Biotransformation of linoleic acid into hydroxy fatty acids and carboxylic acids using a linoleate double bond hydratase as key enzyme

Article abstract of DOI:10.1002/adsc.201400893

Hydroxy fatty acids are used as starting materials for the production of secondary metabolites and signalling molecules as well as in the manufacture of industrial fine chemicals. However, these compounds are usually difficult to produce from renewable biomass by chemical means. In this study, linoleate double bond hydratases of Lactobacillus acidophilus NBRC 13951 were cloned for the first time. These enzymes were highly specific for the hydration of the C-9 or the C-12 double bond of unsaturated fatty acids (e.g., linoleic acid). Thereby, the enzymes allowed the selective production of hydroxy fatty acids such as 13-hydroxy- cis -9-octadecenoic acid and 10-hydroxy- cis -12-octadecenoic acid from linoleic acid. In addition, the hydroxy fatty acids were further converted into industrially relevant carboxylic acids (e.g., 12-hydroxy-cis-9-dodecenoic acid, a, w-tridec-9-enedioic acid) and lactones (i.e., d-decalactone, g-dodecelactone) via whole-cell biocatalysis using an enzyme cascade. This study thus contributes to the preparation of hydroxy fatty acids, unsaturated carboxylic acids, and lactones from renewable unsaturated fatty acids.

Full text of DOI:10.1002/adsc.201400893

FULL PAPERS
DOI: 10.1002/adsc.201400893
Very Important Publication
Biotransformation of Linoleic Acid into Hydroxy Fatty Acids and
Carboxylic Acids Using a Linoleate Double Bond Hydratase as
Key Enzyme
+
a
+b
b
b
a
Hye-Jin Oh, Sae-Um Kim, Ji-Won Song, Jung-Hoo Lee, Woo-Ri Kang,
a
a
c
a,
Ye-Seul Jo, Kyoung-Rok Kim, Uwe T. Bornscheuer, Deok-Kun Oh, *
and Jin-Byung Park *
b,
a
Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea
E-mail: deokkun@konkuk.ac.kr
Department of Food Science and Engineering, Ewha Womans University, Seoul 120-750, Republic of Korea
b
Fax : (+82)-2-3277-4213; phone: (+82)-2-3277-4509; e-mail: jbpark06@ewha.ac.kr
Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, 17487 Greifswald.
Germany
c
+
H.-J. Oh and S.-U. Kim equally contributed to this work.
Received: September 10, 2014; Revised: November 21, 2014; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201400893.
Abstract: Hydroxy fatty acids are used as starting acid and 10-hydroxy-cis-12-octadecenoic acid from li-
materials for the production of secondary metabo- noleic acid. In addition, the hydroxy fatty acids were
lites and signalling molecules as well as in the manu- further converted into industrially relevant carboxyl-
facture of industrial fine chemicals. However, these ic acids (e.g., 12-hydroxy-cis-9-dodecenoic acid, a,w-
compounds are usually difficult to produce from re- tridec-9-enedioic acid) and lactones (i.e., d-decalac-
newable biomass by chemical means. In this study, li- tone, g-dodecelactone) via whole-cell biocatalysis
noleate double bond hydratases of Lactobacillus using an enzyme cascade. This study thus contributes
acidophilus NBRC 13951 were cloned for the first to the preparation of hydroxy fatty acids, unsaturat-
time. These enzymes were highly specific for the hy- ed carboxylic acids, and lactones from renewable un-
dration of the C-9 or the C-12 double bond of unsa- saturated fatty acids.
turated fatty acids (e.g., linoleic acid). Thereby, the
enzymes allowed the selective production of hydroxy Keywords: carboxylic acids; enzyme catalysis; hydra-
fatty acids such as 13-hydroxy-cis-9-octadecenoic tion; hydroxy fatty acids; lactones
Introduction
cants, polymers, and are used as additives in coatings
and paintings.
A variety of hydroxy fatty acids were reported to
[
3–6]
Hydroxy fatty acids, which contain one or more hy-
droxy groups in the carbon skeleton of fatty acids, are be produced by various microbes including lactic acid
found as components of cerebrosides, triacylglycerols, bacteria and ruminal microorganisms. 10-Hydroxy-
waxes, and other essential lipids in living organisms.
ACHTUNGTRENNGNUs tearic acid was produced from oleic acid by myosin
Hydroxy fatty acids are also used as starting materials cross-reactive antigen proteins from Elizabethkingia
[
7]
[8]
for the production of diverse secondary metabolites meningoseptica, Streptococcus pyogenes, and Bifi-
and signalling molecules.
