Combined Biocatalytic and Chemical Transformations of Oleic Acid to ω-Hydroxynonanoic Acid and α,ω-Nonanedioic Acid

Article abstract of DOI:10.1002/adsc.201600216

A practical chemoenzymatic method for the synthesis of 9-hydroxynonanoic acid and 1,9-nonanedioic acid (i.e., azelaic acid) from oleic acid [(9Z)-octadec-9-enoic acid] was investigated. Biotransformation of oleic acid into 9-(nonanoyloxy)nonanoic acid via 10-hydroxyoctadecanoic acid and 10-keto-octadecanoic acid was driven by a C-9 double bond hydratase from Stenotrophomonas maltophilia, an alcohol dehydrogenase from Micrococcus luteus, and a Baeyer–Villiger monooxygenase (BVMO) from Pseudomonas putida KT2440, which was expressed in recombinant Escherichia coli. After production of the ester (i.e., the BVMO reaction product), the compound was chemically hydrolyzed to n-nonanoic acid and 9-hydroxynonanoic acid because n-nonanoic acid is toxic to E. coli. The ester was also converted into 9-hydroxynonanoic acid and the n-nonanoic acid methyl ester, which can be oxygenated into the 9-hydroxynonanoic acid methyl ester by the AlkBGT from P. putida GPo1. Finally, 9-hydroxynonanoic acid was chemically oxidized to azelaic acid with a high yield under fairly mild reaction conditions. For example, whole-cell biotransformation at a high cell density (i.e., 10 g dry cells/L) allowed the final ester product concentration and volumetric productivity to reach 25 mM and 2.8 mM h^?1, respectively. The overall molar yield of azelaic acid from oleic acid was 58%, based on the biotransformation and chemical transformation conversion yields of 84% and 68%, respectively. (Figure presented.).

Full text of DOI:10.1002/adsc.201600216

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DOI: 10.1002/adsc.201600216
Combined Biocatalytic and Chemical Transformations of Oleic
Acid to w-Hydroxynonanoic Acid and a,w-Nonanedioic Acid
Satish Koppireddi,+a Joo-Hyun Seo,+b Eun-Yeong Jeon,b
Partha Sarathi Chowdhury,a Hyun-Young Jang,b Jin-Byung Park,b,*
and Yong-Uk Kwona,*
a
Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760, Republic of Korea
Fax : (+82)-2-3277-2384/4213; phone: (+82)-2-3277-4509/6685; e-mail: yukwon@ewha.ac.kr
Department of Food Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea
b
E-mail: jbpark06@ewha.ac.kr
+
S. Koppireddi and J.-H. Seo equally contributed to this work.
Received: February 27, 2016; Revised: July 26, 2016; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201600216.
Abstract: A practical chemoenzymatic method for nonanoic acid methyl ester, which can be oxygenated
the synthesis of 9-hydroxynonanoic acid and 1,9-no- into the 9-hydroxynonanoic acid methyl ester by the
ACHTUNGTRENNUNGnanedioic acid (i.e., azelaic acid) from oleic acid AlkBGT from P. putida GPo1. Finally, 9-hydroxyno-
[(9Z)-octadec-9-enoic acid] was investigated. Bio- nanoic acid was chemically oxidized to azelaic acid
transformation of oleic acid into 9-(nonanoyloxy)no- with a high yield under fairly mild reaction condi-
nanoic acid via 10-hydroxyoctadecanoic acid and 10- tions. For example, whole-cell biotransformation at
keto-octadecanoic acid was driven by a C-9 double a high cell density (i.e., 10 g dry cells/L) allowed the
bond hydratase from Stenotrophomonas maltophilia, final ester product concentration and volumetric pro-
an alcohol dehydrogenase from Micrococcus luteus, ductivity to reach 25 mM and 2.8 mMhÀ1, respective-
and a Baeyer–Villiger monooxygenase (BVMO) ly. The overall molar yield of azelaic acid from oleic
from Pseudomonas putida KT2440, which was ex- acid was 58%, based on the biotransformation and
pressed in recombinant Escherichia coli. After pro- chemical transformation conversion yields of 84%
duction of the ester (i.e., the BVMO reaction prod- and 68%, respectively.
