Simultaneous Enzyme/Whole-Cell Biotransformation of C18 Ricinoleic Acid into (R)-3-Hydroxynonanoic Acid, 9-Hydroxynonanoic Acid, and 1,9-Nonanedioic Acid

Article abstract of DOI:10.1002/adsc.201701029

Regiospecific oxyfunctionalization of renewable long chain fatty acids into industrially relevant C9 carboxylic acids has been investigated. One example was biocatalytic transformation of 10,12-dihydroxyoctadecanoic acid, which was produced from ricinoleic acid ((9Z,12R)-12-hydroxyoctadec-9-enoic acid) by a fatty acid double bond hydratase, into (R)-3-hydroxynonanoic acid, 9-hydroxynonanoic acid, and 1,9-nonanedioic acid with a high conversion yield of ca. 70%. The biotransformation was driven by enzyme/whole-cell biocatalysts, consisting of the esterase of Pseudomonas fluorescens and the recombinant Escherichia coli expressing the secondary alcohol dehydrogenase of Micrococcus luteus, the Baeyer-Villiger monooxygenase of Pseudomonas putida KT2440 and the primary alcohol/aldehyde dehydrogenases of Acinetobacter sp. NCIMB9871. The high conversion yields and the high product formation rates over 20 U/g dry cells with insoluble reactants indicated that various (poly-hydroxy) fatty acids could be converted into multi-functional products via the simultaneous enzyme/whole-cell biotransformations. This study will contribute to the enzyme-based functionalization of hydrophobic substances. (Figure presented.).

Full text of DOI:10.1002/adsc.201701029

Title: Simultaneous Enzyme/Whole-Cell Biotransformation of C18
Ricinoleic Acid into (<i>R</i>)-3-Hydroxynonanoic Acid, 9-
Hydroxynonanoic Acid, and 1,9-Nonanedioic Acid
Authors: Hee-Jeong Cha, Eun-Ji Seo, Ji-Won Song, Hye-Jin Jo, Ravi
Kumar A, and Jin-Byung Park
This manuscript has been accepted after peer review and appears as an
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content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201701029
Link to VoR: http://dx.doi.org/10.1002/adsc.201701029

10.1002/adsc.201701029
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Simultaneous Enzyme/Whole-Cell Biotransformation of
C18 Ricinoleic Acid into (R)-3-Hydroxynonanoic Acid,
9-Hydroxynonanoic Acid, and 1,9-Nonanedioic Acid
Hee-Jeong Cha+a, Eun-Ji Seo+a, Ji-Won Songa, Hye-Jin Joa, Akula Ravi Kumarb,
and Jin-Byung Park a,c*
a
Department of Food Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea
Fax: (+82)-2-3277-4213; phone: (+82)-2-3277-4509; e-mail: jbpark06@ewha.ac.kr
Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760, Republic of Korea
Institute of Molecular Microbiology and Biosystems Engineering, Ewha Womans University, Seoul 03760,
b
c
Republic of Korea
+ H.-J. Cha and E.-J. Seo equally contributed to this work.
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201######.((Please delete if not appropriate))
monooxygenase of Pseudomonas putida KT2440 and the
Abstract. Regiospecific oxyfunctionalization of renewable
primary
alcohol/aldehyde
dehydrogenases
of
long chain fatty acids into industrially relevant C9 carboxylic
acids has been investigated. One example was biocatalytic
transformation of 10,12-dihydroxyocatadecanoic acid, which
was produced from ricinoleic acid ((9Z,12R)-12-
hydroxyoctadec-9-enoic acid) by a fatty acid double bond
Acinetobacter sp. NCIMB9871. The high conversion
yields and the high product formation rates over 20 U/g
dry cells with insoluble reactants indicated that various
(poly-hydroxy) fatty acids could be converted into multi-
functional products via the simultaneous enzyme/whole-
cell biotransformations. This study will contribute to the
hydratase,
into
(R)-3-hydroxynonanoic
acid,
9-
hydroxynonanoic acid, and 1,9-nonanedioic acid with a high
conversion yield of ca. 70%. The biotransformation was
driven by enzyme/whole-cell biocatalysts, consisting of the
esterase of Pseudomonas fluorescens and the recombinant
Escherichia coli expressing the secondary alcohol
dehydrogenase of Micrococcus luteus, the Baeyer-Villiger
enzyme-based
substances.