[
1,2]
[9]
For instance, 10-hydroxy- dobacterium breve. Linoleic acid was converted to
ACHTUNGTRENNUNGo ctadecenoic acid, 9- and 13-hydroxyoctadecadienoic 10-hydroxy-12-octadecenoic acid by Lactobacillus
[
10]
[11]
acids, and leukotriene B4 are metabolized to lactones acidophilus AKU1137, L. plantarum, and Strep-
[
12]
through the b-oxidation pathway. In the chemical in- tococcus bovis. Linoleic acid was also transformed
dustry, hydroxy fatty acids are widely used to manu- to 13-hydroxy-9-octadecenoic acid and/or 10,13-dihy-
facture flavors, resins, waxes, nylons, plastics, lubri- droxyoctadecanoic acid by L. acidophilus, Pediococ-
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Scheme 1. Linoleic acid biotransformation pathway. Linoleic acid (1) is converted into 13-hydroxy-cis-9-octadecenoic acid
3) by a linoleate C-12 double hydratase, which is further converted into n-hexanoic acid (7) and 12-hydroxydodec-9-enoic
acid (8) or n-pentanol (9) and a,w-tridec-9-enedioic acid (10) by the multistep enzyme reactions. 13-Hydroxy-cis-9-octadec-
enoic acid (3) and 10-hydroxy-cis-12-octadecenoic acid (2) could be also transformed into d-decalactone (11) and g-dodece-
lactone (12) via whole-cell biocatalysis.
(
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
ACHTUNGTRENNUNG
[
13]
[14]
cus pentosaceus, Pediococcus sp. AKU 1080, and lactone (12) via whole-cell biocatalysis using wild-
[
15]
ruminal strain S. bovis JB1.
type yeast cells. 13-Hydroxy-cis-9-octadecenoic acid
Most of the hydroxy fatty acids produced by mi- was also converted to 12-hydroxy-cis-9-dodecenoic
crobes are considered to be synthesized by fatty acid acid (8) and n-hexanoic acid (7) via enzymatic cas-
double bond hydratases (e.g., oleate hydratase), cade reactions (Scheme 1), which was based on our
[
19]
which catalyze the regiospecific, irreversible addition previous study.
of a hydrogen atom and a hydroxy group from water
to the carbon-carbon cis-double bond of unsaturated
[
2,7]
fatty acids.
Until now, a number of C-9 double Results and Discussion
bond hydratases (e.g., myosin cross-reactive antigen
proteins from E. meningoseptica, S. pyogenes, B. Cloning, Expression, and Purification of Putative
[
7]
[8]
[9]
[16]
breve, L. acidophilus,
oleate hydratases from S. Fatty Acid Hydratases
[
17]
[18]
maltophilia,
cloned and characterized in detail, whereas C-12 Screening of a novel putative linoleate double bond
double bond hydratases remained unidentified. hydratase was based on known bacterial genome se-
Macrococcus caseolyticus
)
were
In this study, we cloned linoleate double bond hy- quences. The amino acid sequences of the fatty acid
dratases of L. acidophilus NBRC 13951, which was C-9 double bond hydratases (see the Supporting In-
known to produce 13-hydroxyoctadec-9-enoic acid (3) formation, Table S2 for details) were used as probes
[
13]
from linoleic acid, for the first time. The enzymes to identify putative linoleate double bond hydratases.
were very specific to hydration of the C-9 or the C-12 It was found that 70 genomes appeared to harbor at
double bond of linoleic acid and thereby allowed the least two different genes encoding putative linoleate
selective production of 10-hydroxy-cis-12-octadeceno- double bond hydratases out of 1,851 bacterial ge-
ic acid (2) or 13-hydroxy-cis-9-octadecenoic acid (3) nomes examined (Supporting Information, Table S3).
from linoleic acid. These hydroxy fatty acids were fur- Notably, 9 of the 70 genomes belonged to L. acido-
ther transformed to d-decalactone (11) and g-dodece- philus strains including the strain L. acidophilus
2
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Biotransformation of Linoleic Acid into Hydroxy Fatty Acids
NBRC 13951, which was known to produce 13-hy- DNA of L. acidophilus NBRC 13951. These genes
[
13]
droxyoctadec-9-enoic acid from linoleic acid (Sup- were cloned followed by expression of the putative
porting Information, Table S4 and Figure S1). Next, hydratases in Escherichia coli ER2566. The resulting
the primers were designed to clone the putative lino- soluble enzymes were purified by His-Trap affinity
leate double bond hydratase genes from L. acidophi- chromatography (Supporting Information, Figure S2)
lus NBRC 13951 (Supporting Information, Table S5). and gave single bands in SDS-PAGE at the molecular
This led to the identification of two genes (accession weight of approximately 68 kDa. The amino acid se-
numbers KJ560553, KJ560554) from the genomic quences of the putative linoleate hydratases
(
AHW98239.1, AHW98240.1) from L. acidophilus
NBRC 13951 were 57% identical to each other and
similar to oleate hydratase of Lysinibacillus fusiformis
KCTC 3454 (identity: ca. 60%) (Supporting Informa-
tion, Table S6 and Figure S1).
Catalytic Activity of the Putative Fatty Acid
Hydratases
The substrate specificity of the purified enzymes
AHW98239.1 and AHW98240.1 for fatty acids was
investigated using oleic acid (cis-9-octadecenoic acid)
and linoleic acid (cis-9,12-octadecadienoic acid). Ad-
dition of the purified enzyme AHW98239.1 into
5
1
0 mM citrate-phosphate buffer (pH 7.0) containing
0 mM linoleic acid resulted in accumulation of
a single product in the medium (Figure 1a), whereas
no product was observed from the oleic acid biotrans-
formation. The resulting product was isolated and
identified via GC/MS analysis (Supporting Informa-
tion, Figures S3 and S4). It turned out to be 13-hy-
droxy-cis-9-octadecenoic acid (3). The concentration
of 13-hydroxy-cis-9-octadecenoic acid in the reaction
medium increased up to 8 mM within 3 h. The maxi-
mal specific enzyme reaction rate reached 127 Ug
À1
protein
at t<0.5 h. The enzymatic conversion,
which was calculated based on the substrate depletion
and the product concentration determined by GC/MS,
reached 96%. The isolated yield, measured based on
amount of the products isolated and amount of the
substrates added into the reaction medium, was ca.
