Microbial Synthesis of Linoleate 9 S-Lipoxygenase Derived Plant C18 Oxylipins from C18 Polyunsaturated Fatty Acids

Article abstract of DOI:10.1021/acs.jafc.8b05857

Plant oxylipins, including hydroxy fatty acids, epoxy hydroxy fatty acids, and trihydroxy fatty acids, which are biosynthesized from C18 polyunsaturated fatty acids (PUFAs), are involved in pathogen-specific defense mechanisms against fungal infections. However, their quantitative biotransformation by plant enzymes has not been reported. A few bacteria produce C18 trihydroxy fatty acids, but the enzymes and pathways related to the biosynthesis of plant oxylipins in bacteria have not been reported. In this study, we first report the biotransformation of C18 PUFAs into plant C18 oxylipins by expressing linoleate 9S-lipoxygenase with and without epoxide hydrolase from the proteobacterium Myxococcus xanthus in recombinant Escherichia coli. Among the nine types of plant oxylipins, 12,13-epoxy-14-hydroxy-cis,cis-9,15-octadecadienoic acid was identified as a new compound by NMR analysis, and 9,10,11-hydroxy-cis,cis-6,12-octadecadienoic acid and 12,13,14-trihydroxy-cis,cis-9,15-octadecadienoic were suggested as new compounds by LC-MS/MS analysis. This study shows that bioactive plant oxylipins can be produced by microbial enzymes.

Full text of DOI:10.1021/acs.jafc.8b05857

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Biotechnology and Biological Transformations
Microbial Synthesis of Linoleate 9S-Lipoxygenase derived
Plant C18 Oxylipins from C18 Polyunsaturated Fatty Acids
Jung-Ung An, In-Gyu Lee, Yoon-Joo Ko, and Deok-Kun Oh
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05857 • Publication Date (Web): 27 Feb 2019
Downloaded from http://pubs.acs.org on March 4, 2019
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Page 1 of 42
Journal of Agricultural and Food Chemistry
1 Microbial Synthesis of Linoleate 9S-Lipoxygenase derived
2 Plant C18 Oxylipins from C18 Polyunsaturated Fatty Acids
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Jung-Ung An†, In-Gyu Lee†, Yoon-Joo Ko‡, and Deok-Kun Oh*,†
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†Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of
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Korea
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‡ National Center for Inter-University Research Facilities (NCIRF), Seoul National University,
Seoul 08826, Republic of Korea
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ABSTRACT: Plant oxylipins, including hydroxy fatty acids, epoxy hydroxy fatty acids, and
trihydroxy fatty acids, biosynthesized from C18 polyunsaturated fatty acids (PUFAs), are
involved in pathogen-specific defense mechanisms against fungal infections. However, the
quantitative biotransformation by plant enzymes has not been reported. A few bacteria
produce C18 trihydroxy fatty acids, but the enzymes and pathways related to the biosynthesis
of plant oxylipins in bacteria have not been reported. In this study, we first report the
biotransformation of C18 PUFAs into plant C18 oxylipins by expressing linoleate 9S-
lipoxygenase with or without epoxide hydrolase from the proteobacterium Myxococcus
xanthus in recombinant Escherichia coli. Among nine types of plant oxylipins, 12,13-epoxy-
14-hydroxy-cis,cis-9,15-octadecadienoic acid was identified as a new compound by NMR
analysis, and 9,10,11-hydroxy-cis,cis-6,12-octadecadienoic acid and 12,13,14-trihydroxy-
cis,cis-9,15-octadecadienoic were suggested as new compounds by LC-MS/MS analysis. This
study shows that bioactive plant oxylipins can be produced by microbial enzymes.
KEYWORDS: Biotransformation; Epoxy hydroxy fatty acid; Trihydroxy fatty acid;
Microbial enzyme; Enzyme catalysis
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INTRODUCTION
Oxylipins, oxygenated unsaturated fatty acids, have important biological roles in various
organisms. They are divided into fatty acid aldehydes, divinyl ethers, hydroxy fatty acids
(HFAs), keto fatty acids, epoxy hydroxy fatty acids, epoxy fatty acids, polyhydroxy fatty
acids, diepoxides, tetrahydrofurandiols, and prostanoids.1-5 Plant oxylipins are usually
biosynthesized from C18 polyunsaturated fatty acids (PUFAs), including linoleic acid, α-
linolenic acid, and γ-linolenic acid, as starting materials.
Among oxylipins, HFAs, epoxy hydroxy fatty acids (EHFAs), and trihydroxy fatty acids
(THFAs) are extremely important bioactive molecules in plants. The plant C18 oxylipins, 9S-
and 13S-hydroxyoctadecadienoic acids have antifungal activity,6 9,10-epoxy-18-hydroxy-
octadecanoic acid and 9,10,18-trihydroxy-octadecanoic acid have been described as potential
messengers in plant-pathogen interactions,7 and 9S,12S,13S-trihydroxy-octadecenoic acid
shows strong inhibition activity toward the germination and elongation of rice blast fungus in
plants, and is also used as an adjuvant for vaccines.8,9 The 7,8,9-, 9S,10S,11R-, and 12,13,17-
trihydroxy-octadecenoic acids in plants inhibit the growth of the plant pathogenic fungi
related to wheat powdery mildew, potato late blight, and cucumber botrytis.10-12 These
oxylipins can also be used in the industry as starting materials for resins, waxes, nylons,
plastics, and cosmetics because the hydroxyl group gives fatty acids special properties such
as higher viscosity and reactivity compared to non-hydroxylated fatty acids.6,13
Lipoxygenase (LOX) converts PUFAs having a Z,Z-1,4-pentadiene structure into different
regiospecific hydroperoxy fatty acids (HpFAs), which are readily reduced to HFAs under
physiological conditions. In animals, EHFAs are converted from HpFAs by LOX, undergoing
bi-functional catalytic reactions,14,15 whereas, in plants, this is achieved by different enzymes,
i.e., peroxygenase or epoxy alcohol synthase.11,16 THFAs are converted from EHFAs by the
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hydrolysis of epoxide rings by epoxide hydrolase (EH) in both plants and animals.11,17
Especially, human EH divided into EH1 (microsomal EH), EH2 (soluble EH), EH3, EH4,
cholesterol hydrolase, leukotriene A4 hydrolase, and hepoxilin EH.18 Among them, soluble
EH and hepoxilin EH had activity on the conversion of trioxilin (THFA) from hepoxilin
(EHFA).17,19 The study of the biosynthesis of oxylipins in plants is challenging because
oxylipins exist in trace amounts in vivo and rapidly degrade. Moreover, the quantitative
biotransformation by plant enzymes has not been reported.
