid. The whole cell system of the recombinant Escherichia coli efficiently
produced (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid (7), (Z)-9-hydroxynon-6-enoic acid (15), (Z)-dec-4-enedioic acid (17),
and (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid (2). This study demonstrated that various secondary metabolites of plants can
be produced by implementing artificial biosynthetic pathways into whole-cell biocatalysis.
KEYWORDS: plant oxylipins, γ-linolenic acid, biotransformation, whole-cell biocatalysis, Escherichia coli
structure, which may lead to enhancements or innovations of
different end products.18
INTRODUCTION
■
Biocatalysis is expanding its synthetic capacity by covering the
preparation of large and complex molecules as well as small
molecules with the help of the rapidly increasing number of
enzymes discovered.1−6 Biocatalysis is especially useful in the
synthesis of bioactive functional compounds (e.g., polyphenols,
carotenoids, and terpenoids) found in nature, where their
synthesis is hardly possible by any chemical means or reac-
tions.5,7,8 Furthermore, there are products for which biological
production remains challenging. These compounds include
oxygenated unsaturated carboxylic acids that contain one or
more hydroxyl or carboxyl groups as well as CC double
bonds in the carbon skeleton such as oxylipins like as (6Z,9Z)-
12-hydroxydodeca-6,9-dienoic acid (7), (Z)-9-hydroxynon-6-
enoic acid (15), (4Z,7Z)-trideca-4,7-dienedioic acid (9),
(Z)-dec-4-enedioic acid (17), or (6Z,9Z)-13-hydroxyoctadeca-
6,9-dienoic acid (2) (Figures 1 and 5).
Oxygenated carboxylic acids are found in nature as com-
ponents of diverse lipids (e.g., cerebrosides and waxes)9−13
and as secondary metabolites and signaling molecules (e.g.,
oxylipins).14,15 For instance, 11-hydroxyundec-9-enoic acid,
12-hydroxydodec-9-enoic acid, and 1,9-nonanedioic acid were
identified as antifungal metabolites of the wild rice Oryza
officinalis.14 12-Hydroxydodec-9-enoic acid was reported to
stimulate the growth of the main root and lateral roots of plant
seedlings.15 In view of industrial applications, ω-hydroxycarboxylic
acids can be used for the production of α,ω-dicarboxylic acids and
ω-aminocarboxylic acids, which are the starting materials of
polyamides, polyesters, and other chemical products.16,17 In
particular, a double bond in carboxylic acids allows for cross-
linking or the addition of different functional groups to the
Oxygenated carboxylic acids can be produced from the native
plant biosynthetic pathways, which are composed of lip-
oxygenases, hydroperoxide lyases belonging to the CYP74
family, and alcohol dehydrogenases.15 However, the functional
expression of the required biosynthetic enzymes in conven-
tional microbial cells such as Escherichia coli or Saccharomyces
cerevisiae was reported to be challenging.19,20
In the present study, we investigated oxygenation-based
biocatalysis to produce a variety of oxygenated unsaturated
carboxylic acids from a renewable biomass, which is difficult to
obtain by traditional chemical means or reactions. On the basis
of our previous proof-of-concept study, our approach was to
design a biotransformation pathway consisting of fatty acid
double-bond hydratases, alcohol dehydrogenases, Baeyer−
Villiger monooxygenases (BVMOs), and esterases (Figures 1
and 5).21 Because most target products contain CC bond(s)
in the carbon skeleton, γ-linolenic acid, which is abundant in
nature, was used as the starting material.
MATERIALS AND METHODS
■
Microbial Strains and Culture Media. The recombinant
Escherichia coli ER2566 harboring the linoleate C12 double-bond
hydratase gene from Lactobacillus acidophilus22 was grown at 37 °C
with vigorous shaking in a Luria−Bertani (LB) medium supplemented
with 50 μg mL−1 ampicillin. The expression of the recombinant
Received: December 5, 2014
Revised: February 16, 2015
Accepted: February 18, 2015
Published: February 25, 2015
© 2015 American Chemical Society
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Figure 1. Designed biotransformation pathway 1. γ-Linolenic acid (1) was converted into n-hexanoic acid (6) and (6Z,9Z)-12-hydroxydodeca-6,9-
dienoic acid (7) or n-pentanol (8) and (4Z,7Z)-trideca-4,7-dienedioic acid (9) by the multistep enzyme reactions.
hydratase gene was induced by adding 0.1 mM isopropyl-β-D-
thiogalactopyranoside (IPTG) at an optical density (OD600) of 0.5.