60% having a purity of 96% (Supporting Information,
Figure 1. Time course of the biotransformations. (a) Linoleic
acid (1) was converted into 13-hydroxyoctadec-9-enoic acid
(
(
(
3) by the linoleate C-12 double bond hydratase
AHW98239.1) isolated from L. acidophilus NBRC 13951.
b) Oleic acid and (c) linoleic acid were converted into 10-
hydroxyoctadecanoic acid and 10-hydroxy-cis-12-octadece-
noic acid (2), respectively, by the linoleate C-9 double bond
hydratase (AHW98240.1) isolated from L. acidiphilus
NBRC 13951. The biotransformations were carried out in
5
0 mM citrate-phosphate buffer (pH 7.0) containing 10 mM
of individual unsaturated fatty acid and 2% (v/v) ethanol.
The experiments were carried out in triplicate and the error
bars indicate standard deviation. The symbols indicate con-
centration of linoleic acid (a, c), oleic acid (b) (*) and 13-
hydroxyoctadec-9-enoic acid (a), 10-hydroxyoctadecanoic
acid (b), 10-hydroxy-cis-12-octadecenoic acid (c) (*).
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[a]
Table 1. Products accessible through biocatalysis.
[b]
Starting material
Final products
Product yield [%]
[
c]
linoleic acid (1)
13-hydroxyoctadeca-9-enoic acid (3)
96Æ2 (61%, 96%)
94Æ4
1
0-hydroxyoctadeca-12-enoic acid (2)
[
c]
1
3-hydroxyoctadeca-9-enoic acid (3)
0-hydroxyoctadeca-12-enoic acid (2)
12-hydroxydodeca-9-enoic acid (8)
d-decalactone (11)
g-dodecelactone (12)
91Æ4 (54%, 71%)
[d]
[c]
73Æ4 (60%, 97%)
[c]
1
86Æ3 (50%, 90%)
[
a]
Linoleic acid was converted into 13-hydroxyoctadeca-9-enoic acid (3) and 10-hydroxy-cis-12-octadecenoic acid (2) by the
linoleate C-12 and C-9 double bond hydratases, respectively, isolated from L. acidophilus NBRC 13951. Biotransforma-
tion of 13-hydroxyoctadec-9-enoic acid (3) into 12-hydroxydodeca-9-enoic acid (8) was carried out by the recombinant E.
coli BL21(DE3) pACYC-ADH, pET-BmoF1, pCOLA-PFE1. Transformation of 13-hydroxyoctadeca-9-enoic acid (3) and
1
0-hydroxyoctadeca-12-enoic acid (2) into the corresponding lactones were conducted via whole-cell reactions using Wal-
tomyces lipofer KCTC 17657 or Candida boidinii KCTC 17776, respectively.
The product yield was calculated based on the substrate depletion and the product concentration, which was determined
by GC/MS. All experiments were carried out in triplicate.
The numbers in parenthesis indicate the isolated yield and purity, respectively. The isolated yields were calculated based
on amount of the products isolated and amount of the substrates added into the reaction medium.
The product yield was calculated based on the substrate depletion and the product concentration measured at t=18 h in
Figure 3a.
[
[
[
b]
c]
d]
Figure S4 and Table 1). The high regioselectivity of the reaction medium (Supporting Information, Fig-
the enzyme toward a double bond between C-12 and ure S7). This result indicates that 10,13-dihydroxyoc-
C-13 of linoleic acid indicated that the enzyme tadecanoic acid, which was observed in the fermenta-
[
13,14]
AHW98239.1 could be a linoleate C-12 double bond tion products of lactic acid bacteria
and ruminal
[
15]
hydratase. Bioconversion of 1 to 3 has been also ex- bacteria, could be produced by a combined reaction
amined by using the wild-type strain of Pediococcus of a linoleate C-9 double bond hydratase and a lino-
[14]
sp. AKU 1080. However, significant amounts of by- leate C-12 double bond hydratase (Supporting Infor-
products such as 10-hydroxy-cis-12-octadecenoic acid mation, Figure S8).
and 10,13-dihydroxyoctadecanoic acid were formed
during the biotransformation of linoleic acid. The
product yield was less than 20%. This indicated that Biotransformation of 13-Hydroxy-cis-9-octadecenoic
our enzyme process is quite efficient in terms of selec- acid (3) into Carboxylic Acids
tivity of the biotransformation.
The other enzyme AHW98240.1 was able to selec- Hydroxy fatty acids are used as starting materials for
[
1–6]
tively catalyze hydration of a double bond between the production of fine chemicals.