Plant oxylipins have also been found in corals, fungi, and bacteria.20 A few types of
bacteria have been found to produce plant THFAs,21,22 however, the biosynthetic pathways
and related enzymes have not been reported to date. Recently, we have discovered that
arachidonate 11S- and 12S-LOXs and EH from the proteobacterium Myxococcus xanthus
DK1622 convert C20 and C22 PUFAs into the EHFAs, hepoxilins, and the THFAs,
trioxilins.23 Arachidonate 11S- and 12S-LOXs from M. xanthus, corresponding linoleate 9S-
and 13S-LOX, respectively, catalyze first a two-step reaction of peroxidation and epoxidation
to date among bacterial LOXs. The two-step reactions enable the conversion of C20 and C22
PUFAs into EHFAs. EHFAs are converted into THFAs by M. xanthus EH because its activity
is similar to human soluble EH (EH2). In this study, we attempted the biotransformation for
the production of plant C18 oxylipins (e.g., HFAs, EHFAs, and THFAs) from C18 PUFAs
(e.g., linoleic acid, α-linolenic acid, and γ-linolenic acid) by expressing only linoleate 9S-
LOX or both linoleate 9S-LOX and EH from M. xanthus in Escherichia coli ER2566 (Figure
1).
MATERIALS AND METHODS
Preparation of Standards. The PUFA standards linoleic acid (1), α-linolenic acid (6),
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and γ-linolenic acid (11) were purchased from Sigma-Aldrich. The HFA standards 9S-
hydroxy-trans,cis-10,12-octadecadienoic acid (5) and 9R-hydroxy-trans,cis-10,12-
octadecadienoic acid were purchased from Cayman Chemical. The other HFA, EHFA, and
THFA standards were prepared as follows: The reactions were performed for 3 h at 35 °C in
50 mM 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (HEPPS, pH 8.5) buffer
containing 100 mg L−1 PUFA as a substrate and 20 g L−1 recombinant E. coli expressing only
linoleate 9S-LOX or both linoleate 9S-LOX and EH from M. xanthus. The reaction solution
was extracted with an equal volume of ethyl acetate, the solvent layer was harvested, and the
solvent in the layer was removed using a rotary evaporator. The solvent-free extract was
applied to a preparative HPLC (Agilent 1260, Santa Clara, CA, USA) equipped with a
Nucleosil C18 column (10×250 mm, 5-μm particle size; Phenomenex). The column was
eluted at 30 °C at a flow rate of 6 mL min−1, and the product fractions were collected by
detecting the absorbance at 202 nm. The adsorbent resin SP825 (Ion Technology, Sungnam,
Republic of Korea), which bound specifically hydrophilic compounds like fatty acid
derivatives, at 10 g L−1 was added to 10 mM of the collected products. The absorption of
products to the resin was performed in shaking at 200 rpm at 40 °C during 3 h. The absorbed
resin was recovered by filtration through a 45-µm pore-size filter and the purified products
were obtained by extraction of the resin with ethyl acetate. To investigation of purity of
reaction products, the purified 9-HODE as an example was estimated to be 98% purity by
comparing with the standard 9-HODE, which was purchased from Cayman Chemical (See
the Supporting Information, Figure S1). All the collected samples were purified to ≥93%
purity (See Table 2 and Supporting Information, Figures S2−S4 for product purification), and
were used as standards after identification by LC-MS/MS.
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Microbial Strains, Plasmids, and Gene Cloning. M. xanthus DK1622 (KCCM
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44251, Seoul, Republic of Korea) was used as the source of the genomic DNA for cloning
linoleate 9S-LOX (Genbank accession number ABF88826.1) and EH (Genbank accession
number ABF91519.1). E. coli ER2566 and pET-28a (Takara) and pACYC duet vectors
(Novagen) were used as host cells and expression vectors, respectively (Supporting
Information Table S1). The genes encoding linoleate 9S-LOX and EH of M. xanthus were
amplified by polymerase chain reaction (PCR) with the primers as shown in the Supporting
Information Table S2. The DNA fragments were cloned into pET-28a or the pACYC duet
vector. The plasmid was transformed into E. coli ER2566.
Culture Conditions. M. xanthus DK1622 was cultivated in a 500-mL flask containing
100 mL of Casitone medium at 30 °C with shaking at 200 rpm for 24 h. Recombinant E. coli
ER2566 was cultivated in a 2-L flask containing 500 mL Luria–Bertani medium
supplemented with 0.1 mM kanamycin for pET-28a vector or chloramphenicol for the
pACYC duet vector at 37 °C and shaken at 200 rpm. When the optical density of the bacterial
culture at 600 nm reached 0.6, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was
added, and incubation was continued at 16 °C with shaking at 150 rpm for 18 h to induce
enzyme expression.
Enzyme Purification. After cultivation, recombinant E. coli ER2566 cells were
harvested from culture broth by centrifugation at 6000×g for 30 min at 4 °C and washed with
0.85% NaCl by centrifugation at 6,000 g for 30 min at 4 °C. The washed cells were
suspended in 50 mM phosphate buffer (pH 7.0) containing 10 mM imidazole, 300 mM NaCl,
and 0.1 mM phenylmethylsulphonyl fluoride as a protease inhibitor and the suspended cells
were disrupted by sonication and kept on ice for 30 min. Cell debris was removed by
centrifugation at 13000×g for 20 min at 4 °C, and the supernatant was applied to an
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immobilized metal-ion affinity chromatography cartridge (GE Healthcare) equilibrated with
50 mM phosphate buffer (pH 8.0) containing 300 mM NaCl. The bound protein in the
cartridge was eluted by the same buffer with a linear gradient of 10−250 mM imidazole. The
active fractions were collected and dialyzed against 50 mM HEPPS (pH 8.5) buffer. After
dialysis, the resulting solution was used as the purified enzyme. The purification was
performed in a cold chamber at 4 °C using a fast protein liquid chromatography system (Bio-
Rad).