functionalization
of
hydrophobic
Keywords: Enzyme catalysis; Oxygenation; Carboxylic
acids; Biotransformation; Fatty acids
Hydroxynonanoic acid and 1,9-nonanedioic acid,
Introduction
which are used as monomers for the production of
[3]
polyesters and polyamides
,
are currently
[4]
Recent developments in synthetic biology, metabolic manufactured via ozonolysis of oleic acid
[3c]. 3-
engineering, and enzyme engineering enable us to Hydroxynonanoic acid, which can be used as an
[5]
expand the repertory of biocatalysis [1]. For instance, antimicrobial agent
and a building block for the
[6] [7]
the engineered whole-cell based biocatalysis synthesis of polyhydroxyalkanoates
and
permitted the production of a variety of chemicals rhamnolipids [8], is difficult to produce by chemical
including medium chain ω-hydroxycarboxylic acids, means.
α,ω-dicarboxylic acids, and ω-aminocarboxylic acids
According to previous studies [2a,2e], oleic acid
from renewable oils and fatty acids [2]. Here, we have could be converted into n-nonanoic acid and 9-
included another type of biocatalysis to provide hydroxynonanoic acid by multi-step enzyme cascade
industrially relevant C9 carboxylic acids (i.e., (R)-3- reactions; oleic acid was transformed into an ester
hydroxynonanoic acid (5), 9-hydroxynonanoic acid (i.e., 9-(nonanoyloxy)-nonanoic acid) via 10-
(6), and 1,9-nonanedioic acid (8)) from renewable hydroxyoctadecanoic acid and 10-ketooctadecanoic
long chain fatty acids (i.e., ricinoleic acid (1)) acid by a fatty acid double bond hydratase, a long
(Scheme S1) to a high conversion yield. 9- chain secondary alcohol dehydrogenase, and a
1
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10.1002/adsc.201701029
Advanced Synthesis & Catalysis
Results and Discussion
Baeyer-Villiger monooxygenase (BVMO). The ester
was then hydrolyzed into n-nonanoic acid and 9-
hydroxynonanoic acid by a lipase. Alternatively, the
ester was converted into n-nonanoic acid and 9-
hydroxynonanoic acid or 1,9-nonanedioic acid by
The first step for the biotransformation of ricinoleic
acid into 3-hydroxynonanoic acid (5) and 9-
hydroxynonanoic acid (6) (Scheme S1) was to
discover suitable enzymes for the cascade reactions.
Hydration of ricinoleic acid (1) into 10,12-dihydroxy-
octadecanoic acid (2) has been reported in an earlier
study [11]. However, the other steps were not described
chemical reactions [2a,9]
.
In the present study, the biocatalytic system to
produce the C9 carboxylic acids was established on a
basis of a fatty acid double bond hydratase, a long
chain secondary alcohol dehydrogenase (SADH), a
BVMO, an esterase, a primary alcohol dehydrogenase
(PADH), and an aldehyde dehydrogenase (AlDH)
(Scheme 1). The nicotinamide cofactor-dependent
enzymes (i.e., SADH, BVMO, PADH, and AlDH)
have been expressed in Escherichia coli cells, while
cofactor-independent enzymes (i.e., esterases) were
directly added into the reaction medium to obtain high
yet. The long chain secondary alcohol dehydrogenase
[2a,2e]
of Micrococcus luteus NCTC2665
was
10,12-
examined
for
oxidation
of
dihydroxyoctadecanoic acid (2). The enzyme was
able to oxidize the substrate into the target compound
(3) with high regioselectivity (Fig. S1). The BVMOs
from Pseudomonas putida KT2440 [12], Pseudomonas
fluorescens DSM 50106 [13], and Rhodococcus josti
[14], which were reported to catalyze oxygenation of
long chain keto-fatty acids into esters [2a,2e], were
investigated with 10-oxo-12-hydroxyoctadecanoic
acid (3) as the substrate (Fig. S2). Remarkably, the
BVMO from P. putida KT2440 catalyzed the Baeyer-
Villiger oxygenation of 10-oxo-12-hydroxyoctadeca-
noic acid (3) into the ester (4) (Fig. S2), whereas the
BVMOs from P. fluorescens DSM 50106 and R. josti
were not active with the compound (3). A lipase from
Thermomyces lanuginosus, which was active with 9-
(nonanoyloxy)-nonanoic acid [2e], was not so active in
hydrolysis of the BVMO reaction product (i.e., 9-(12-
hydroxynonanoyloxy)-nonanoic acid (4)). However,
an esterase from P. fluorescens SIK WI, which was
shown to hydrolyze long chain linear esters (e.g., n-
product formation rates per whole-cell biocatalyst.