Here, we investi-
C-9 and C-10 of oleic acid and linoleic acid under gated the biotransformation of 13-hydroxy-cis-9-octa-
identical reaction conditions as given in Figure 1a decenoic acid (3) into a C-12 carboxylic acid [i.e., 12-
(
Figure 1b and c). 10-Hydroxystearic acid and 10-hy- hydroxy-cis-9-dodecenoic acid (8)], which can be used
droxy-cis-12-octadecenoic acid (2) were the major for the production of polyester and/or polyamide
products from oleic and linoleic acid, respectively monomers such as 1,12-dodecanedioic acid and 12-
[
20]
(
see Supporting Information, Figures S5 and S6 for aminododecanoic acid. The Baeyer–Villiger mono-
product identification). The maximal specific enzyme oxygenase (BVMO) enzymes, which determine the
À1
reaction rate of 1 to 2 reached 14 Ug protein at t< chemical structure of the final products due to their
1
h. The biotransformation rate was greater with lino- regioselectivity, were first examined. The BVMOs
[21–23]
leic acid than oleic acid, indicating that the enzyme that are active with long chain aliphatic ketones
AHW98240.1 might be a linoleate C-9 double bond were chosen as candidates. When the recombinant E.
hydratase. Most fatty acid C-9 double bond hydratas- coli BL21(DE3) pACYC-ADH, pET-BmoF1,
es reported so far including myosin cross-reactive an- pCOLA-PFE1 expressing an alcohol dehydrogenase
tigen (MCRA) showed greater activity with oleic acid (ADH) from Micrococcus luteus NCTC2665,
[
2]
compared to linoleic acid. The higher activity of
AHW98240.1 with linoleic acid is one of the most 50106 and an esterase from P. fluorescens SIK WI,
striking characteristics of the enzyme. was added to the reaction medium containing 13-hy-
The biotransformation of linoleic acid with both droxy-cis-9-octadecenoic acid (3), the compound was
AHW98239.1 and AHW98240.1 resulted in the accu- selectively converted into 12-hydroxy-cis-9-dodeceno-
mulation of 10,13-dihydroxyoctadecanoic acid (Sup- ic acid (8) and n-hexanoic acid (7) via 13-oxo-cis-9-oc-
porting Information, S4 in Figure S8) as a product in tadecenoic acid (4) and ester (5) (Scheme 1) (Fig-
a BVMO from Pseudomonas fluorescens DSM
[23]
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
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Biotransformation of Linoleic Acid into Hydroxy Fatty Acids
PFE1 in one-pot (Supporting Information, Fig-
ure S11). Overall, 12-hydroxy-cis-9-dodecenoic acid
8), which was reported to stimulate growth of the
(
main root and lateral roots of plant seedlings and to
act as antifungal metabolites of the wild rice Oryza
[
24]
officinalis,
could be synthesized from renewable
biomass with a high yield for the first time.
On the other hand, 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
[
22]
KT2440
and an esterase from P. fluorescens SIK
WI resulted in the production of not only 12-hydroxy-
cis-9-dodecenoic acid (8) and n-hexanoic acid (7), but
also a,w-tridec-9-enedioic acid (10) and n-pentanol
(
9) (Figure 2b and Supporting Information, Fig-
ure S12). It was assumed that the ester compound (6)
was produced via the formation of the abnormal
ester, which is driven by migration of the less-substi-
[
25]
tuted carbon center during BVMO catalysis.
The
was
[
21]
BVMO from Pseudomonas veronii MEK700
poorly active with 13-ketooctadec-9-enoic acid (4).
Overall, 12-hydroxy-cis-9-dodecenoic acid (8) and
a,w-tridec-9-enedioic acid (10) were produced via li-
noleate cis-C-12 hydratase- and BVMO-based bioca-
talysis from linoleic acid (Table 1).
Biosynthesis of d-Decalactone (11) and g-
Dodecelactone (12)
Figure 2. Time course of the biotransformation of 13-hy-
droxyoctadec-9-enoic acid (2). 13-Hydroxyoctadec-9-enoic
acid (2) was converted into carboxylic acids by (a) the re-
combinant E. coli BL21(DE3) pACYC-ADH, pET-BmoF1,
pCOLA-PFE1 and (b) the recombinant E. coli BL21
1
3-Hydroxy-cis-9-octadecenoic acid (3) was then used
for the production of d-decalactone (11), which was
reported to be suitable for use in flavor composi-
tions.