Specific Activity and Kinetic Parameters. The specific activity of the purified
linoleate 9S-LOX from M. xanthus for C18 PUFA substrates was determined by measuring
the increase in absorbance at 234 nm using a Beckman Coulter DU-700 spectrophotometer
after incubation at 35 °C in 50 mM HEPPS (pH 8.5) buffer containing 0.1 mM substrate and
3.0–6.0 μg mL−1 enzyme for 2 min. Only the part of each reaction showing a linear
correlation between product concentration and time was used, and the enzyme activity was
calculated with the extinction coefficient of 25000 M–1 cm–1 for conjugated fatty acid
products. The enzyme activity (U mg−1) was defined as the amount of product concentration
(µmol) per amount of protein (mg) per reaction time (min). To determine the kinetic
parameters, the reactions were conducted at 35 °C in 50 mM HEPPS (pH 8.5) buffer by
varying the concentration of C18 PUFA from 10 to 900 μM for 1 min, and the enzyme
activity was determined by measuring the absorbance. The kinetic parameters Km (mM) and
kcat (min−1) were determined by non-linear regression method using a GraphPad software
(See the Supporting Information, Figure S5). To calculate kcat, the amount of protein was
divided by the total molecular mass.
Biotransformation of C18 PUFAs into Plant C18 Oxylipins. The concentrations of
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cells and substrate for the maximal production of 9,10,11-trihydroxy-cis-12-octadecenoic
acid (4) were determined with 3 mM linoleic acid (1) by varying the cell concentration from
0.5 to 6 g L−1 and with 3 g L−1 cell by varying substrate concentration from 0.5 to 3.5 mM,
respectively. The cell concentration was measured from the optical density at 600 nm of the
cell suspension and converted into the dry cell weight using a linear calibration curve of the
optical density at 600 nm versus dry cell weight. The reactions for the biotransformation of
C18 PUFAs into plant C18 oxylipins by recombinant E. coli expressing only LOX or both
LOX and EH from M. xanthus were performed at 35 °C in 50 mM HEPPS (pH 8.5) buffer
containing 1−3 mM substrate and 3−15 g L cells for 60 and 90 min, respectively. The
reactions were conducted in 100-mL baffled flasks containing 10 mL reaction solution in an
incubator shaken at 200 rpm.
Quantification of the biotransformation products by HPLC analysis. The
reaction products were extracted using an equal volume of ethyl acetate, the solvent was then
removed with a rotary evaporator, and methanol was added to the dried extract. All fatty
acids and their derivatives were quantitatively analyzed using an HPLC system (Agilent) with
a UV detector at a wavelength of 202 nm and a reverse-phase Nucleosil C18 column
(3.2×150 mm, 5-μm particle size; Phenomenex). The column was eluted at 35 °C with a
gradient of solvent A (acetonitrile/water/acetic acid, 50:50:0.1, v/v/v) and solvent B
(acetonitrile/acetic acid, 100:0.1, v/v). CP HPLC was run using a Chiralcel OD-H column
(2.1 × 150 mm, 5-μm particle size; Daicel) with solvent system of n-hexane/2-propanol/acetic
acid (98: 2:0.1, v/v/v).
The concentrations of PUFAs, HFAs, EHFAs, and THFAs were calculated by calibrating
the peak areas to the concentrations of standards (Supporting Information Table S3 for
calibration curves). For an example, the calibration curve of linoleic acid using determination
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methods was provided in Supporting Information Figure S6. The concentrations of PUFAs,
including linoleic acid (1), α-linolenic acid (6), and γ-linolenic acid (11), and the HFA 9S-
hydroxy-trans,cis-10,12-octadecadienoic acid (5) were determined by commercially available
standards. The calibration curves of HFAs, including 12-hydroxy-cis,trans,cis-9,13,15-
octadecatrienoic acid (10) and 9-hydroxy-cis,trans,cis-6,10,12-octadecatrienoic acid (15),
were made after linoleate 9S-LOX from M. xanthus converted completely 6 and 11 into 10
and 15, without any byproducts, respectively. The reactions were performed in 50 mM
HEPES buffer (pH 7.5) containing 400 mM cysteine as a reducing agent and 1 mg mL−1
enzyme at 35 °C by varying the substrate concentration from 0.2 to 0.8 mM with 0.2 mM
increase for 90 min. The calibration curves of EHFAs were made after linoleate 9S-LOX
from M. xanthus converted completely PUFAs, including 1, 6, and 11, into HFAs, including
5, 10, and 15, and EHFAs, including 9,10-epoxy-11-hydroxy-cis-12-octadecenoic acid (3),
12,13-epoxy-14-hydroxy-cis,cis-9,15-octadecadienoic acid (8), and 9,10-epoxy-11-hydroxy-
cis,cis-6,12-octadecadienoic acid (13), without other byproducts, respectively. The
concentrations of EHFAs were calculated by subtracting the concentrations of HFAs from the
concentrations of PUFAs, which were known from the already made calibration curves. The
calibration curves of THFAs were made after linoleate 9S-LOX from M. xanthus converted
completely PUFAs, including 1, 6, and 11, into HFAs, including 5, 10, and 15, and THFAs,
including 9,10,11-trihydroxy-cis-12-octadecadienoic acid (4), 12,13,14-trihydroxy-cis,cis-
9,15-octadecadienoic acid (9), and 9,10,11-trihydroxy-cis,cis-6,12-octadecadienoic acid (14),
without other byproducts, respectively. The concentrations of THFAs were calculated by
subtracting the concentrations of HFAs from the concentrations of PUFAs because the
concentrations of HFAs and PUFAs were known from the already made calibration curves.