Afterward, the culture was further incubated at 16 °C for 16 h. The
recombinant E. coli BL21(DE3), used for hydration of γ-linolenic acid
and biotransformation of hydroxy fatty acids, was cultivated in a
Riesenberg medium, which was supplemented with 10 g L−1 glucose
and the appropriate antibiotics for plasmid maintenance (Supporting
Information Table S1). The Riesenberg medium consisted of 4 g L−1
(NH4)2HPO4, 13.5 g L−1 KH2PO4, 1.7 g L−1 citric acid, 1.4 g L−1
MgSO4, and 10 mL L−1 trace metal solution (10 g L−1 FeSO4,
2.25 g L−1 ZnSO4, 1.0 g L−1 CuSO4, 0.5 g L−1 MnSO4, 0.23 g L−1 Na2B4O7,
2.0 g L−1 CaCl2, and 0.1 g L−1 (NH4)6Mo7O24). Stenotrophomonas
nitritireducens (Table S1) used for the hydration of the C9 double
bond in γ-linolenic acid was cultivated in a nutrient medium as
described previously.23
Reagents. γ-Linolenic acid (1), palmitic acid, n-hexanoic acid (6),
n-pentanol (8), and ethyl acetate were obtained from Sigma-Aldrich.
(6Z,9Z)-13-Hydroxyoctadeca-6,9-dienoic acid (2) and (6Z,12Z)-10-
hydroxyoctadeca-6,12-dienoic acid (10) were prepared via hydration
of γ-linolenic acid with a linoleate C12 double-bond hydratase or a
linoleate C9 double-bond hydratase in our laboratory (see Purification
of Hydroxy Fatty Acids below for details). (6Z,9Z)-12-Hydroxydo-
deca-6,9-dienoic acid (7), (Z)-9-hydroxynon-6-enoic acid (15), (Z)-
dec-4-enedioic acid (17), and (4Z,7Z)-trideca-4,7-dienedioic acid (9)
were synthesized from (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid
(2) or (6Z,12Z)-10-hydroxyoctadeca-6,12-dienoic acid (10) via the re-
combinant E. coli-based biocatalysis in our laboratory (see Purification
of Unsaturated Carboxylic Acids below for details).
supernatant was applied to a His-Trap HP chromatography column
(GE Healthcare) equilibrated with 50 mM phosphate buffer (pH 8.0)
containing 300 mM NaCl. The active fractions were collected and
dialyzed against 50 mM citrate−phosphate buffer (pH 7.0).
Hydration of γ-Linolenic Acid. The enzymatic transformation of
γ-linolenic acid into (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid was
performed in 50 mM citrate−phosphate buffer (pH 7.0) containing
10 mM substrate and 2% (v/v) ethanol. In a 15 mL polypropylene
tube with a screw cap, 3 mL of reaction mixture was incubated at
35 °C for 6 h with agitation at 200 rpm. The purified hydratases were
applied to 3 mg mL−1. One unit (U) of the hydratase activity is
defined as 1 μmol of (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid
produced per minute at 35 °C.
The whole-cell transformation of γ-linolenic acid into (6Z,9Z)-13-
hydroxyoctadeca-6,9-dienoic acid was performed in 50 mM Tris-HCl
buffer (pH 7.0) containing 38 mM substrate and 0.05% (v/v) Tween 80.
In a 15 mL polypropylene tube with a screw cap, 3 mL of reaction
mixture was incubated at 35 °C for 24 h with agitation at 200 rpm.
The recombinant E. coli BL21(DE3) expressing the linoleate C12
double-bond hydratase of L. acidophilus NBRC 13951 was added
to 14.4 g L−1.