[10]
The long chain fatty acid transporter FadL
was
also overexpressed in the outer membrane of the E.
coli to facilitate mass transport of the insoluble
reactants. In theory, the SADH and BVMO reactions
will occur inside cells. The reaction products (4) will
be excreted out of the cells and subjected to
hydrolysis by the extracellular esterases (Scheme 1).
The hydrolysis products (6) will enter into the E. coli
cells and be further oxidized to 1,9-nonanedioic acid
(8) by the intracellular PADH and AlDH. Ultimately,
3-hydroxynonanoic acid (5) and 1,9-nonanedioic acid
(8) will accumulate in the reaction medium.
OH OH
OH OH
OH
O
OH
OH
5
OH
5
2
5
O
O
Alcohol
Hydratase
1
Dehydrogenase
OH
O
OH
5
3
O
Baeyer-Villiger
Monooxygenase
O
OH
O
O
OH
O
O
OH
5
O
OH
4
5
4
Esterase
OH
O
OH
OH
5
6
+
O
O
HO
OH
HO
6
Alcohol Dehydrogenase (ChnD)
O
O
H
OH
7
Aldehyde Dehydrogenase (ChnE)
O
O
O
O
HO
OH
HO
OH
8
8
Scheme 1. Designed biotransformation pathway. Ricinoleic acid (1) is converted into (R)-3-hydroxynonanoic acid (5) and
1,9-nonanedioic acid (8) via 9-hydroxynonanoic acid (6).
2
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10.1002/adsc.201701029
Advanced Synthesis & Catalysis
10
8
20
15
10
5
6
4
2
0
0
0
2
4
6
0
2
4
6
8
10
Reaction time (h)
Reaction time (h)
Figure 1. Time course of the biotransformation of Figure 2. Time course of the biotransformation of
ricinoleic acid by the recombinant E. coli BL21(DE3) ricinoleic acid into the ester (4) by two-whole cell systems,
pACYC-ADH-FadL-OhyA, pJOE-E6BVMO, expressing a consisting of the recombinant E. coli BL21(DE3) pET-
fatty acid double bond hydratase (OhyA) of OhyA and the recombinant E. coli BL21(DE3) pACYC-
Stenotrophomonas maltophilia, an alcohol dehydrogenase ADH-FadL, pJOE-E6BVMO. The recombinant E. coli
(ADH) of Micrococcus luteus, the engineered BVMO from BL21(DE3) pACYC-ADH-FadL, pJOE-E6BVMO was
Pseudomonas putida KT2440 (E6BVMO), and a long added into the culture medium of the recombinant E. coli
chain fatty acid transporter (FadL). The biotransformation BL21(DE3) pET-OhyA after over 90% of the starting
was initiated by adding 15 mM ricinoleic acid and 0.5 g/L material (i.e., ricinoleic acid) was transformed into the
Tween80 at t = 0 into the recombinant E. coli culture broth dihydroxy fatty acid (2) (final cell density: 3 g dry cells/L).
(cell density: 3 g dry cells/L). Symbols indicate the Symbols indicate the concentrations of ricinoleic acid (1)
concentrations of ricinoleic acid (1) (●), 10,12- (●), 10,12-dihydroxyoctadecanoic acid (2) (◆), 10-keto-
dihydroxyoctadecanoic acid (2) (◆), 10-keto-12-hydroxy- 12-hydroxyoctadecanoic acid (3) (▽), and ester 4 (■). The
octadecanoic acid (3) (▽), ester 4 (■), 12-ketooctadec-9- error bars indicate standard deviations.
enoic acid (9) (◇), and ester 10 (□). The error bars indicate
standard deviations.
pJOE-E6BVMO was added into the culture medium
octylacetate) [15], turned out to be quite active with the of the recombinant E. coli BL21(DE3) pET-OhyA just
ester (4) (Fig. S3).
after approximately 90% of the starting material (i.e.,
The first trial to produce 3-hydroxynonanoic acid ricinoleic acid) was transformed into the dihydroxy
(5) and 9-hydroxynonanoic acid (6) from ricinoleic fatty acid (2) (final cell density: 3 g dry cells/L) (Fig.
acid (1) (Scheme 1) was to use the recombinant E. 2). Ricinoleic acid was ultimately converted into the
coli BL21(DE3) pACYC-ADH-FadL-OhyA, pJOE- ester (4) via 10,12-dihydroxyoctadecanoic acid (2)
E6BVMO as a biocatalyst based on our previous and 10-oxo-12-hydroxyoctadecanoic acid (3) to a
study [2e]. The recombinant strain expresses not only final concentration of 12.5 mM in the culture medium.