uct), the compound was chemically hydrolyzed to n-
nonanoic acid and 9-hydroxynonanoic acid because
n-nonanoic acid is toxic to E. coli. The ester was also Keywords: carboxylic acids; enzyme catalysis; fatty
converted into 9-hydroxynonanoic acid and the n- acids; oxidation
Introduction
acid) is also used in the preparation of flavors, plasti-
cizers, and lacquers.
Azelaic acid (1,9-nonanedioic acid) is widely used as
n-Nonanoic acid, 9-hydroxynonanoic acid, and aze-
a building block and/or intermediate for the produc- laic acid are manufactured via the ozonolysis of C9
tion of polyamides and polyesters, pharmaceuticals, and C10 unsaturated fatty acids (e.g., oleic acid) fol-
plasticizers, lubricants, or hydraulic fluids.[1] It is also lowed by the reduction of the intermediate ozonide-
utilized for food packaging (e.g., paper, film, and foil s.[1a,3] A biological synthesis of azelaic acid has also
laminates) and in the electronics, textiles, and auto- been reported.[1a] Azelaic acid is naturally produced
motive industries. 9-Hydroxynonanoic acid can be in small amounts from oleic acid by Malassezia furfur
used for the production of nonanolactone, which is on human skin. It is biotechnologically produced from
a 10-membered ring lactone, and is an important in- nonane via fermentation with Candida tropicalis.[3]
termediate and a useful monomer for producing ali- However, C. tropicalis is known as a pathogen and
phatic polyesters or polylactones via ring-opening nonane should be obtained from non-renewable bio-
polymerization.[2] n-Nonanoic acid (i.e., pelargonic mass and petrochemical feedstocks. Recently, the bio-
synthesis of 9-hydroxynonanoic acid and azelaic acid
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verted into 9-hydroxynonanoic acid and n-nonanoic
acid methyl ester (8), which can be oxygenated into
the 9-hydroxynonanoic acid methyl ester (9) by the
AlkBGT from P. putida GPo1.[7] Finally, 9-hydroxyno-
nanoic acid was chemically oxidized to azelaic acid
with a high yield under fairly mild reaction condi-
tions.
Results and Discussion
Production of the Ester 4 from 10-Hydroxystearic
Acid
The preparation of 10-hydroxystearic acid (i.e., 10-hy-
droxyoctadecanoic acid) with a high concentration by
biotransformation of oleic acid with an oleate hydra-
tase from Stenotrophomonas maltophilia has been re-
ported previously.[5a] Therefore, the bioconversion of
10-hydroxystearic acid into the ester (4) (Scheme 1)
was first investigated by using the recombinant E. coli
BL21(DE3):pACYC-ADH, pJOE-BVMO expressing
the ADH from M. luteus and the BVMO from P.
putida KT2440, which has been previously reported
(Figure 1).[8]
The addition of 5 mM 10-hydroxystearic acid into
the recombinant E. coli culture at the stationary
growth phase led to the initiation of the biotransfor-
mation. The ester 4 was produced with a final concen-
tration of 4.1 mM in the medium, 12 h after the initia-
tion of the biotransformation (Figure 1A). The con-
centration of the reaction intermediate [i.e., 10-keto-
Scheme 1. Designed biotransformation pathway. Oleic acid
(1) is enzymatically converted into ester 4 (see the Results
section for details), which can be hydrolyzed into n-nonano-
ic acid (5) and 9-hydroxynonanoic acid (6). Adopted from
our previous study.[4]
AHCTUNGTREGUNsNN tearic acid (3)] in the reaction medium remained
below 0.1 mM until t=8 h. After this period, it began
from oleic acid was reported.[4] Oleic acid was first hy- to accumulate in the medium, indicating that the
drated to 10-hydroxystearic acid by an oleate hydra- BVMO reaction rate was gradually decreasing.