The concentrations of HpFAs were the same as those of HFAs. The reactions for EHFAs and
THFAs were performed in 50 mM HEPES buffer (pH 7.5) containing 1 mg mL−1 enzyme at
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35 °C by varying the substrate concentration from 0.2 to 0.8 mM with 0.2 mM increase for
120 min and 150 min, respectively. The purity of all products was estimated by the
calibration curves.
LC-MS/MS Analysis for the Identification of Products. LC-MS/MS analysis of
nine types of the biotransformation products plant C18 oxylipins, including HFAs, EHFAs,
and THFAs, was performed using a Thermo-Finnigan LCQ Deca XP Plus ion trap mass
spectrometer (Thermo Scientific). The instrument consisted of an LC pump, an auto sampler,
and a photodiode array detector. Ionization of the samples was carried out through
electrospray ionization at 15 V fragmentor voltage in negative mode, 46 V capillary voltage
in positive ionization mode, 0.01 min average scan time, 0.02 min average time to change
polarity, 5kV ion source voltage, nebulizer gas of 30 psi, 275 °C capillary temperature, and
35 % abundant precursor ions at collision energy.
NMR Analysis for the Identification of Product. The structure of compound 8 was
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identified by recording 1D (1H and C) and 2D (COSY, correlation spectroscopy; TOCSY,
total correlation spectroscopy; ROESY, rotating frame nuclear over-hauser effect
spectroscopy; HSQC, hetero-nuclear single-quantum correlation spectroscopy; HMBC,
hetero-nuclear multiple-bond correlation spectroscopy) NMR spectra on a Bruker Avance
HD (850 MHz) equipped with TCI cryoprobe (NCIRF, Seoul National University). CDCl3
was used as a solvent and TMS was used as an internal standard. All chemical shifts are
quoted in δ (ppm).
RESULTS AND DISCUSSION
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Gene Cloning, Expression, and Biochemical Properties of LOX and EH from
M. xanthus. Linoleate 9S-LOX, the same as arachidonate 11S-LOX, and EH from M.
xanthus DK1622, which had been already used in our previous study, were applied in the
biotransformation of C18 PUFAs into plant oxylipins. The genes encoding LOX (2,028 bp)
and EH (957 bp) from M. xanthus DK1622 were cloned and expressed in E. coli ER2566.
The recombinant LOX and EH, which were expressed from the pACYC duet vector, showed
two distinct bands in crude extract, having molecular masses of approximately 76 and 35
kDa, respectively (Supporting Information Figure S7). LOX, which was expressed from the
pET-28a vector, was purified as a soluble protein from crude extract through His-Trap
affinity chromatography, and showed a single band in sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) at a molecular mass of approximately 76
kDa,23 which is almost consistent with the molecular mass calculated from 675 amino acids
with the hexa-histidine tag.
The amino acid sequence of M. xanthus LOX exhibited 6%, 25%, and 34% identities with
the reported LOXs from Cyanothece sp.,24 Pseudomonas aeruginosa,25 and humans,26
respectively. Interestingly, M. xanthus LOX exhibited the highest identity to human LOX but
a low identity to bacterial LOXs. Thus, M. xanthus LOX probably converts PUFAs to EHFAs
by a two-step reaction in a similar way to human LOX. The amino acid sequence of M.
xanthus EH showed 21% identity to that of human soluble EH (EH2).27
Catalytic Activity of LOX from M. xanthus. To identify the reaction products of M.
xanthus LOX, the purified enzyme was reacted with C18 PUFAs. The total molecular masses
of the reaction products obtained from the conversion of linoleic acid (cis,cis-9,12-
octadecadienoic acid) (1), α-linolenic acid (cis,cis,cis -9,12,15-octadecatrienoic acid) (6), and
γ-linolenic acid (cis,cis,cis-6,9,12-octadecatrienoic acid) (11) are represented by peaks at
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mass-to-charge ratios (m/z) of 295.3, 293.2, and 293.2, respectively, in the liquid
chromatography (LC)-tandem mass spectrometry (MS/MS) spectra. A peak at m/z 171.2 for
the product obtained from the conversion of 1 resulted from the cleavage of the hydroxyl
group at the C9 position (Figure 2A). The peaks at m/z 183.1 and 211.0 for the product of 6
resulted from the cleavage of the hydroxyl group at the C12 position (Figure 2B). The peaks
at m/z 141.1 and 169.0 for the product of 11 resulted from the cleavage of the hydroxyl group
at the C9 position (Figure 2C). Based on the LC-MS/MS data, the reaction products were
suggested as 9-hydroxy-trans,cis-10,12-octadecadienoic acid (5), 12-hydroxy-cis,trans,cis-
9,13,15-octadecatrienoic acid (10), and 9-hydroxy-cis,trans,cis-6,10,12-octadecatrienoic acid
(15), respectively.
The chirality of 5, which was obtained from the conversion of 1 by M. xanthus LOX, was
identified as the S-form by chiral-phase (CP) high-performance liquid chromatography
(HPLC) analysis with the 9S- and 9R- standards because it showed the same retention time as
the 9S-standard (Supporting Information Figure S8). The specific activity and catalytic
efficiency (kcat/Km) of LOX from M. xanthus were the highest for 1, followed by 6 and 11
(Table 1). These results indicated that LOX from M. xanthus is a linoleate 9S-LOX. 9S-LOX
from M. xanthus showed high catalytic efficiency for C20 and C22 PUFAs, including
arachidonic acid, EPA and DHA, as arachidonate 11S-LOX.28 The proteobacterium M.
xanthus had two LOXs, and the other LOX, known as arachidonate 12S-LOX,29 was
identified as linoleate 13S-LOX. Most of bacterial LOXs were found only several gram-
negative bacteria. LOXs from cyanobacteria including Nostoc sp.,30 Anabaena sp.,31 and
Acaryochloris marina,32 are linoleate 9R-LOXs, whereas LOXs of two strains in
proteobacteria, P. aeruginosa33 and Burkholderia thailandensis,34 are linoleate 13S-LOX.