[
26]
This was accomplished by whole-cell bio-
pACYC-ADH-BVMO, pCOLA-PFE1 (Scheme 1). The bio- transformation. A variety of yeasts were examined
transformation was initiated by adding 2 mM of 13-hydroxy- with the substrate. It turned out that the yeast Walto-
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
octadec-9-enoic acid into 50 mM Tris-HCl buffer (pH 8.0) myces lipofer KCTC 17657 was one of the best strains
À1
À1
containing 0.5 gL Tween 80 and 7.2 g dry cells L of the
recombinant E. coli BL21(DE3) cells. The experiments were
carried out in triplicate and the error bars indicate standard
deviation. The symbols indicate concentration of 13-
hydroxy
acid (3) (!), the ester (4, 5) (
enoic acid (7) (~), a,w-tridec-9-enedioic acid (9) (*).
to produce the target compound. When the W. lipofer
cells were added into 50 mM citrate-phosphate buffer
(
pH 7.0) containing 8 mM 13-hydroxy-cis-9-octadece-
noic acid (3) and 0.05% (w/v) Tween 80, d-decalac-
tone (11) was produced to a concentration of 8 mM in
the medium at t=15 h (Figure 3a and Supporting In-
formation, Figure S13). The maximal specific reaction
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
octadec-9-enoic acid (2) (~), 13-ketooctadec-9-enoic
), and 12-hydroxydodec-9-
&
rate of the yeast cells increased up to 1.1 Ug dry
À1
ure 2a and Supporting Information, Figures S9 and cells at t<12 h. The isolated yield of d-decalactone
S10). Notably, the final product 12-hydroxy-cis-9-do- reached 60% with a purity of 97% (Table 1 and Sup-
decenoic acid (8) was produced to a concentration of porting Information, Figure S14). This is to the best
1
9
.8 mM from 2.0 mM of the substrate 13-hydroxy-cis- of our knowledge the first report for the synthesis of
-octadecenoic acid. The maximal specific reaction d-decalactone (11) from renewable biomass.
rate of the recombinant cells reached 1.4 Ug dry
Biosynthesis of g-dodecelactone (12) from 10-hy-
À1
cells at t<2 h. The target product (8) was also pro- droxy-cis-12-octadecenoic acid (2) (Scheme 1 and
duced from linoleic acid by serial reactions of re- Supporting Information, Figure S15), which had been
combinant E. coli expressing the linoleate C-12 produced from linoleic acid by the linoleate C-9
double bond hydratase and recombinant E. coli double bond-hydratase, was also carried out with vari-
BL21(DE3) pACYC-ADH, pET-BmoF1, pCOLA- ous yeasts. Candida boidinii KCTC 17776 showed the
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reactions of the linoleate C-9 double bond hydratase
and C. boidinii KCTC 17776 in one-pot (Supporting
Information, Figure S17). Overall, g-dodecelactone
was synthesized from linoleic acid with a yield of ca.
43%.
Conclusions
The bacterial linoleate double bond hydratases from
L. acidophilus, which are highly specific for the hydra-
tion of the C-9 or the C-12 double bond of unsaturat-
ed fatty acids, were cloned for the first time. The re-
combinant enzymes were successfully used for the
production of hydroxy fatty acids such as 13-hydroxy-
cis-9-octadecenoic acid (3) and 10-hydroxy-cis-12-oc-
tadecenoic acid (2) from linoleic acid. Furthermore,
the industrially relevant carboxylic acids [e.g., 12-hy-
droxy-cis-9-dodecenoic acid (8), a,w-tridec-9-enedioic
acid (10)] and lactones [i.e., d-decalactone (11), g-do-
decelactone (12)] could be produced from the hy-
droxy fatty acids via the whole-cell biocatalysis.
Experimental Section
Cultivation of Microbial Strains
Lactobacillus acidophilus NBRC 13951 (Supporting Infor-
mation, Table S1), which was used to clone the putative hy-
dratase genes, was cultivated in de Man, Rogosa and Sharpe
[10]
(
MRS) medium as described elsewhere. Recombinant Es-
cherichia coli ER2566 harboring the linoleate hydratase
genes from L. acidophilus was grown at 378C with vigorous
shaking in Luria–Bertani (LB) medium supplemented with
À1
5
0 mgmL of ampicillin. Expression of the recombinant hy-
Figure 3. Time course of the transformation of hydroxy fatty
acids into lactones. (a) 13-Hydroxy-cis-9-octadecenoic acid
dratase genes was induced by adding 0.1 mM of isopropyl b-
d-thiogalactopyranoside (IPTG) and/or 2 gL rhamnose at
À1
(
3) was converted into d-decalactone (11) by Waltomyces
an optical density (OD600) of 0.5. Afterwards, the culture
was further incubated at 168C for 16 h. Recombinant E. coli
BL21(DE3), used for the biotransformation of hydroxy fatty
acids, was cultivated in Riesenberg medium, which was sup-
lipofer KCTC 17657. (b) 10-Hydroxy-cis-12-octadecenoic
acid (2) was transformed into g-dodecelactone (12)
(
Scheme 1 and Supporting Information, Figure S15) by Can-
À1
dida boidinii KCTC 17776. See the production of lactones
from hydroxy fatty acids in the Experimental Section for de-
tails. The experiments were carried out in triplicate and the
error bars indicate standard deviation. The symbols indicate
concentration of 13-hydroxy-cis-9-octadecenoic acid (a), 10-
plemented with 10 gL glucose and the appropriate antibi-
otics for plasmid maintenance (Supporting Information,
À1
Table S1). The Riesenberg medium consisted of 4 gL
À1
À1
(
1
NH ) HPO , 13.5 gL
KH PO , 1.7 gL
citric acid,
4
2
4
2
4
À1
À1
À1
.4 gL
MgSO4, and 10 mLL
trace metal solution
hydroxy-cis-12-octadecenoic acid (b) (*
) and d-decalactone
À1
À1
À1
À1
[
10 gL FeSO , 2.25 gL ZnSO , 1.0 gL CuSO , 0.5 gL
4
4
4
a), g-dodecelactone (b) (*
).