tase[4a,5] and then further oxidized to 9-(nonanoyloxy)-
The ester (4) was hydrolyzed by an esterase to and biocatalyst concentrations in the reaction
yield n-nonanoic acid and 9-hydroxynonanoic acid. 9- medium. The biotransformation was carried out after
Hydroxynonanoic acid was then further oxidized to fed-batch cultivation in a 1-L scale bioreactor, as re-
azelaic acid by the alcohol dehydrogenase (AlkJ) of ported previously.[8] When the recombinant E. coli
Pseudomonas putida GPo1.[4b,6] The multistep bio- BL21(DE3):pACYC-ADH, pJOE-BVMO had grown
transformation was carried out by recombinant Es- to 10 g cell dry weight (CDW)/L, 30 mM 10-hydroxy-
cherichia coli expressing the catalytic enzymes in
ACHTUNGTRENsNGNU tearic acid were added into the culture broth (Fig-
whole-cell assay conditions after cell cultivation, har- ure 1B). The reaction dynamics were similar to those
vest, and resuspension into a buffer solution. The of the whole-cell biotransformation in the flask; 10-
final product concentration was a few mM in the reac- hydroxystearic acid was stoichiometrically converted
tion medium.
into the ester 4 via 10-ketostearic acid (3). However,
The goal of this study was to develop a chemo-en- the final product concentration and volumetric pro-
zymatic process that allows the efficient production of ductivity were increased up to 21 mM and 1.4 mMhÀ1,
high concentrations of n-nonanoic acid, 9-hydroxyno- respectively, which were 5- and 4-fold higher than in
nanoic acid, and azelaic acid from renewable biomass the experiment shown in Figure 1A.
(i.e., oleic acid). To realize this goal, the ester (4) was
chemically hydrolyzed into n-nonanoic acid (5) and 9-
hydroxynonanoic acid (6). The ester (4) was also con-
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a solid in water. The solubility of the compound in
solvents is also quite low. As a consequence, it can be
very difficult to distribute into an aqueous reaction
medium. Therefore, we investigated the bioconversion
of oleic acid (1) into the ester (4). The first approach
was to use the recombinant E. coli BL21(DE3):pE-
T28a-OhyA, pACYC-ADH-BVMO, expressing the
hydratase from S. maltophilia, the ADH from M.
luteus, and the BVMO from P. putida KT2440. The
recombinant biocatalysts were able to catalyze the
conversion of oleic acid into the ester (4) (Table 1
and Supporting Information, Figure S2A). However,
the rate of ester formation was very low, probably be-
cause of low enzyme activity of the BVMO and ADH
in the recombinant cells. Therefore, the target gene
expression system was changed to pACYC-OhyA-
ADH, pJOE-BVMO to reduce the expression of the
OhyA while enhancing the expression of the BVMO
and ADH. The newly constructed recombinant E.