Therefore, this is the first 9S-form of bacterial linoleate LOXs.
Unfortunately, M. xanthus linoleate 9S-LOX has lower turnover number as 7.2 s−1 for
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linoleic acid than those of the previously reported bacterial LOXs. Linoleate 13S-LOX, which
are reported as arachidonic 12S-LOX form M. xanthus LOX, showed 9.2 s−1 turnover number,
showed 1.3-fold higher turnover number29 and linoleate 13S-LOXs from P. aeruginosa35 and
B. tailandensis34 belonging to the same proteobacteria showed 2.8- and 13.0-fold higher
turnover number than that of M. xanthus linoleate 9S-LOX. Also, linoleate 9R-LOX, other
stereochemical LOX from cyanobacterium A. marina shown 13.4 s−1, showing 1.9-folds
higher.32 However, this is the first 9S-form of bacterial linoleate LOXs although the activity
was lower than those of the reported LOXs.
Biotransformation of PUFAs (1, 6, 11) into HFAs (5, 10, 15). The specific
productivity of recombinant E. coli to produce HFAs using PUFAs during the production of
EHFAs was higher than that during the production of THFAs (Table 2). Thus, only the
biotransformation activity of PUFAs to HFAs during the production of EHFAs is described
in this sections.
Linoleic acid (1) was transformed into 9S-hydroxy-trans,cis-10,12-octadecenoic acid (5)
via 9S-hydroperoxy-trans,cis-10,12-octadecadienoic acid (2) by linoleate 9S-LOX from M.
xanthus, which had been expressed in E. coli ER2566, and 2 was readily reduced to 5 without
reductant (Figure 1A). The 5 was produced to a concentration of 0.28 mM in the reaction
medium from 1 mM linoleic acid with a specific and volumetric productivity of 123 μmol h−1
g-cells−1 and a 370 μM h−1, respectively (Figure 3A). The isolated yield of 5 reached 28%
with a purity of 99% (Table 2). Although the conversion of 1 to 5 by LOXs of plants and
fungi has been reported, the compound 5 could be prepared by using the bacterial 9S-LOX.
The biosynthesis of 12-hydroxyoctadecatrienoic acid was very rare in nature. Among
bacterial LOXs, linoleate 9R-LOX from A. marina converts α-linolenic acid (6) into 12R-
hydroxy-cis,trans,cis-9,13,15-octadecatrienoic acid as a stereoisomer,32 whereas M. xanthus
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linoleate 9S-LOX converted 6 into 12S-hydroxy-cis,trans,cis-9,13,15-octadecatrienoic acid
(10) (Figure 1B). Recombinant E. coli at 9 g L−1 produced 0.35 mM 10 from 1 mM 6 via
12S-hydroperoxy-cis,trans,cis-9,13,15-octadecatrienoic acid (7) in 60 min (Figure 3B).
Recombinant E. coli at 15 g L−1 converted 1 mM γ-linolenic acid (11) into 0.60 mM 9S-
hydroxy-cis,trans,cis-6,10,12-octadecatrienoic acid (15) via 9S-hydroperoxy-cis,trans,cis-
6,10,12-octadecatrienoic acid (12) in 45 min (Figure 1C and 3C). CYP443D1 (CYP74 family)
from Nematostella vectensis, a sea anemone, has been shown to convert 11 into 15. However,
no quantitative data was shown.36 The yield and specific productivity of the recombinant E.
coli expressing LOX for the production of HFAs followed the orders 11 > 6 > 1 and 1 > 11 >
6, respectively (Table 2).
Biotransformation of PUFAs (1, 6, 11) into EHFAs (3, 8, 13). EHFAs were
qualitatively identified in mostly plants and marine organisms and were converted from C18
PUFAs by epoxy alcohol synthase, kind of CYP74 family. However, the biotransformation of
PUFAs into EHFAs with two-step reaction by bacterial LOX has not been reported to date.
Thus, linoleate 9S-LOX from M. xanthus was used for the production of EHFAs from C18
PUFAs.
9,10-Epoxy-11-hydroxy-cis-12-octadecenoic acid (3) has been qualitatively identified from
the reaction product of linoleic acid (1) by epoxy alcohol synthase (CYP74-related enzyme)
from the brown algae Ectocarpus siliculosus, However, the quantitative production of 3 has
not been reported. Thus, the quantitative production of 3 by recombinant E. coli expressing M.
xanthus 9S-LOX was attempted. The cells converted 1 mM 1 into 0.72 mM 3 (Figure 4A) via
9-hydroperoxy-trans,cis-10,12-octadecadienoic acid (2) in 45 min with a specific
productivity of 321 μmol h−1 g-cells−1, a productivity of 960 μM h−1, and a purity of 97%
(Figure 3A and Table 2). α-Linolenic acid (6) (1 mM) was converted into 0.65 mM 12,13-
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epoxy-14-hydroxy-cis,cis-9,15-octadecadienoic acid (8) (Figure 4B) via 7 in 60 min by
recombinant E. coli (Figure 3B). Compound 8 is a new compound, and thus the structure was
additionally identified through NMR analysis (Supporting Information Figure S9−S11). γ-
Linolenic acid (11) (1 mM) was converted into 0.40 mM 9,10-epoxy-11-hydroxy-cis,cis-
6,12-octadecadienoic acid (13) (Figure 4C) by recombinant E. coli via 9-hydroperoxy-
cis,trans,cis-6,10,12-octadecatrienoic acid (12) in 45 min (Figure 3C). The CYP74 family
from N. vectensis also converts 11 into 13.36 The yield and specific productivity of
recombinant E. coli for EHFAs described in Table 2.