The hydration of γ-linolenic acid into (6Z,12Z)-10-hydroxyocta-
deca-6,12-dienoic acid with S. nitritireducens was carried out in 10 mL
of 50 mM citrate−phosphate buffer (pH 7.0). γ-Linolenic acid was
added to a reaction medium containing 20 g dry cells L−1 and 0.05%
(v/v) Tween 80. The reaction mixture was incubated at 30 °C and
sparged with a gas mixture consisting of 95% N2 and 5% CO2 at
0.02 vvm to maintain anaerobic conditions.23
Purification of a Linoleate C12 Double-Bond Hydratase. The
linoleate C12 double bond-hydratase of L. acidophilus was purified as
previously reported.22 In brief, after cultivation of recombinant E. coli
ER2566 harboring the linoleate C12 double-bond hydratase gene of
L. acidophilus, the cell mass was harvested, washed, and resuspended
into 50 mM phosphate buffer (pH 8.0) containing 300 mM NaCl,
10 mM imidazole, and 0.1 mM phenylmethanesulfonyl fluoride as a
protease inhibitor. The resuspended cells were disrupted, and the
Biotransformation of Hydroxy Fatty Acids. The biotransfor-
mation of the hydroxy fatty acids with recombinant E. coli cells was
carried out on the basis of our earlier work.21 Briefly, the recombinant
cells were cultivated in the Riesenberg medium at 30 °C, and the
expression of the target genes was induced with 0.1 mM IPTG and/or
2.0 g L−1 rhamnose at an OD600 of 0.5. Thereafter, the cultivation tem-
perature was reduced to 20 °C to facilitate the soluble expression of
the target genes (Supporting Information Figure S4). When the added
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glucose was consumed, the recombinant cells were harvested by
centrifugation at 3500g for 15 min at 4 °C and used as biocatalysts. The
biotransformation was initiated by adding 2 mM of a hydroxy fatty acid
to 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 g L−1 Tween 80 and
7.2 g dry cells L−1 of the recombinant E. coli BL21(DE3) that expressed
the ADH from Micrococcus luteus, the esterase from P. fluorescens SIK
WI, and the BVMO from either Pseudomonas putida KT2440 or
Pseudomonas fluorescens DSM 50106. The biotransformations were
conducted in a shaking incubator (35 °C and 200 rpm).
Table 1. Products Accessible through the Multistep
Biocatalysis
product
yield (%)
a
starting material
final product
γ-linolenic acid (1)
(6Z,9Z)-13-hydroxyoctadeca- 92 2 (60%,
b
6,9-dienoic acid (2)
96%)
(6Z,12Z)-10-
97
4
hydroxyoctadeca-6,12-
dienoic acid (10)
Product Analysis by GC-MS. The concentrations of the
remaining fatty acids and accumulating carboxylic acids in the medium
(i.e., γ-linolenic acid (1), (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid
(2), (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid (7), (Z)-9-hydrox-
ynon-6-enoic acid (15), (4Z,7Z)-trideca-4,7-dienedioic acid (9), and
(Z)-dec-4-enedioic acid (17)) were determined as described pre-
viously.21 The reaction medium was mixed with an equal volume of
ethyl acetate containing 2 or 5 g L−1 palmitic acid as an internal stan-
dard. The organic phase was harvested after vigorous vortexing and
then derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide
(MSTFA). The trimethylsilyl (TMS) derivatives were analyzed using a
Thermo Ultra Trace GC system connected to an ion trap mass
detector (Thermo ITQ1100 GC-ion Trap MS; Thermo Scientific).
The derivatives were separated on a nonpolar capillary column (30 m
length, 0.25 μm film thickness, HP-5MS; Agilent, Santa Clara, CA,
USA). A linear temperature gradient was programmed as follows:
90 °C, 15 °C min−1 to 200 °C; 200 °C, 5 °C min−1 to 250 °C; 90 °C,
15 °C min−1 to 200 °C; and 200 °C, 5 °C min−1 to 280 °C. The injec-
tion port temperature was 230 °C. Mass spectra were obtained by
electron impact ionization at 70 eV. Scan spectra were obtained within
the range of m/z 100−600. Selected ion monitoring (SIM) was used
for the detection and fragmentation analysis of the reaction products.
Product Analysis by GC. The obtained hydroxy fatty acids were
derivatized by using a 3:1 mixture of pyridine and MSTFA. TMS-
derivatized hydroxy fatty acids were analyzed by GC (Agilent 6890N)
equipped with a flame ionization detector and an SPB-1 capillary
column (15 m × 0.32 mm inside diameter, 0.25 μm thickness;
Supelco) using palmitic acid as an internal standard.23 The column
temperature increased from 150 to 210 °C at an initial rate of
4 °C min−1 and then at rate of 30 °C min−1 until 280 °C, and this tem-
perature was maintained at 280 °C for 5 min. The injector and
detector temperatures were held at 260 and 250 °C, respectively.