À1
À1
(
MnSO , 0.23 gL Na B O , 2.0 gL CaCl , and 0.1 gL
4
2
4
7
2
(
NH ) Mo O ]. Waltomyces lipofer KCTC 17657 and Candi-
4 6 7 24
da boidinii KCTC 17776 used for the production of lactones
highest productivity among the strains tested. g-Dode-
celactone was produced to 6.5 mM within 5 h bio-
transformation in the set-up identical to that for the
production of d-decalactone (Figure 3b). The produc-
tivity during biotransformation reached over 4 Ug dry
cells and 1.2 mMh . The isolated yield of g-dodece-
lactone reached 50% with a purity of 90% (Table 1
were cultivated in yeast malt (YM) broth as described previ-
[27]
ously.
Screening of Linoleate Hydratase Genes
À1
À1
The fatty acid double bond hydratase genes originating from
Elizabethkingia meningoseptica, Macrococcus caseolyticus,
and Supporting Information, Figure S16). g-Dodece- Streptococcus pyogenes NZ131, and L. acidophilus NCFM
lactone was also produced from linoleic acid by serial were chosen as probes to detect the putative linoleate hy-
6
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Biotransformation of Linoleic Acid into Hydroxy Fatty Acids
dratase genes in public databases (Supporting Information,
Table S2). A total 2,875 of bacterial oleate hydratase homo-
logues were found in InterPro (http://www.ebi.ac.uk/
interpro/entry/IPR010354/taxonomy) and 1,018 homol-
ogous in 897 bacterial species were found in Pfam
acid (2), 10-hydroxy-cis-12-octadecenoic acid, 10-hydroxyoc-
tadecanoic acid] was performed in 50 mM citrate-phosphate
buffer (pH 7.0) containing 10 mM of individual unsaturated
fatty acid and 2% (v/v) ethanol. In a 15-mL polypropylene
tube with a screw cap, 3 mL of reaction mixture were incu-
bated at 358C for 6 h with agitation at 200 rpm. For bio-
transformation of linoleic acid into 13-hydroxy-cis-9-octade-
(
http://pfam.xfam.org/family/PF06100.6#tabview=tab7) reg-
istered as PF01600 (67 kDa myosin cross-reactive antigen).
After removing overlapping sequences, the genomes that
harbored at least two different genes encoding putative lino-
leate double bond hydratases were selected using the
organism-specific genome search option in BLAST
À1
cenoic acid, 1.5 mgmL of purified hydratase was applied.
To produce 10-hydroxyoctadecanoic acid and 10-hydroxy-
cis-12-octadecenoic acid from oleic acid and linoleic acid,
À1
2 mgmL of purified hydratase was used. One unit (U) of
(
http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Supporting Informa-
the hydratase activity is defined as mmol of 10-hydroxy-12-
octadecenoic acid or 13-hydroxy-9-octadecenoic acid pro-
duced per min at 258C by enzymes.
tion, Table S3). The genes encoding protein sequences too
short (smaller than 400 amino acids in length) or fragment-
ed were excluded for functional candidates at this study be-
cause existing functional hydratases were reported to consist
Biotransformations of Hydroxy Fatty Acids into
Carboxylic Acids
[7]
of 564 to 732 amino acids in length.
Biotransformations of the hydroxy fatty acids with recombi-
Cloning of Linoleate Hydratase Genes
nant E. coli cells were carried out on the basis of our earlier
[19,27]
Among the 70 genomes containing at least two different
genes encoding putative linoleate double bond hydratases,
the genome of L. acidophilus NBRC 13951, which is known
to produce 13-hydroxyoctadec-9-enoic acid from linoleic
work.
Briefly, the recombinant cells were cultivated in
Riesenberg medium at 308C, and expression of the target
À1
genes was induced with 0.1 mM IPTG and/or 2.0 gL rham-
nose at an OD600 of 0.5. Thereafter, the cultivation tempera-
ture was reduced to 208C to facilitate the soluble expression
of the target genes. When the added glucose was consumed,
the recombinant cells were harvested by centrifugation at
3,500 g for 15 min at 48C and used as biocatalysts. The bio-
transformation was initiated by adding 2 mM of a hydroxy
fatty acid to 50 mM Tris-HCl buffer (pH 8.0) containing
[13]
acid
was
(Supporting Information, Table S4 and Figure S1),
used for gene cloning. The two genes
(
LACIP7613_00033 and LACIP7613_01498) encoding puta-
tive linoleate hydratases were amplified by polymerase
chain reaction (PCR) with the primers shown in the Sup-
porting Information, Table S5. The PCR products were de-
signed to include the NdeI and XhoI restriction sites. Each
PCR product was subcloned into the pET-15b plasmid (No-
vagen). The resulting plasmid was used to transform E. coli
ER2566 as an expression host.