Biotransformation of PUFAs (1, 6, 11) into THFAs (4, 9, 14). P. aeruginosa
converts 35.6 mM linoleic acid into total 13.3 mM of 9,10,13- and 9,12,13-trihydroxy-cis-11-
octadecenoic acid from after 72 h fermentation with a productivity of 184 μmol h−1 and a
yield of 37%.37 Clavibacter sp. converts 25.3 mM linoleic acid into 6.1 mM 12,13,17-
trihydroxy-trans-9-octadecenoic acid in 140 h fermentation with a productivity of 43 μmol
h−1 and a yield of 24%.21,22 However, the enzymes involved in the production of these
THFAs were not determined. The bacteria produced 9,10,13-, 9,12,13-, and 12,13,17-THFAs,
whereas recombinant E. coli expressing linoleate 9S-LOX and EH from M. xanthus produced
9,10,11- and 12,13,14-THFAs.
The time course reactions for the biotransformation of C18 PUFAs into THFAs were
performed using recombinant E. coli expressing linoleate 9S-LOX and EH from M. xanthus.
The conversion of THFA from EHFA by EH was confirmed by the reactions of recombinant
E. coli expressing linoleate 9S-LOX with and without EH under the same conditions
(Supporting Information Figure S12). Recombinant E. coli at 3 g L−1 converted 1 mM
linoleic acid (1) into 0.75 mM 9,10,11-trihydroxy-cis-12-octadecadienoic acid (4) (Figure 5A)
via 9-hydroperoxy-trans,cis-10,12-octadecadienoic acid (2) and 9,10-epoxy-11-hydroxy-cis-
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12-octadecenoic acid (3) sequentially in 60 min with a specific productivity of 250 μmol h−1
g-cells−1 and a purity of 99% (Figure 6A and Table 2). Although 4 has been isolated from
Boehmeria nivea root and potato leaf,11,12 this is the first quantitative biotransformation. The
concentrations of cells and substrate for the production of 4 were determined to be 3 g L−1
cells and 3 mM 1 (Supporting Information Figure S13). Under these conditions, 3 mM 1 was
converted into 2.11 mM 4 in 90 min by 3 g L−1 recombinant E. coli with a specific
productivity of 469 μmol h−1 g-cells−1, a productivity of 1,410 μM h−1, and a yield of 70%
(Figure 7 and Table 2), which were 2.0- and 1.9-fold increases and 5% decrease, compared to
those of 1 mM 1. The productivity and yield of recombinant E. coli for 9,10,11-trihydroxy-
cis-12-octadecadienoic acid (4) were 7.7-times and 33% higher than those of P. aeruginosa
for 9,10,13- and 9,12,13-trihydroxy-cis-11-octadecenoic acid, respectively, which showed the
previously highest productivity and yield. The recombinant E. coli at 9 g L−1 converted 1 mM
α-linolenic acid (6) into 0.63 mM 12,13,14-trihydroxy-cis,cis-9,15-octadecadienoic acid (9)
(Figure 5B) via 12-hydroperoxy-cis,trans,cis-9,13,15-octadecatrienoic acid (7) and 12,13-
epoxy-14-hydroxy-cis,cis-9,15-octadecadienoic acid (8) sequentially in 90 min (Figure 6B).
Recombinant E. coli at 15 g L−1 converted 1 mM γ-linolenic acid (11) into 0.29 mM 9,10,11-
trihydroxy-cis,cis-6,12-octadecadienoic acid (14) (Figure 5C) in 90 min via 9-hydroperoxy-
cis,trans,cis-6,10,12-octadecatrienoic acid (12) and 9,10-epoxy-11-hydroxy-cis,cis-6,12-
octadecadienoic acid (13) sequentially (Figure 6C and Table 2). The yield and specific
productivity of recombinant E. coli for THFAs followed the order 1 > 6 > 11. Compounds 9
and 14 have not been reported to date and, thus, are newly synthesized in this study. However,
unfortunately, two of new THFA compounds were not analyzed by NMR because a sufficient
amount with high purity was not secured for NMR analysis.
In conclusion, plant C18 oxylipins with antifungal activity have not been commercialized
because studies of their quantitative biotransformation and function have little been carried
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out. In the present study, E. coli expressing linoleate 9S-LOX and EH from M. xanthus were
used to synthesize plant C18 oxylipins, including HFAs, EHFAs, and THFAs, from C18
PUFAs. This is the first 9S-form of bacterial linoleate LOXs although the activity was lower
than those of the reported LOXs. The biosynthesis of plant EHFAs and THFAs by microbial
enzymes has been demonstrated for the first time. Moreover, the productivity and yields of
the recombinant E. coli expressing enzymes for the production of THFAs were significantly
higher than those of other bacteria. Our results will contribute to the industrial production of
plant oxylipins from C18 PUFAs.
ASSOCIATED CONTENT
Supporting Information
Tables S1: Strains and plasmids used in this study. Table S2: Primers designed for PCR
analysis. Table S3: Calibration curves of all fatty acids and their derivatives in this study.