the fatty acid double bond hydratase of The resulting ester product (4) was hydrolyzed into 3-
Stenotrophomonas maltophilia [16], the SADH of M. hydroxynonanoic acid (5) and 9-hydroxynonanoic
luteus, and the BVMO of P. putida KT2440 in the acid (6) (Scheme 1) by adding the esterase from P.
cytoplasm for the multi-step biotransformation of fluorescens SIK WI into the reaction medium (Fig.
ricinoleic acid (1) into the ester (4), but also the long S4), because the ester compound (4) was excreted out
chain fatty acid transporter FadL in the outer of the cells. Approximately 85% of the reaction
membrane for the efficient substrate transport into the intermediates including 3 and 4 were found in the
cascade enzymes. The whole-cell biotransformation extracellular space.
of ricinoleic acid produced the ester (10) (Scheme S2)
Overall, the results indicated that the industrially
as the major product rather than the ester (4) targeted relevant C9 carboxylic acids could be produced from
(Fig. 1). This was due to greater activity of the SADH C18 ricinoleic acid by the serial reactions of whole-
of M. luteus with ricinoleic acid as compared to the cell and enzyme catalysts.
fatty acid hydratase of S. maltophilia in the cytoplasm
of E. coli.
The next experiment was focused on the
production of the ester (4) to a high concentration in
Our strategy has been changed to use a serial the reaction medium. This was exploited by using
combination of two-whole cell systems, consisting of high cell density cultivation of the recombinant E.
the recombinant E. coli BL21(DE3) pET-OhyA for coli BL21(DE3) pET-OhyA and the recombinant E.
the biotransformation of ricinoleic acid into 10,12- coli BL21(DE3) pAPTm-E6BVMO-ADH, pCon-
dihydroxyoctadecanoic acid (2) and the recombinant
FadL on a basis of our previous studies [2e,17]. The first
E. coli BL21(DE3) pACYC-ADH-FadL, pJOE- step was the fed-batch cultivation of the recombinant
E6BVMO for the conversion of the dihydroxy fatty E. coli BL21(DE3) pET-OhyA to a cell density of 10
acid (2) into the ester (4) (Scheme 1). The g dry cells/L. The whole-cell biotransformation of
recombinant E. coli BL21(DE3) pACYC-ADH-FadL, ricinoleic acid into 10,12-dihydroxyoctadecanoic acid
3
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10.1002/adsc.201701029
Advanced Synthesis & Catalysis
80
A
mmol) ester (Fig. S5). These results indicated that the
ester could be produced to a high concentration with a
high productivity and a high product yield.
60
40
20
0
We have next investigated the biotransformation of
10,12-dihydroxyoctadecanoic acid (2) into 3-
hydroxynonanoic acid (5) and 1,9-nonanedioic acid (8)
(Scheme 1). This was carried out by using the
alcohol/aldehyde dehydrogenases (ChnDE) of
[18]
Acinetobacter sp. NCIMB9871
in addition to the
SADH of M. luteus, the BVMO of P. putida KT2440,
and the esterase of P. fluorescens. The
alcohol/aldehyde dehydrogenases of Acinetobactersp.
NCIMB9871 was chosen because the enzymes have
shown the much greater activity with 9-
hydroxynonanoic acid (6) as compared to the PADH
(AlkJ) of P. putida GPo1[19], which had been used for
0
1
2
3
4
5
Reaction time (h)
B
oxidation of 9-hydroxynonanoic acid in our earlier
30
25
20
15
10
5
[2b]
study
(Fig. S6). The biotransformation of the
dihydroxy fatty acid (2) into 3-hydroxynonanoic acid
(5) and 1,9-nonanedioic acid (8) was conducted by
the recombinant E. coli BL21(DE3) pACYC-ADH-
FadL, pJOE-E6BVMO, pCOLA-ChnDE and the
esterase of P. fluorescens (Scheme 1). The esterase
was not expressed in cytoplasm of E. coli but just
added into reaction medium, because the ester product
(4) was secreted into extracellular space before
hydrolysis into 5 and 6 by esterases in the cytoplasm.