coli BL21(DE3):pACYC-OhyA-ADH, pJOE-BVMO
showed a rather higher BVMO reaction rate com-
pared to the E. coli BL21(DE3):pET28a-OhyA,
pACYC-ADH-BVMO (Table 1 and Supporting Infor-
mation, Figure S2B). To further improve the rate of
ester formation by increasing the expression levels of
both ADH and hydratase, E. coli BL21(DE3):pCO-
LA-ADH-OhyA, pJOE-BVMO was constructed; the
pCOLA vector has a ca. 3-fold higher copy number
(i.e., 20–40) than pACYC.[9] The enhanced expression
of ADH and OhyA in the recombinant cells allowed
the increase of the biotransformation rate (Table 1
and Supporting Information, Figure S2C). Another
approach was to use a two-cell system, as previously
reported for the production of e-caprolactone from
cyclohexanol via cyclohexanone[10] and of perillyl ace-
tate from glucose via limonene.[11] E. coli
BL21(DE3):pET28a-OhyA and E. coli BL21(DE3):-
pACYC-ADH, pJOE-BVMO were independently
grown to enhance the expression levels of the cascade
enzymes. Following this, E. coli BL21(DE3):pET28a-
OhyA was added into culture of E. coli BL21(DE3):-
pACYC-ADH, pJOE-BVMO at a ratio of 1 to 3. No-
tably, the rate of ester formation from oleic acid was
significantly increased up to 4.6 U/g CDW, which was
ca. 7 times greater than that of E. coli BL21(DE3):-
pET28a-OhyA, pACYC-ADH-BVMO (Table 1 and
Supporting Information, Figure S2D). These results
indicate that the rate of biotransformation of oleic
acid to the ester (4) was dependent upon the expres-
Figure 1. Time course of the biotransformation of 10-hy-
droxystearic acid (2) by the recombinant E. coli
BL21(DE3):pACYC-ADH, pJOE-BVMO, expressing the
ADH from M. luteus and the BVMO from P. putida
KT2440. The biotransformation was initiated at the station-
ary growth phase of the batch culture (cell density: 3 g
CDW/L) (A) or at the cell density of 10 g CDW/L (B) after
fed-batch cultivation in a Riesenberg medium. 5 mM (A) or
30 mM (B) of the reaction substrate was added into the cul-
ture broth, to which 0.5 gLÀ1 of Tween 80 was then added.
The symbols indicate the concentrations of 10-hydroxystea-
!
!
&
ric acid (2) ( ), 10-ketooleic acid (2) ( ), and 3 ( ). The
error bars indicate standard deviations.
Biosynthesis of the Ester 4 from Oleic Acid
The whole-cell biotransformation rate in the experi- sion level of the cascade enzymes in the E. coli cells.
ment shown in Figure 1B was 2.3 U/g CDW, which is Next, the oleic acid biotransformation was conduct-
ca. 4-fold lower than the whole-cell biotransformation ed at a high cell density, based on the experiment
rate of ricinoleic acid under similar conditions.[8] One shown in Figure 1B (Figure 2). Mixing of the E. coli
of the factors influencing the biotransformation rate BL21(DE3):pET28a-OhyA and E. coli BL21(DE3):-
might include substrate transport to the catalytic en- pACYC-ADH, pJOE-BVMO strains in a ratio of 1 to
zymes inside cells because the starting material (i.e., 5 in the reaction medium allowed the whole-cell ester
10-hydroxystearic acid) is barely soluble and exists as formation rate to increase up to 2.8 mMhÀ1 and
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Table 1. Catalytic performance of recombinant E. coli-based biocatalysts[a]
Strains/biocatalysts
Initial ester (4) production rate Final ester (4) concentra-
[U/g CDW][b]
tion [mM][c]
E. coli BL21(DE3):pET28a-OhyA, pACYC-ADH-BVMO
E. coli BL21(DE3):pACYC-OhyA-ADH, pJOE-BVMO
E. coli BL21(DE3):pCOLA-ADH-OhyA, pJOE-BVMO
E. coli BL21(DE3):pET28a-OhyA and
0.6
1.1
1.7
4.6
0.8
1.5
2.8
5.2
E. coli BL21(DE3):pACYC-ADH, pJOE-BVMO
[a]
The cell cultivation and reaction conditions were the same as the experiment shown in Figure 1A, except for the substrate
used. The cell density was 3 g CDW/L. Oleic acid was added instead of 10-hydroxystearic acid into the culture broth at
the stationary growth phase.
The initial ester (4) production rates were calculated based on the product concentration, which was measured at t=1.0
or 2.0 h by GC/MS (see the reaction profile in the Supporting Information, Figure S2).