Figure S1: HPLC profile of the standard and purified compounds of 9-HODE. Figure S2:
(A) HPLC profiles of products obtained from the conversion of linoleic acid (LA). (B)
HPLC profile of purified 9-hydroxy-trans,cis-10,12-octadecadienoic acid (9-HODE). (C)
HPLC profile of purified 9,10-epoxy-11-hydroxy-cis-12-octadecenoic acid (9,10-EP-11-
HOD). (D) HPLC profile of purified 9,10,11-trihydroxy-cis-12-octadecenoic acid (9.10.11-
THOD). Figure S3: HPLC profiles of the purified products obtained from the conversion of
α-linolenic acid. (A) HPLC profile of products obtained from the conversion of α-linolenic
acid (ALA). (B) HPLC profile of purified 12-hydroxy-cis,trans,cis-9,13,15-octadecatrienoic
acid (12-HOTrE). (C) HPLC profile of purified 12,13-epoxy-14-hydroxy-cis,cis-9,15-
octadecadienoic acid (12,13-EP-14-HODE). (D) HPLC profile of purified 12,13,14-
trihydroxy-cis,cis-9,15-octadecadienoic acid (12,13,14-THODE). Figure S4: (A) HPLC
profiles of products obtained from the conversion of γ-linolenic acid (GLA). (B) HPLC
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profiles of purified 9-hydroxy-cis,trans,cis-6,10,12-octadecatrienoic acid (9-HOTrEγ). (C)
HPLC profiles of purified 9,10-epoxy-11-hydroxy-cis,cis-6,12-octadecadienoic acid (9,10-
EP-11-HODE). (D) HPLC profiles of purified 9,10,11-trihydroxy-cis,cis-6,12-
octadecadienoic acid (9,10,11-THODE). Figure S5: Determination for kinetics parameter of
M. xanthus LA 9S-LOX by non-linear regression method using a GraphPad software. (A)
LA (B) ALA (C) GLA. Figure S6: Determination for the concentration of LA by HPLC. (A)
HPLC profile of LA standard at each concentration ranging from 0.2 to 0.8 mM. (B)
Calibration curve of LA standard between peak area and concentration. Figure S7: SDS-
PAGE analysis of recombinant proteins, linoleate 9S-LOX and EH, expressed in
recombinant E. coli ER2566. Figure S8: CP HPLC analysis of the reaction product obtained
from the conversion of linoleic acid. Figure S9: The structure of 12,13-EP-14-HODE. (A)
Chemical structure of (Z)-11-((2S,3R)-3-((R,Z)-1-hydroxypent-2-en-1-yl)oxiran-2-yl)undec-
9-enoic acid. (B) Chemical structure of (Z)-11-((2S,3R)-3-((S,Z)-1-hydroxypent-2-en-1-
1
yl)oxiran-2-yl)undec-9-enoic acid. Figure S10: NMR data of 12,13-EP-14-HODE. (A) H
NMR peak of compound 8. (B) 13C NMR peak of compound 8. Figure S11: 2D NMR data
of 12,13-EP-14-HODE. (A) COSY spectrum of compound 8. (B) TOCSY spectrum of
compound 8. (C) ROESY spectrum of compound 8. (D) HSQC spectrum of compound 8.
(E) HMBC spectrum of compound 8. Figure S12: HPLC profiles of the reaction of PUFA
(linoleic acid) into THFA (9,10,11-THOD, 9,10,11-trihydroxy-cis-12-octadecenoic acid) by
E. coli expressing 9S-LOX with and without EH. Figure S13: Determination of the
concentrations of cells and substrate for the biotransformation of 9,10,11-trihydroxy-cis-12-
octadecenoic acid from linoleic acid by recombinant E. coli expressing linoleate 9S-LOX
and EH from M. xanthus. (A) Determination of cell concentration. (B) Determination of
substrate concentration.
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AUTHOR INFORMATION
Corresponding Author
* *Phone: (822) 454-3118. Fax: (822) 444-5518. E-mail: deokkun@konkuk.ac.kr
ORCID
Deok-Kun Oh: 0000-0002-6886-7589
Funding
This study was supported by the Mid-Career Researcher Program through the National
Research Foundation grant funded by the Ministry of Science and ICT, Republic of Korea
(No. 2019R1A2C3002781).
Notes
The
authors
declare
no
competing
financial
interest.
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Figure captions
Figure 1. Biotransformation pathways of C18 PUFAs into plant C18 oxylipins. (A) Linoleic
acid (1) is converted into 9-hydroperoxy-trans,cis-10,12-octadecadienoic acid (2) by linoleate
9S-LOX, which is sequentially converted into 9,10-epoxy-11-hydroxy-cis-12-octadecenoic
acid (3) and 9,10,11-trihydroxy-cis-12-octadecenoic acid (4) by linoleate 9S-LOX and EH.
Compound 2 is transformed into 9-hydroxy-trans,cis-10,12-octadecadienoic acid (5) via auto
reduction. (B) α-Linolenic acid (6) is converted into 12-hydroperoxy-cis,trans,cis-9,13,15-
octadecatrienoic acid (7) by linoleate 9S-LOX, which is sequentially converted into 12,13-
epoxy-14-hydroxy-cis,cis-9,15-octadecadienoic acid (8) and 12,13,14-trihydroxy-cis,cis-
9,15-octadecadienoic acid (9) by linoleate 9S-LOX and EH, respectively. Compound 7 is
transformed into 12-hydroxy-cis,trans,cis-9,13,15-octadecatrienoic acid (10) via auto
reduction. (C) γ-Linolenic acid (11) is converted into 9-hydroperoxy-cis,trans,cis-6,10,12-
octadecatrienoic acid (12) by linoleate 9S-LOX, which is sequentially converted into 9,10-
epoxy-11-hydroxy-cis,cis-6,12-octadecadienoic acid (13) and 9,10,11-trihydroxy-cis,cis-
6,12-octadecadienoic acid (14) by linoleate 9S-LOX and EH. Compound 12 is transformed
into 9-hydroxy-cis,trans,cis-6,10,12-octadecatrienoic acid (15) via auto reduction.
Compounds 8, 9, and 14 are newly discovered in this study.
Figure 2. LC-MS/MS analysis of the HFA products obtained from the conversion of C18
PUFAs. The reactions were carried out using recombinant E. coli ER2566 containing
linoleate 9S-LOX from M. xanthus. (A) LC-MS/MS fragments of the HFA obtained from the
conversion of linoleic acid (1). The HFA was identified as 9-hydroxy-trans,cis-10,12-
octadecadienoic acid (5). (B) LC-MS/MS fragments of the HFA obtained from the
conversion of α-linolenic acid (6). The HFA was identified as 12-hydroxy-cis,trans,cis-
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9,13,15-octadecatrienoic acid (10). (C) LC-MS/MS fragments of the HFA obtained from the
conversion of γ-linolenic acid (11). The HFA was identified as 9-hydroxy-cis,trans,cis-
6,10,12-octadecatrienoic acid (15). The asterisks (*) and numbers underlined with red
indicate the molecular masses of key fragments and total molecular mass of the compound,
respectively, used for the identification of the structure.