0
0
1
2
3
4
5
Reaction time (h)
Figure 3. Time course of the biotransformation of
ricinoleic acid at high cell density cultures. The
biotransformation of ricinoleic acid into 10,12-
dihydroxyoctadecanoic acid (2) was conducted by adding
70 mM ricinoleic acid into the recombinant E. coli
BL21(DE3) pET-OhyA culture (A). Afterwards, the
resulting reaction mixture was transferred into the culture
of the recombinant E. coli BL21(DE3) pAPTm-E6BVMO-
ADH, pCon-FadL (final cell density: 10 g dry cells/L) (B).
Symbols indicate the concentrations of ricinoleic acid (1)
(●), 10,12-dihydroxyoctadecanoic acid (2) (◆), 10-keto-
12-hydroxyoctadecanoic acid (3) (▽), and ester 4 (■). The
error bars indicate standard deviations.
10
8
6
4
2
0
0
2
4
6
8
10
Reaction time (h)
Figure 4. Time course of the biotransformation of 10,12-
dihydroxy-octadecanoic acid (2) into 3-hydroxynonanoic
acid (5) and 1,9-nonanedioic acid (8) by the recombinant E.
coli BL21(DE3) pACYC-ADH-FadL, pJOE-E6BVMO,
pCOLA-ChnDE, expressing the ADH of M. luteus, the
E6BVMO of P. putida KT2440, the alcohol/aldehyde
(2) was conducted by adding 70 mM ricinoleic acid
into the recombinant E. coli culture (Fig. 3A). 10,12-
Dihydroxyoctadecanoic acid (2) was produced to 60
mM in the medium at t = 3 h. The resulting reaction
mixture was then added into the culture broth of the
recombinant E. coli BL21(DE3) pAPTm-E6BVMO-
ADH, pCon-FadL (final cell concentration: 10 g dry
cells/L). The dihydroxy fatty acid (2) was converted
into the ester (4) to a concentration of 25 mM (8.3 g/L)
in the culture medium with the volumetric
productivity of 8.3 mM/h (2.8 g/L/h) (Fig. 3B). The
molar conversion yield from ricinoleic acid, which
was estimated by GC/MS analysis of the reactants,
reached 68%. The isolation yield of the ester (4) from
the reaction medium was ca. 70%; 320 mg ester
(purity > 90%) was obtained as a colorless solid from
50 mL reaction medium containing 410 mg (1.24
dehydrogenases
(ChnDE)
of
Acinetobacter
sp.
NCIMB9871, and a long chain fatty acid transporter
(FadL). The biotransformation was initiated by adding 10
mM 10,12-dihydroxyoctadecanoic acid (2) and 0.5 g/L
Tween80 at t = 0 into the recombinant E. coli culture broth
(cell density: 3 g dry cells/L). Symbols indicate the
concentrations of 10,12-dihydroxy-octadecanoic acid (2)
(◆), 10-keto-12-hydroxyocta-decanoic acid (3) (▽), ester 4
(■), 3-hydroxynonanoic acid (5) (▼), 9-hydroxynonanoic
acid (6) (▲), and 1,9-nonanedioic acid (8) (○). The error
bars indicate standard deviations.
4
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10.1002/adsc.201701029
Advanced Synthesis & Catalysis
Table 1. Comparison of the specific whole-cell biotransformation rates of fatty acids
Biotransformation
of 2 to ester (4)
Biotransformation
Biotransformation
of ricinoleic acid into esters b)
a)
of 6 to 8 a)
Substrate
concentrations (mM)
10 to 1.4
0 to 1.0
21
10 to 4.5
27
(16 to 4.8)
Specific whole-cell
biotransformation rates
(U/g dry cells) c)
37 (33)
a) The biotransformation shown in Fig 4. The numbers in the parenthesis indicate the values obtained from the experiment
shown in Fig. 2.
b) The biotransformation of ricinoleic acid into esters shown in Fig. 1.
c) The specific whole-cell biotransformation rates were estimated with the cell concentrations and the initial product
formation rates at t = < 1 h, which were measured by GC/MS analysis.
In more detail, the whole-cell biotransformation
was initiated by adding the dihydroxy fatty acid (2)
Conclusion
and the esterase of P. fluorescens into culture medium
of the recombinant E. coli BL21(DE3) pACYC-
ADH-FadL, pJOE-E6BVMO, pCOLA-ChnDE (Fig.
4). The dihydroxy fatty acid (2) was transformed into
3-hydroxynonanoic acid (5) and 1,9-nonanedioic acid
(8) to over 20 U/g dry cells (Table 1). The molar
conversion yield, which was estimated by GC/MS
analysis of the reactants, reached approximately 80%.