Identification of the Biotransformation Products. The
biotransformation products were identified by GC-MS or NMR
analyses. (Z)-9-Hydroxynon-6-enoic acid (15), (4Z,7Z)-trideca-4,7-
dienedioic acid (9), and (Z)-dec-4-enedioic acid (17) were identified
by GC-MS analysis and comparison to pure reference compounds
(Figures 4 and 7). (6Z,9Z)-13-Hydroxyoctadeca-6,9-dienoic acid (2)
and (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid (7) were identified by
GC-MS and NMR analyses (see Figure S3 in the Supporting
Information for identification of (6Z,9Z)-13-hydroxyoctadeca-6,9-
dienoic acid and Figure S5 for identification of (6Z,9Z)-12-
hydroxydodeca-6,9-dienoic acid). Identification of compounds 2, 7,
9, 15, and 17 was further confirmed by the GC-MS analysis of their
hydrogenated products, which were obtained by catalytic hydro-
genation (Figures 4, 7, and S1B).
(6Z,9Z)-13-hydroxyoctadeca- (6Z,9Z)-12-hydroxydodeca-
81 4 (43%,
b
6,9-dienoic acid (2)
6,9-dienoic acid (7)
71%)
c
(4Z,7Z)-trideca-4,7-
dienedioic acid (9)
34
6
(6Z,12Z)-10-
hydroxyoctadeca-6,12-
dienoic acid (10)
(Z)-9-hydroxynon-6-enoic
acid (15)
72
7
4
(Z)-dec-4-enedioic acid (17) 73
a
The product yield was calculated on the basis of the substrate
depletion and the product concentration, which were determined by
b
GC-MS. All experiments were carried out in triplicate. The numbers
in parentheses indicate the isolated yield and purity, respectively. The
isolated yields were calculated on the basis of the amount of the
products isolated and the amount of the substrates added into the
c
reaction medium. The product yields were low compared to those of
the other products because (6Z,9Z)-12-hydroxydodeca-6,9-dienoic
acid (7) was coproduced during biotransformation (Figure 3B).
C-18 semiprep column as described in a previous study24 (see
Figure S2 in the Supporting Information for purification of (6Z,9Z)-
13-hydroxyoctadeca-6,9-dienoic acid).
Purification of Unsaturated Carboxylic Acids. The medium-chain,
unsaturated carboxylic acids (e.g., 12-hydroxydodeca-6,9-dienoic
acid, 9-hydroxynona-6-enoic acid, and 1,10-deca-6-enedioic acid)
were isolated from the whole-cell reaction medium as described
previously.21,25 In brief, the biotransformation products were extracted
with ethyl acetate three times. The extract was dried over MgSO4 and
concentrated via evaporation in vacuo. The impurities (e.g., n-alkanoic
acids coproduced during biotransformations and long-chain fatty acids
originating from the starting materials) were removed by washing with
hexane (see Figure S6 in the Supporting Information for purification
of (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid). (6Z,9Z)-12-Hydrox-
ydodeca-6,9-dienoic acid was also purified by HPLC (Figure S5).
RESULTS
■
The fatty acid double-bond hydratases and BVMOs were the
key enzymes in our designed biotransformation pathways
because the structures of the final products were determined
by the regioselectivity of both enzymes (Figures 1 and 5).
Thereby, the first step was to select the appropriate enzymes in
terms of substrate scope and regioselectivity for the production
of the target compounds.
Screening of a γ-Linolenic Acid C12 Double-Bond
Hydratase. Recently, a linoleate C12 double-bond hydratase
of L. acidophilus NBRC 13951 was cloned and characterized.22
Thereby, the enzyme was first examined for hydration of the
C12 double bond in γ-linolenic acid. The enzyme was produced
in E. coli ER2566 and subjected to purification via His-Trap
affinity chromatography. Afterward, the purified enzyme was
added to 50 mM citrate−phosphate buffer (pH 7.0) containing
10 mM γ-linolenic acid. A single product was accumulated in
the reaction mixture (Figure 2A and Supporting Information
Figure S1A). The product (2) was purified to a purity of >96%
via solvent extraction and column chromatography (Figure S2).