The final ester (4) concentrations were measured at t=11 or 9 h by GC/MS (see the reaction profile in the Supporting In-
formation, Figure S2).
[b]
[c]
4.6 U/g CDW. The specific ester formation rate was
identical to that achieved in the flask experiment
shown in the Supporting Information, Figure S2D.
However, this value is two-fold greater than that ob-
tained in the experiment shown in Figure 1B
(Table 2). In situ generation of the 10-hydroxystearic
acid appeared to contribute to the substantial increase
of the specific product formation rate.
In summary, overexpression of the cascade enzymes
in whole-cell biocatalysts, in situ generation 10-hy-
droxystearic acid via a two-strain approach, and the
application of higher cell densities allowed a high
volumetric productivity and high product concentra-
tion in the culture medium.
Regarding the regeneration of the nicotinamide co-
factors (Scheme 1) during whole-cell biotransforma-
tion, we assume that a small fraction of the fatty acid
Figure 2. Time course of the biotransformation of oleic acid
by the recombinant E. coli BL21(DE3):pET-OhyA express-
ing the oleate hydratase of S. maltophilia and E. coli
BL21(DE3):pACYC-ADH, pJOE-BVMO. The biotransfor-
mation was initiated by adding 30 mM oleic acid, 0.5 gLÀ1
Tween 80, and 1 g CDW/L of E. coli BL21(DE3):pET-
OhyA into the E. coli BL21(DE3):pACYC-ADH, pJOE-
BVMO culture broth (cell density: 10 g CDW/L). Symbols
substrates were used as a carbon and energy source
for E. coli. Cofactor regeneration by the intracellular
transhydrogenases (e.g., PntAB) is another possibility.
The NADH cofactor, which is generated from the
second reaction by the alcohol dehydrogenase, could
be converted into NADPH by the PntAB, which is
oxidized during the third reaction by the BVMO. The
serial reactions by the alcohol dehydrogenase, PntAB,
and BVMO might allow the coupling of cofactor re-
generation and maintenance of redox balance in cells.
*
indicate the concentrations of oleic acid (1) ( ), 10-hydrox-
!
!
&
ystearic acid (2) ( ), 10-ketostearic acid (3) ( ), and 4 ( ).
The error bars indicate standard deviations.
Table 2. Biocatalytic performance of recombinant E. coli-based biocatalyst[a]
Reaction at fed-batch cultivation I
Reaction at fed-batch cultivation II
Substrate concentration [mM]
Biocatalyst concentration [g CDW/L]
Biotransformation time [h]
Final product concentration [mM]
Spedific product formation rate [U/g CDW]
Volumetric productivity [mMhÀ1]
30
10
15
21
2.3
1.4
30
10
9
25
4.6
2.8
[a]
Reaction in fed-batch cultivation I and II was shown in Figure 1B and Figure 2, respectively.
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Optimization of the transhydrogenase activity would cause the actual conversion efficiency might be more
lead to further increases of the whole-cell biotransfor- than 95% before recrystallization. However, these ox-
mation rates.
idation conditions are relatively expensive and the
chromium-based oxidant is generally considered eco-
unfriendly. Thus, other oxidation conditions were in-
vestigated with the aim to develop a green catalytic
process that is feasible for large-scale applications.
Many oxidation conditions were tried by employing
Chemical Conversion of the Ester Intermediate to
Azelaic Acid
One of the major factors limiting the fatty acid bio- different oxidants. The two best oxidation conditions
transformation productivity of whole-cell biocatalysts were successfully developed, and further modified
may include the toxic effects of fatty acids on the host and optimized by many screening reactions from the
cells (e.g., E. coli).[8,12] For instance, the specific reported conditions.[13] At first, hydroxynonanoic acid
growth rate of E. coli was reduced by approximately (6) was easily oxidized to azelaic acid (7) with 94%
50% in the presence of 5 mM n-nonanoic acid (Sup- yield with NaClO2 (1.2 equiv.), 2,2,6,6-tetramethyl-pi-
porting Information, Figure S1). Therefore, we inves- peridin-1-yl oxyl (TEMPO) (4 mol%), and NaOCl
tigated the chemical hydrolysis of the ester into n- (2 mol%) in aqueous acetonitrile at room tempera-
nonanoic acid and 9-hydroxynonanoic acid.