The isolation yield of 3-hydroxynonanoic acid (5)
and 1,9- nonanedioic acid (8) was ca. 70%,
respectively; 220 mg of 3-hydroxynonanoic acid and
240 mg of 1,9-nonedioic acid (purity > 90%) were
obtained as a colorless solid from 200 mL reaction
medium containing 282 mg (1.6 mmol) of 3-
hydroxynonanoic acid and 308 mg (1.6 mmol) of 1,9-
nonedioic acid (Fig. S7 and S8). These results
indicated that the ester (4), which had been produced
in the cytoplasm of the recombinant E. coli
BL21(DE3) pACYC-ADH-FadL, pJOE-E6BVMO,
pCOLA-ChnDE, was excreted out of cells and then
converted into 3-hydroxynonanoic acid (5) and 9-
hydroxynonanoic acid (6) by the esterase of P.
The novel enzyme cascades (Scheme 1) including
newly developed enzymes (e.g., ChnDE) enabled us
to produce the industrially relevant C9 carboxylic
acids
(i.e.,
3-hydroxynonanoic
acid,
9-
hydroxynonanoic acid, and 1,9-nonanedioic acid)
from the renewable long chain fatty acid (i.e.,
ricinoleic acid) with
a
high product yield.
specific whole-cell
Furthermore, the high
biotransformation rates with the insoluble reactants in
a solid form indicated that solid-type fatty acids could
be good substrates even for the complex
biotransformations
like
the
simultaneous
enzyme/whole-cell biotransformation of dihydroxy
fatty acid. This study can be used for the
biotransformation of poly-hydroxy fatty acids such as
[2d,21]
10,13-dihydroxyoctadecanoic acid
,
7,8-
dihydroxy-9,12-octadecadienoic acid [22], 7, 10-
[23]
dihydroxy-8-octadecenoic acid
,
and 5,8-
.
dihydroxy-9,12,15-octadecatrienoic acid [24]
Experimental Section
fluorescens
(Scheme
1).
Afterwards,
9-
hydroxynonanoic acid (6) entered into the cytoplasm Microbial strains and culture media
of the recombinant E. coli BL21(DE3) and then
oxidized into 1,9-nonanedioic acid (8) via 9-oxo-
nonanoic acid (7) by ChnDE in the cytoplasm. The
high product formation rates over 20 U/g dry cells
(Table 1) pointed to the great performance of the
simultaneous enzyme/whole-cell biocatalsysis.
Recombinant E. coli BL21(DE3) cells were grown in
Lysogeny broth (LB) medium (5 g/L yeast extract, 10 g/L
tryptone, and 10 g/L NaCl) supplemented with appropriate
antibiotics (Table S1) for seed cultivation. Riesenberg
[25]
medium
supplemented with 10 g/L glucose and the
appropriate antibiotics was used for the main cultivation
and biotransformation. The Riesenberg medium consisted
The dihydroxy fatty acid (2) and other reaction
intermediates are almost insoluble in the aqueous of 4 g/L (NH4)2HPO4, 13.5 g/L KH2PO4, 1.7 g/L citric acid,
1.4 g/L MgSO4, and 10 mL/L trace metal solution (10 g/L
FeSO4, 2.25 g/L ZnSO4, 1.0 g/L CuSO4, 0.5 g/L MnSO4,
0.23 g/L Na2B4O7, 2.0 g/L CaCl2, and 0.1 g/L
(NH4)6Mo7O24). Recombinant gene expression was induced
by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside
(IPTG) and/or 2 g/L rhamnose into the cultivation medium.
The recombinant genes in the pAPTm-E6BVMO-ADH
was constitutively expressed by the synthetic promoter
(J23100 (http://parts.igem.org/Part:BBa_J23100)).
cultivation medium. They are present in a solid form
in the culture medium, suggesting that mass transport
can be problematic during the simultaneous
enzyme/whole-cell biotransformation. However, the
specific whole-cell biotransformation rates were
comparable to those with liquid substrates (e.g.,
ricinoleic acid and its derivatives [17a] [20]) (Table 1).
Therefore, it was assumed that solid-type fatty acid
substrates could be also good substrates for the
complex biotransformations.
5
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10.1002/adsc.201701029
Advanced Synthesis & Catalysis
Chemicals and materials
5°C/min to 280°C. Injection port temperature was 230°C.
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 was used for
detection and the fragmentation analysis of the reaction
products.