The mass spectrum contained peaks at m/z 173 and 369 due to
the α-cleavages at both sides of the hydroxy-TMS function at
Purification of the Biotransformation Products. Purification
of Hydroxy Fatty Acids. The hydroxy fatty acids (i.e., (6Z,9Z)-13-
hydroxyoctadeca-6,9-dienoic acid (2) and (6Z,12Z)-10-hydroxyocta-
deca-6,12-dienoic acid (10)) were isolated from the reaction medium
by solvent extraction and column chromatography as described
previously.23 In brief, the biotransformation products were isolated by
the extraction using two volumes of ethyl acetate two times. After the
organic layer was separated from the aqueous phase, the organic
extracts were dried with Na2SO4 and concentrated by the evaporation
in vacuo. The concentrate was purified by the silicic acid column
chromatography with methanol gradient (0−5%, v/v) in CH3Cl
(50 mL). Any impurities and the remaining substrate were washed off
with CH3Cl and 1% methanol in CH3Cl. In the case of (6Z,9Z)-13-
hydroxyoctadeca-6,9-dienoic acid, the concentrate was purified with
a semipreparative HPLC system (Agilent) equipped with a Nucleosil
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Figure 3. Biotransformation of (6Z,9Z)-13-hydroxyoctadeca-6,9-
dienoic acid. (6Z,9Z)-13-Hydroxyoctadeca-6,9-dienoic acid (2) was
converted into (A) (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid (7) or
(B) (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid and (4Z,7Z)-trideca-
4,7-dienedioic acid (9). The first biotransformation was conducted
with the recombinant E. coli BL21(DE3) pACYC-ADH, pET-BmoF1,
pCOLA-PFE1 expressing the ADH from M. luteus, the BVMO from
P. fluorescens DSM 50106, and the esterase from P. fluorescens SIK WI.
The second biotransformation was conducted with the recombinant
E. coli BL21(DE3) pACYC-ADH-BVMO, pCOLA-PFE1, expressing
the ADH from M. luteus, the BVMO from Ps. putida KT2440, and the
esterase from P. fluorescens SIK WI. The experiments were carried out
in triplicate, and the error bars indicate the standard deviation.
Figure 2. Biotransformation of γ-linolenic acid. γ-Linolenic acid (1)
was converted into (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid (2)
by (A) the isolated linoleate C12 double-bond hydratase of
L. acidiphilus NBRC 13951 or (B) the recombinant E. coli BL21(DE3)
expressing the linoleate C12 double-bond hydratase of L. acidiphilus
NBRC 13951. The experiments were carried out in triplicate, and the
error bars indicate the standard deviation.
C13, whereas the mass spectrum of the hydrogenation product
showed peaks at m/z 173 and 373 (Figure S1B). The resonance
signals at 3.63 ppm (m, 1H) and four olefinic protons at 5.38
hydratase of L. acidophilus NBRC 13951, cell harvest via
centrifugation, and resuspension into 50 mM Tris-HCl buffer
(pH 7.0), γ-linolenic acid was added to a concentration of
38 mM in the reaction medium. γ-Linolenic acid was converted
into the target product at a rate of 2.9 μmol g dry cells−1 min−1
(i.e., 2.9 U g dry cells−1) at t < 10 h, thus resulting in a final
product concentration of ∼30 mM (8.9 g L−1) in the reaction
medium (Figure 2B). The bioconversion yield, which was
calculated on the basis of the substrate depletion and the
product concentration determined by GC-MS, reached >95%.
The byproduct formation was hardly observed (Supporting
Information Figure S1B). These results indicated that the
whole-cell biotransformation system was quite efficient in terms
of reaction selectivity and rate.
1
(m, 4H, H-6, H-7, H-9, and H-10) in H NMR indicated the
existence of a hydroxyl group and a double bond (Figure S3).
The observed signal at 71.8 ppm (C-13) of 13C NMR also
supported the existence of one hydroxyl group (Figure S3). On
the basis of these data, the product was identified as (6Z,9Z)-
13-hydroxyoctadeca-6,9-dienoic acid (2). The concentration of
(6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid in the reaction
medium increased to 6 mM. The hydration rate was increased
to 21 U g protein−1 at t < 1 h. The isolated yield of the
compound was ∼60% with a purity of 96% (Table 1). This
result indicated that the linoleate C12 double-bond hydratase
also exhibited very high regioselectivity with γ-linolenic acid.