Figure 3. Time-course reactions for the biotransformation of C18 PUFAs into EHFAs by
recombinant E. coli ER2566:pET28a-MXLOX. (A) Biotransformation of linoleic acid (1)
into 9,10-epoxy-11-hydroxy-cis-12-octadecenoic acid (3). (B) Biotransformation of α-
linolenic acid (6) into 12,13-epoxy-14-hydroxy-cis,cis-9,15-octadecadienoic acid (8). (C)
Biotransformation of γ-linolenic acid (11) into 9,10-epoxy-11-hydroxy-cis,cis-6,12-
octadecadienoic acid (13). The biotransformation was performed in 50 mM HEPPS buffer
(pH 8.5) containing 3−15 g L−1 cells and 1 mM PUFA at 35 °C for 60 min. The data
represent the mean of three experiments, and the error bars represent the standard deviations.
The symbols indicate the concentrations of PUFAs (1, 6, and 11) (●), HpFAs (2, 7, and 12)
(■), HFAs (5, 10, and 15) (▲), and EHFAs (3, 8, and 13) (♦).
Figure 4. LC-MS/MS analysis of EHFA products obtained from the conversion of C18
PUFAs. The reactions were carried out using recombinant E. coli ER2566 containing
linoleate 9S-LOX from M. xanthus. (A) LC-MS/MS fragments of the EHFA obtained from
the conversion of linoleic acid (1). The EHFA was identified as 9,10-epoxy-11-hydroxy-cis-
12-octadecenoic acid (3). (B) LC-MS/MS fragments of the epoxy hydroxy fatty acid obtained
from the conversion of α-linolenic acid (6). The EHFA was identified as 12,13-epoxy-14-
hydroxy-cis,cis-9,15-octadecadienoic acid (10). (C) LC-MS/MS fragments of the EHFA
obtained from the conversion of γ-linolenic acid (11). The EHFA was identified as 9,10-
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epoxy-11-hydroxy-cis,cis-6,12-octadecadienoic acid (15). The asterisks (*) and numbers
underlined in red indicate the molecular masses of key fragments and the total molecular
mass of the compound, respectively, used for structure identification.
Figure 5. LC-MS/MS analysis of THFA products obtained from the conversion of C18
PUFAs. The reactions were carried out using recombinant E. coli ER2566 containing
linoleate 9S-LOX and EH from M. xanthus. (A) LC-MS/MS fragments of THFA obtained
from the conversion of linoleic acid (1). The THFAwas identified as 9,10,11-trihydroxy-cis-
12-octadecenoic acid (4). (B) LC-MS/MS fragments of THFA obtained from the conversion
of α-linolenic acid (6). The THFA was identified as 12,13,14-trihydroxy-cis,cis-9,15-
octadecadienoic acid (10). (C) LC-MS/MS fragments of THFA obtained from the conversion
of γ-linolenic acid (11). The THFA was identified as 9,10,11-hydroxy-cis,cis-6,12-
octadecadienoic acid (15). The asterisks (*) and numbers underlined in red indicate the
molecular masses of key fragments and the total molecular mass of the compound,
respectively, used for structure identification.
Figure 6. Time-course reactions for the biotransformation of C18 PUFAs into THFAs by
recombinant E. coli ER2566:pACYC-MXLOX-MXEH. (A) Biotransformation of linoleic
acid (1) into 9,10,11-trihydroxy-cis-12-octadecenoic acid (4). (B) Biotransformation of α-
linolenic acid (6) into 12,13,14-trihydroxy-cis,cis-9,15-octadecadienoic acid (9). (C)
Biotransformation of γ-linolenic acid (11) was converted into 9,10,11-trihydroxy-cis,cis-6,12-
octadecadienoic acid (14). The biotransformation was performed in 50 mM HEPPS buffer
(pH 8.5) containing 3−15 g L−1 cells and 1 mM PUFA at 35 °C for 90 min. The data
represent the mean of three experiments, and the error bars represent the standard deviations.
The symbols indicate the concentrations of PUFAs (1, 6, and 10) (●), HpFAs (2, 7, and 11)
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(■), HFAs (5, 10, and 15) (▲), EHFAs (3, 8, and 13) (♦), and THFAs (4, 9, and 14) (▼).
Figure 7. Time-course reactions for the biotransformation of linoleic acid (1) into 9,10,11-
trihydroxy-cis-12-octadecenoic acid (4) by recombinant E. coli ER2566:pACYC-MXLOX-
MXEH. The biotransformation was performed in 50 mM HEPPS buffer (pH 8.5) containing
3 g L−1 cells and 3 mM linoleic acid at 35 °C for 90 min. The data represent the mean of three
experiments, and error bars represent the standard deviations. The symbols indicate the
concentrations of linoleic acid (1) (●), 9-hydroperoxy-trans,cis-10,12-octadecadienoic acid (2)
(■), 9-hydroxy-trans,cis-10,12-octadecadienoic acid (5) (▲), 9,10-epoxy-11-hydroxy-cis-12-
octadecenoic acid (3) (♦), and 9,10,11-trihydroxy-cis-12-octadecenoic acid (4) (▼).
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Table 1. Specific activity and kinetic parameters of M. xanthus LOX for C18 PUFAs
Specific activityb
kcat/Km
(min−1 mM−1)
c
Km
kcat
Substrate
Producta
(mM) (min−1)
0.330 477.1
−1
(U mg )
Linoleic acid
(C18:2Δ9,12/ω-6
9-Hydroperoxy-trans,cis-10,12-octadecadienoic
acid
12-Hydroperoxy-cis,trans,cis-9,13,15-
octadecatrienoic acid
9-Hydroperoxy-cis,trans,cis-6,10,12-
octadecatrienoic acid
11.4 ± 0.2
3.4 ± 0.1
1.9 ± 0.3
1,446
)
α-Linolenic acid
0.081
0.073
76.1
48.4
940
663
(C18:3Δ9,12,15/ω-6
)
γ-Linolenic acid
(C18:3Δ6,9,12/ω-3
)
aThese products were the first-step reaction products of M. xanthus linoleate 9S-LOX.
bUnit is micromoles per minute (μmol min−1).
cThe kinetics parameters were calculated by nonlinear regression analysis.
Data represent the mean of three experiments, and the error bars represent the standard deviations.
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