Ricinoleic
acid,
rhamnose,
and
N-methyl-N-
(trimethylsilyl)-trifluoroacetamide (TMS) were purchased
from Tokyo Chemical Co (Tokyo, Japan). Glucose was
obtained from Junsei Chemical Co (Tokyo, Japan).
Antibiotics, trace elements for cell cultivation, IPTG, and
Tween80 were purchased from Sigma (St. Louis, MO, Isolation of the ester (4) after
USA). Ethyl acetate was obtained from Duksan Pure biotransformation
Chemical Co. (Ansan, Republic of Korea).
The ester intermediate was isolated by simple extraction
Gene cloning
with ethyl acetate, based on our earlier studies [9,20]. The
combined organic layer was washed with saline, dried over
anhydrous Na2SO4, filtered, and evaporated in vacuo. The
crude ester product was purified by silica gel column
chromatography using 20% ethyl acetate-hexane as the
eluent. The isolation/purification yield of the ester (4) was
over 70%, based on the concentration of the ester
intermediate after whole-cell biotransformation. The
purified ester (320 mg) was a colorless solid; Mp 46-48°C;
1H NMR (500 MHz, DMSO-d6) δ 11.86 (broad, COOH),
4.64 (broad, OH), 3.98-4.02 (2H, m), 3.79-3.81 (1H, m),
2.20-2.34 (2H, m), 2.17 (2H, m), 1.48-1.58 (4H, m), 1.18-
1.26 (20H, m), 0.82-0.87 (3H, m).13C NMR (125 MHz;
DMSO-d6) δ 174.40 (COOH), 171.32 (-COOR), 67.09 (C-
OH), 63.57 (H2C-OH), 42.62, 36.86, 33.65, 31.29, 28.71,
28.66, 28.54, 28.50, 28.14, 25.36, 24.98, 24.47, 22.07
(CH3) (Fig. S5).
For construction of pCOLA-ChnDE, genomic DNA was
extracted from Acinetobacter johnsonii NCIMB 9871
grown in LB medium using the Qiagen Genomic DNA
Purification kit (Valencia, CA, USA). The chnD gene
(GenBank accession code: BAC80217.1) was amplified
from genomic DNA by using primer 1 & 2 and sub-cloned
into the NdeI and BglII site of pCOLAduet vector resulting
in pCOLA-ChnD. Afterward, the chnE gene (GenBank
accession code: BAA86294.1) was amplified from
genomic DNA by using primer 3 & 4 and inserted into
EcoRI and SalI site of pCOLA-ChnD resulting in pCOLA-
ChnDE. For constructio of pCon-FadL, the full length fadL
gene (GenBank accession code: CAQ32746.1) was PCR-
amplified by using pACYC-ADH-FadL as template and
primer 5 & 6 and inserted into BamHI and Eco88I site of
pCon plasmid with p15A origin and synthetic promoter
(J23100). The plasmids and primers used in this study are Isolation of 3-hydroxynonanoic acid (5) and
presented in Table S1 and S2.
1,9-nonanedioic acid (8) after
biotransformation
Whole-cell biotransformation
3-Hydroxynonanoic acid (5) and 1,9-nonedioic acid (8)
were isolated from the biotransformation medium by
simple extraction with ethyl acetate on a basis of our
previous studies [9,20]. The combined organic layer was
washed with saline, dried over sodium sulfate, filtered, and
evaporated in vacuo. The crude compounds were separated
and purified by column chromatography (silica gel) eluting
with varying concentration of ethyl acetate-hexane. 3-
Hydroxynonanoic acid (5) and 1,9-nonedioic acid (8) were
recovered when 25% and 30% ethyl acetate-hexane,
Biotransformation was carried out as reported previously
[2a,2e]. Briefly, the biotransformation was initiated at the
stationary growth phase (cell density: 3 g dry cells/L),
usually 8 h after inducing gene expression with 0.1 mM
IPTG and/or 2 g/L rhamnose. After changing the culture
broth pH to 8.0 and shift of culture temperature to 35°C,
ricinoleic acid (1) or 10,12-dihdyroxyoctadecanoic acid (2)
and 0.5 g/L Tween80 were added to the culture medium.
Cell cultivation and biotransformation were carried out in a
250 mL flask (working volume: 20 mL) in a shaking
incubator (200 rpm). The high cell density culture of
respectively,
was
used
as
the
eluent.