À1
À1
0.5 gL Tween 80 and 7.2 g dry cells L of the recombinant
E. coli BL21(DE3) that expressed the ADH from Micrococ-
cus luteus, the BVMO from Pseudomonas putida KT2440 or
Pseudomonas fluorescens DSM 50106, and the esterase from
P. fluorescens SIK WI. The biotransformations were con-
ducted in a shaking incubator (358C and 200 rpm). One unit
Purification of Linoleate Hydratases
(
U) of whole cell activity was defined as mmol of the prod-
After cultivation of recombinant E. coli ER2566 harboring
the putative linoleate hydratase genes, the cell mass was har-
vested, washed, and resuspended into 50 mM phosphate
buffer (pH 8.0) containing 300 mM NaCl, 10 mM imidazole,
and 0.1 mM phenylmethylsulfonyl fluoride as a protease in-
hibitor. The resuspended cells were disrupted using an ultra-
sonicator (Fisher Scientific) with the samples kept on ice.
The cell debris was removed by centrifugation at 13,000 g
for 20 min at 48C, and the supernatant was filtered through
a 0.45 mm filter. The filtrate was applied to a His-Trap HP
affinity chromatography column (GE Healthcare) equili-
brated with 50 mM phosphate buffer (pH 8.0) containing
uct produced per min at 358C by whole cells.
Production of Lactones from Hydroxy Fatty Acids
Lactones were produced from 13-hydroxy-cis-9-octadeceno-
ic acid or 10-hydroxy-cis-12-octadecenoic acid on the basis
[28]
of previous studies. A variety of yeasts (e.g., W. lipofer, C.
boidinii, Yarrowia lipolytica, and Candida tropicalis) were
cultivated and used for biotransformation of the hydroxylat-
ed fatty acids into lactones. A single colony was inoculated
into 15 mL of YM medium and cultivated at 288C with
200 rpm of agitation for 12 h. The culture was transferred
into a 2-L baffled flask containing 500 mL of production
300 mM NaCl. The column was washed extensively with the
À1
À1
À1
same buffer, and the bound protein was eluted with a linear
medium consisting of 5.0 gL glucose, 2.5 gL ammonium
À1
gradient of 10 to 250 mM imidazole at a flow rate of
chloride, 0.1 gL
yeast extract, 2.1 gL
KH
2
PO
4
,
À1
À1
À1
À1
1
mLmin . The active fractions were collected and dialyzed
4.51 gL
K
HPO
2
4
, 0.1 gL NaCl, 0.2 gL MgSO
·7H
4
O,
2
À1
À1
À1
against 50 mM citrate-phosphate buffer (pH 7.0). After dial-
ysis, the solution was used as the purified enzyme. The pu-
rification step was carried out in a cold room at 48C using
a fast protein liquid chromatography system (Bio-Rad Labo-
ratories).
9.14 mgL FeSO
·7H
4
2
O, 0.5 mgL ZnCl
, and 1.56 mgL
2
CuSO
·5H
O. Lactone production was induced by supple-
4
2
À1
mentation of 5 gL oleic acid, and the cells were further
cultivated at 288C with agitation at 200 rpm for 12 h. The
cells were harvested by centrifugation at 13,000 g for 20 min
at 48C and washed twice with 50 mM citrate/phosphate
buffer (pH 5.5) to prepare concentrated cell suspensions for
the production of lactones from hydroxy fatty acids.
Enzymatic Hydration of Unsaturated Fatty Acids
Enzymatic biotransformations of oleic acid and linoleic acid
into hydroxy fatty acids [e.g., 13-hydroxy-cis-9-octadecenoic
d-Decalactone (11) was produced in 10 mL reaction
medium [i.e., 50 mM citrate-phosphate buffer (pH 7.0)] in
Adv. Synth. Catal. 0000, 000, 0 – 0
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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7
ÞÞ
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Hye-Jin Oh et al.
FULL PAPERS
a 100-mL baffled flask containing 10 mM 13-hydroxy-9-octa-
decenoic acid, 5 gL whole cells (as dry cell weight), and
tone (11), and g-dodecelactone (12) were identified via GC/
MS analysis and comparison to authentic reference com-
pounds (Supporting Information, Figures S3, S5, S6, S7, S9,
S12, S13, and S15).
À1
0.05% (w/v) Tween 80 at 288C for 15 h with 200 rpm. After-
wards, the reaction solution was acidified at 1008C for
[
27]
1
0 min with 10% (v/v) HCl for lactonization. g-Dodece-
lactone (12) was produced in 10 mL of reaction medium
i.e., 50 mm citrate-phosphate buffer (pH 5.5)] in a 100-mL
baffled flask containing 10 mM 10-hydroxy-9-octadecenoic
Purification of the Biotransformation Products
[
Purification of hydroxy fatty acids: The hydroxy fatty acids
(e.g., 13-hydroxy-cis-9-octadecenoic acid) were isolated
from the enzyme reaction medium by solvent extraction and
À1
À1
acid, 5 gL cells (as dry cell weight), 3.4 gL yeast nitrogen
base, and 0.05% (w/v) Tween 80 at 258C with agitation at
[15,29]
column chromatography as described previously.
In
200 rpm. For lactonization, the reaction solution was acidi-
brief, the biotransformation products were isolated via ex-
traction using two volumes of ethyl acetate two times. After
the organic layer was separated from the aqueous phase, the
organic extracts were dried with Na SO and concentrated
fied at 1008C for 10 min with 10% HCl.