Whole-Cell Production of (6Z,9Z)-13-Hydroxyoctade-
ca-6,9-dienoic Acid. The microbial production of (6Z,9Z)-
13-hydroxyoctadeca-6,9-dienoic acid (2) from γ-linolenic acid
was also investigated. After batch cultivation of recombinant
E. coli BL21(DE3) expressing the linoleate C12 double-bond
Biotransformation of (6Z,9Z)-13-Hydroxyoctadeca-
6,9-dienoic Acid (2). Hydroxy fatty acids are used as starting
materials for the production of diverse secondary metabolites
and signaling molecules in living organisms.12,13 Thus, we
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Figure 4. GC-MS analysis of (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid biotransformation products. (A) GC-MS of the biotransformation
products, which were produced with the recombinant E. coli BL21(DE3) pACYC-ADH, pET-BmoF1, pCOLA-PFE1. The bottom panel shows the
mass spectrum of the hydrogenated product of (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid, which is identical to that of the authentic compound
12-hydroxydodecanoic acid. (B) GC-MS of the biotransformation products, which were produced with the recombinant E. coli BL21(DE3) pACYC-
ADH-BVMO, pCOLA-PFE1. The bottom panel shows the mass spectrum of the hydrogenated product of (4Z,7Z)-trideca-4,7-dienedioic acid,
which is identical to that of the authentic compound tridecane-1,13-dioic acid.
Figure 5. Designed biotransformation pathway 2. γ-Linolenic acid (1) is converted into 2-nonenoic acid (14) and (Z)-9-hydroxynon-6-enoic acid
(15) or 2-octenol (16) and (Z)-dec-4-enedioic acid (17) by the multistep enzyme reactions.
investigated the biotransformation of (6Z,9Z)-13-hydroxyocta-
deca-6,9-dienoic acid (2) into C12 oxylipins (e.g., (6Z,9Z)-12-
hydroxydodeca-6,9-dienoic acid (7)). The substrate (2) was
prepared after the whole-cell reaction of γ-linolenic acid,
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isolation via ethyl acetate extraction, and concentration via
evaporation in vacuo. The BVMO enzymes, which determine
the chemical structure of the final products due to their
regioselectivity, were first examined. The BVMOs that are
active with long-chain aliphatic ketones26−29 were chosen as
candidates. When the recombinant E. coli BL21(DE3) pACYC-
ADH, pET-BmoF1, pCOLA-PFE1 expressing an alcohol
dehydrogenase (ADH) from Micrococcus luteus NCTC2665, a
BVMO from Pseudomonas fluorescens DSM 50106,30 and an
esterase from P. fluorescens SIK WI was added to the reaction
medium containing (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic
acid (2), the compound was selectively converted into
(6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid (7) and n-hexanoic
acid (6) via (6Z,9Z)-13-oxooctadeca-6,9-dienoic acid (3) and
the ester (4) (Figures 3A and 4A). (6Z,9Z)-12-Hydroxydo-
deca-6,9-dienoic acid (7) was identified via GC-MS and NMR
analyses after purification (Figures 4A and S5). Notably, the
target product (7) was produced with a final concentration
of 1.6 mM from 2.0 mM of the substrate (6Z,9Z)-13-
hydroxyoctadeca-6,9-dienoic acid.
On the other hand, the addition of the recombinant E. coli
BL21(DE3) pACYC-ADH-BVMO, pCOLA-PFE1 expressing
an ADH from M. luteus NCTC2665, a BVMO from Pseudomonas
putida KT2440,27 and an esterase from P. fluorescens SIK WI
resulted in a production of not only (6Z,9Z)-12-hydroxydodeca-
6,9-dienoic acid (7) and n-hexanoic acid (6) but also (4Z,7Z)-
trideca-4,7-dienedioic acid (9) and n-pentanol (8) (Figures 3B
and 4B). (4Z,7Z)-Trideca-4,7-dienedioic acid was identified on the
basis of the GC-MS analysis data of the pure compound 1,12-
dodec-9-enedioic acid,31 which has the same functional group in
the fatty acid skeleton. It was assumed that the ester compound
(5) was produced via the formation of the abnormal ester,
which was driven by migration of the less-substituted carbon
center during BVMO catalysis.32,33
Biotransformation of (6Z,12Z)-10-Hydroxyoctadeca-
6,12-dienoic Acid (10). The biotransformation of (6Z,12Z)-
10-hydroxyoctadeca-6,12-dienoic acid (10) (Figure 5), which
had been produced from γ-linolenic acid by using the hydratase
of S. nitritireducens23 (Supporting Information Figure S7), was
also carried out using the setup identical to the biotransforma-
tion of (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid (2). (Z)-
9-Hydroxynon-6-enoic acid (15) and 2-nonenoic acid (14)
were produced as the major products by recombinant E. coli
BL21(DE3) pACYC-ADH-BVMO, pCOLA-PFE1 (Figures 6A
and 7A). 9-Hydroxynona-6-enoic acid was identified on the
basis of the GC-MS analysis data of the pure compound 12-
hydroxyundec-9-enoic acid,15 which has the same functional
group in the fatty acid skeleton. Notably, the BVMO of
P. putida KT2440 showed quite high selectivity with oxygen-
ation of (6Z,12Z)-10-oxooctadeca-6,12-dienoic acid (11), but it
showed low selectivity with (6Z,9Z)-13-oxooctadeca-6,9-
dienoic acid (3) (Figure 3B).