The
isolation/purification yield of 3-hydroxynonanoic acid (5)
and 1,9-nonedioic acid (8) were both over 70%, based on
the concentrations of the compounds after enzyme/whole-
cell biotransformation. The purified products (220 mg of 3-
hydroxynonanoic acid and 240 mg of 1,9-nonedioic acid)
were colorless solids. The structure of 1,9-nonedioic acid
(8) was thoroughly characterized by GC/MS analysis using
the authentic compound, which was purchased from Sigma
(Fig. S8); Mp 109110°C (lit.[9] Mp 109110°C). The
structure of 3-hydroxynonanoic acid (5) was characterized
by GC/MS and NMR analysis. 3-Hydroxynonanoic acid
recombinant E. coli was conducted in a 1 L scale reactor
[17a]
(Biotron, Bucheon, Korea), as previously reported
.
Agitation speed and aeration rate were 400 to 1000 rpm
and 1 vvm, respectively, to avoid oxygen limitation.
Product analysis by gas chromatography/
mass spectrometry (GC/MS)
The concentrations of the remaining fatty acids and
accumulating carboxylic acids in the medium (e.g.,
ricinoleic acid (1), 3-hydroxynonanoic acid (5), 9-
hydroxynonanoic acid (6), 1,9-nonedioic acid (8)) were
determined as described previously [2a,2e]. The reaction
medium was mixed with an equal volume of ethyl acetate
containing 0.5 g/L methyl palmitate as an internal standard.
The organic phase was harvested after vigorous vortexing
and subjected to derivatization with N-methyl-N-
(trimethylsilyl) trifluoroacetamide. The trimethylsilyl
derivatives were analyzed using a Thermo Ultra Trace GC
system connected to an ion trap mass detector (Thermo
1
(5): Mp 53-55°C; H NMR (500 MHz; DMSO-d6) δ 3.80-
3.78 (m, 1H), 2.26 (dd, 1H, J= 15, 5 Hz), 2.21(dd, 1H, J=
15, 10 Hz), 1.34-1.32 (m, 4H), 1.27-1.20 (m, 6H), 0.85 (t,
J=15 Hz, 3H); 13C NMR (125 MHz; DMSO-d6) δ 173.03
(COOH), 67.1 (C-OH), 42.72, 36.96, 31.34, 28.76, 25.06,
22.10, 13.95 (Fig. S7).
Acknowledgements
ITQ1100 GC-ion Trap MS, Thermo Scientific, Indianapolis, This study was supported by the Korea Research Foundation
(KRF) grant funded by the Korea government and the Marine
Biomaterials Research Center grant from the Marine
Biotechnology Program funded by the Ministry of Oceans
IN, USA). The derivatives were separated on a non-polar
capillary column (30 m length, 0.25 m film thickness,
HP-5MS, Agilent Technologies, Palo Alto, CA, USA). A
linear temperature gradient was programmed as 90°C,
and
Fisheries,
Republic
of
Korea
(No.
D11013214H480000100).
6
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10.1002/adsc.201701029
Advanced Synthesis & Catalysis
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7
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10.1002/adsc.201701029
Advanced Synthesis & Catalysis
FULL PAPER
Escherichia coli
FadL
Simultaneous Enzyme/Whole-Cell
OH OH
OH OH
OH
OH
O
5
5
O
Biotransformation of C18 Ricinoleic Acid into (R)-
3-Hydroxynonanoic Acid, 9-Hydroxynonanoic
Acid, and 1,9-Nonanedioic Acid
SADH
BVMO
OH
O
O
OH
O
O
O
OH
O
OH
5
5
Esterase
Adv. Synth. Catal. Year, Volume, Page – Page
OH
O
OH
O
O
+
HO
OH
HO
OH
PADH
AlDH
Hee-Jeong Cha, Eun-Ji Seo, Ji-Won Song, Hye-Jin
Jo, Akula Ravi Kumar, and Jin-Byung Park*
O
O
O
O
HO
OH
HO
Up to ca.O7H0% yield
FadL : Long chain fatty acid transporter
SADH : Long chain secondary alcohol dehydrogenase
BVMO : Baeyer-Villiger monooxygenase
PADH : Primary alcohol dehydrogenase
AlDH : Aldehyde dehydrogenase
Regiospecific oxyfunctionalization of renewable long chain fatty acids into industrially relevant C9 carboxylic acids was
examined. (R)-3-Hydroxynonanoic acid, 9-hydroxynonanoic acid and 1,9-nonanedioic acid for polyesters and polyamides
could be produced to a high yield from C18 ricinoleic acid by simultaneous enzyme/whole cell biocatalysis.
8
This article is protected by copyright. All rights reserved.