Product Analysis by GC/MS
2
4
Concentrations of remaining fatty acids and accumulating
carboxylic acids in the medium [i.e., oleic acid, linoleic acid,
via evaporation under vacuum. The concentrate was purified
via silicic acid column chromatography with methanol gradi-
13-hydroxy-cis-9-octadecenoic acid (3), 10-hydroxyoctadeca-
ent (0–5%, v/v) in CH
ing substrate were washed off with CH
in CH Cl (seethe Supporting Information, Figure S4 for pu-
Cl (50 mL). Impurities and remain-
3
noic acid, 10-hydroxy-cis-12-octadecenoic acid (2), 10,13-di-
hydroxyoctadecanoic acid, n-hexanoic acid (7), n-pentanol
Cl and 1% methanol
3
3
(
9), 12-hydroxy-cis-9-dodecenoic acid (8), a,w-tridec-9-ene-
rification of 13-hydroxy-cis-9-octadecenoic acid).
Purification of unsaturated carboxylic acids: The
medium chain unsaturated carboxylic acids [e.g., 12-
dioic acid (10), and a,w-tridec-6,9-dienedioic acid] were de-
[20]
termined as described previously.
The reaction medium
was mixed with an equal volume of ethyl acetate containing
hydroxy
whole-cell reaction medium, as described previously.
brief, the biotransformation products were extracted with
ethyl acetate three times. The extract was dried over MgSO
ACHTUNGTNERNGNUd odec-9-enoic acid (8)] were isolated from the
À1
[19]
2
or 5 gL palmitic acid as an internal standard. The organ-
In
ic phase was harvested after vigorous vortexing and then de-
rivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide
4
(
TMS). The TMS derivatives were analyzed using a Thermo
and concentrated via evaporation under vacuum. The impur-
ities (e.g., n-alkanoic acids coproduced during biotransfor-
mations, long chain fatty acids originating from the starting
materials) were removed by washing with hexane (see the
Supporting Information, Figure S10 for purification of 12-
hydroxydodec-9-enoic acid (8)).
Ultra Trace GC system connected to an ion trap mass detec-
tor (Thermo ITQ1100 GC-ion Trap MS; Thermo Scientific).
The derivatives were separated on a non-polar capillary
column (30 m length, 0.25 mm film thickness, HP-5MS; Agi-
lent, Santa Clara, CA, USA). A linear temperature gradient
was programmed as follows; 908C, 15 8C min to 2008C;
2
2
À1
Purification of lactones: g-Dodecelactone and d-decalac-
tone were isolated from whole cell biotransformation
À1
À1
008C, 58C min to 2508C or 908C, 15 8C min to 2008C;
À1
[27]
008C, and 58C min to 2808C. The inject port temperature
medium, as described previously. In short, the biotransfor-
was 2308C. Mass spectra were obtained by electron impact
ionization at 70 eV. Scan spectra were obtained within the
range of 100–600 m/z. Selected ion monitoring (SIM) was
used for the detection and fragmentation analysis of the re-
action products.
mation products were isolated via extraction with a half
volume of mineral oil (Sigma–Aldrich). The organic layer
was separated and boiled at 1808C in an oil bath. The lac-
tones were evaporated and collected in a receiving flask
(see the Supporting Information, Figures S14 and S16 for
the purification).
Product Analysis by GC
The obtained hydroxy fatty acids were derivatized by 3:1
mixture of pyridine and TMS. The TMS-derivative hydroxy Acknowledgements
fatty acids, lactones, and unsaturated fatty acids were ana-
lyzed by GC (Agilent 6890N) equipped with a flame ioniza-
tion detector and a SPB-1 capillary column (15 mꢁ0.32 mm
inside diameter, 0.25 mm thickness; Supelco) using palmitic
This study was supported by the Marine Biomaterials Re-
search Center grant from Marine Biotechnology Program
funded by the Ministry of Oceans and Fisheries, Korea, and
the Bio-industry Technology Development Program funded
by the Ministry for Food and Agriculture, Republic of Korea
(No. 112002-3).
[15,27]
acid as an internal standard.
The column temperature
À1
increased from 150 to 2108C at 48C min and then 308C
À1
min until 2808C, and was maintained at 2808C for 5 min.
The injector and detector temperatures were held at 260
and 2508C, respectively.
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Adv. Synth. Catal. 0000, 000, 0 – 0
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
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ÞÞ
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FULL PAPERS
1
0 Biotransformation of Linoleic Acid into Hydroxy Fatty
Acids and Carboxylic Acids Using a Linoleate Double
Bond Hydratase as Key Enzyme
Adv. Synth. Catal. 2015, 357, 1 – 10
Hye-Jin Oh, Sae-Um Kim, Ji-Won Song, Jung-Hoo Lee,
Woo-Ri Kang, Ye-Seul Jo, Kyoung-Rok Kim,
Uwe T. Bornscheuer, Deok-Kun Oh,* Jin-Byung Park*
10
asc.wiley-vch.de
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Adv. Synth. Catal. 0000, 000, 0 – 0
ÝÝ
These are not the final page numbers!