Figure 6. Biotransformation of (6Z,12Z)-10-hydroxyoctadeca-6,12-
dienoic acid. (6Z,12Z)-10-Hydroxyoctadeca-6,12-dienoic acid (10)
was converted by (A) the recombinant E. coli BL21(DE3) pACYC-
ADH-BVMO, pCOLA-PFE1, and (B) the recombinant E. coli
BL21(DE3) pACYC-ADH, pET-BmoF1, pCOLA-PFE1. The experi-
ments were carried out in triplicate, and the error bars indicate
standard deviation.
DISCUSSION
■
A family of oxygenated unsaturated carboxylic acids (e.g., oxylipins)
that play important roles in plant signaling and defense are usually
produced via a serial reaction of lipoxygenases and hydroperoxide
lyases belonging to CYP74 family.15 For instance, 12-hydroxydodec-
9-enoic acid, 12-oxododec-9-enoic acid, and 1,12-dodec-9-enedioic
acid, which were reported to stimulate growth of the roots of
plant seedlings, were synthesized from α-linolenic acid by 13-
lipoxygenases, hydroperoxide lyases, and alcohol dehydro-
genases. 9-Hydroxynonanoic acid and 1,9-nonanedioic acid,
which are involved in defense against infection, were produced
from α-linolenic acid by 9-lipoxygenases, hydroperoxide lyases,
and alcohol dehydrogenases.15
Because the oxygenated carboxylic acids have a great
potential not only as agrochemicals but also as building blocks
and/or additives for the production of plastics (e.g., polyamides
and polyesters), resins, hot melt adhesives, powder coatings,
corrosion inhibitors, lubricants, plasticizers, greases, and
perfumes,14,18,34,35 their biosyntheses have been extensively
investigated. For instance, 9-oxononanoic acid was produced
from linoleic acid via an enzymatic two-step reaction cata-
lyzed by the 9S-lipoxygenase of Solanum tuberosum and the
9/13-hydroperoxide lyase of Cucumis melo.19 Another example
was the biotransformation of α-linoleic acid into 3(Z)-hexenal
The biotransformation of (6Z,12Z)-10-hydroxyoctadeca-
6,12-dienoic acid (10) with recombinant E. coli BL21(DE3)
pACYC-ADH, pET-BmoF1, pCOLA-PFE1 expressing the BVMO
from P. fluorescens DSM50106 instead of the BVMO from
P. putida KT2440 resulted in the production of (Z)-dec-4-enedioic
acid (17) and 2-octenol as the major products (Figures 6B
and 7B). These results indicated that a variety of plant oxylipins
could be produced via combined biocatalysis using fatty acid
double-bond hydratases and BVMOs.
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Figure 7. GC-MS analysis of (6Z,12Z)-10-hydroxyoctadeca-6,12-dienoic acid biotransformation products. (A) GC-MS of the biotransformation
products, which were produced with the recombinant E. coli BL21(DE3) pACYC-ADH-BVMO, pCOLA-PFE1. The bottom panel shows the mass
spectrum of the hydrogenated product of (Z)-9-hydroxynon-6-enoic acid, which is identical to that of the authentic compound 9-hydroxynonanoic
acid. (B) GC-MS of the biotransformation products, which were produced with the recombinant E. coli BL21(DE3) pACYC-ADH, pET-BmoF1,
pCOLA-PFE1. The bottom panel shows the mass spectrum of the hydrogenated product of (Z)-dec-4-enedioic acid, which is identical to that of the
authentic compound decane-1,10-dioic acid.
and 12-oxododec-9-enoic acid by a recombinant S. cerevisiae
expressing a soybean lipoxygenase and watermelon hydro-
peroxide lyase.20 However, the productivity of the biocatalysts
appeared to be limited due to the low expression level of the
plant enzymes in the microbial cells.
This study herein demonstrated that a variety of the
industrially relevant fatty acids and carboxylic acids (i.e.,
(6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid (2), (6Z,9Z)-12-
hydroxydodeca-6,9-dienoic acid (7), (Z)-9-hydroxynon-6-enoic
acid (15), (4Z,7Z)-trideca-4,7-dienedioic acid (9), and (Z)-dec-
4-enedioic acid (17)) could be produced from renewable fatty
acids (i.e., γ-linolenic acid) by using artificial multistep enzyme
reaction pathways (Figures 1 and 5). The synthetic pathways
were composed of enzymes originating solely from bacteria,
which could be overexpressed easily in functional forms in
bacterial cells.
Some of the key enzymes in the biotransformations reported
herein are the BVMOs. The regiospecificity of the enzymes was
crucial for providing access to the desired reaction products
(Figures 1 and 5). The BVMOs (EC 1.4.13.22) catalyzed the
insertion of oxygen into the carbon skeletons adjacent to the
carbonyl groups.36−38 The regiochemistry of the reaction was
governed by predictable conformational, steric, and electronic
effects as the rearrangement process of the tetrahedral peroxo
Criegee intermediate proceeded with strict retention of
configuration. Common representative substrates were simple
cyclic ketones (e.g., cyclobutanone, cyclopentanone, cyclo-
hexanone, and cycloheptanone), chiral cyclic ketones (e.g.,
2-methylcyclopentanone and bicyclo[3.2.0]hept-2-en-6-one),
and aliphatic ketones (e.g., 2-octanone).38 Most BVMOs
discovered so far showed catalytic activity for the oxygenation
of simple cyclic ketones and chiral cyclic ketones, and only a
small number of BVMOs including the BVMO from P. fluorescens
DSM 50106,30 P. putida KT2440,27,39 R. jostii RHA1,40 P. veronii
MEK700,26 P. cepacia,28 and Mycobacterium tuberculosis29 were
found to be active with aliphatic ketones.
The present study herein demonstrated that product profiles
of the novel biosynthetic pathways (Figures 1 and 5) were
dependent on the catalytic activity, but they were especially de-
pendent on the regioselectivity of the BVMOs (Figures 3 and 6).
The BVMO originating from P. fluorescens DSM 50106 catalyzed
the normal ester formation with (6Z,9Z)-13-oxooctadeca-6,9-
dienoic acid (3), which was driven by migration of the more-
substituted carbon center during BVMO catalysis (Figure 3A).
However, the BVMO from P. putida KT2440 showed a quite high
activity toward the abnormal ester formation32,33 in the case of
(6Z,9Z)-13-oxooctadeca-6,9-dienoic acid (3) (Figure 3B). Nota-
bly, the BVMO from P. fluorescens DSM 50106 catalyzed the
abnormal ester formation of (6Z,12Z)-10-oxooctadeca-6,12-
dienoic acid (11), thus resulting in the production of 1,10-
dec-6-enedioic acid (Figure 6B). Unfortunately, the BVMOs from
P. fluorescens DSM 50106 and P. putida KT2440 were too un-
stable to allow for the proper investigation of their kinetic properties
and their 3-D structures. As a result, a detailed investigation of their
regioselectivity is currently severely hampered.
In summary, we demonstrated that a variety of plant
oxylipins (i.e., (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic
acid (2), (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid (7),
(Z)-9-hydroxynon-6-enoic acid (15), (4Z,7Z)-trideca-4,7-diene-
dioic acid (9), and (Z)-dec-4-enedioic acid (17)) could be
produced from a renewable fatty acid (i.e., γ-linolenic acid) via the
regioselective BVMO-based whole-cell biocatalysis. This study will
contribute to the preparation of industrially relevant unsaturated
carboxylic acids.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Table S1 and Figures S1−S7. This material is available free of
charge via the Internet at http://pubs.acs.org.
(16) Griehl, W.; Ruestem, D. Nylon-12 preparation, properties and
applications. Ind. Eng. Chem. 1970, 62, 16−22.
AUTHOR INFORMATION
Corresponding Author
*(J.-B.P.) E-mail: jbpark06@ewha.ac.kr.
Funding
This study was supported by a Marine Biomaterials Research
Center grant from the Marine Biotechnology Program funded
by the Ministry of Oceans and Fisheries, Korea.
■
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The authors declare no competing financial interest.
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