ture for 8 h (Method B).[13a] In addition, hydroxynona-
The ester intermediate (4) was purified by extrac- noic acid (6) was also oxidized to azelaic acid (7) with
tion and column chromatography to approximately 93% yield by using trichloroisocyanuric acid (TCCA)
85%, based on the measured concentration of the (1.5 equiv.), TEMPO (2 mol%), NaBr (10 mol%), and
ester after biotransformation. Then, the ester inter- 10% aqueous NaHCO3 in acetone at room tempera-
mediate (4) was successfully converted to azelaic acid ture for 4 h (Method C).[13b] The TEMPO-catalyzed
(7) by successive two-step reactions, including the hy- oxidation procedure allowed excellent conversion
drolysis of the ester functionality and the oxidation of yields under mild reaction conditions and easy isola-
the alcohol functionality (Scheme 2). At first, the tion of the target products. Additionally, the proce-
ester functional group was efficiently hydrolyzed with dure required only cheap reagents and a catalytic
sodium hydroxide in a co-solvent of methanol/water amount of TEMPO under no buffer conditions, indi-
(4/1) at 608C to afford 9-hydroxynonanoic acid (6) in cating a rather low production cost. Method B with
approximately 85% yield. Finally, the oxidation of the NaClO2, TEMPO, and NaOCl is preferable because it
terminal hydroxyl group of compound 6 to carboxylic might be more advantageous in terms of economic
acid was examined.
cost and the product (azelaic acid), which was very
Initially, periodic acid in the presence of a catalytic pure after simple extractive work-up without any pu-
amount of chromium trioxide was used, as in rification techniques such as column chromatography
a method previously developed by us.[8] 9-Hydroxyno- and recrystallization. The overall yields were approxi-
nanoic acid (6) was treated with periodic acid (H5IO6) mately 80% over two chemical steps and approxi-
(2.5 equiv.) in the presence of a catalytic amount of mately 68% based on both purification of the ester in-
chromium trioxide (CrO3) (0.01 equiv.) in wet aceto- termediate after biotransformation and the two-step
nitrile (0.75% water) at 08C to give azelaic acid (7) in chemical syntheses, respectively. All the intermediates
approximately 80% yield after recrystallization from were fully confirmed by their NMR spectra (see the
ethyl acetate-hexane (Method A). The periodic acid- Experimental Section and the Supporting Information
mediated oxidation conditions are likely useful be- for details).
Scheme 2. Chemical conversion of the ester intermediate (4) to azelaic acid (7).
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Similarly to the preparation of undecanedioic acid Conclusions
through biotransformation and chemical reactions,[8]
azelaic acid (7) was prepared via direct coupling of A practical chemo-enzymatic synthesis of C9 carbox-
hydrolysis and oxidation, followed by simple recrys- ylic acids, including 9-hydroxynonanoic acid (6) and
tallization from the crude ester intermediate after bio- azelaic acid (7), from oleic acid was examined. The
transformation. The overall yield of azelaic acid (7) chemo-enzymatic process allowed the production of
was approximately 58% by recrystallization, which is the target compounds with rather high concentrations
expected to be improved in a large-scale preparation. by circumventing the toxic effects of medium-chain
fatty acids on the host cells; after biotransformation
of oleic acid into 9-(nonanoyloxy)nonanoic acid (4)
Production of Methyl Nonanoate and 9-
Hydroxynonanoic Acid from the Ester (4)
by the novel whole-cell biocatalytic process developed
in this study (Figure 2), the ester was chemically hy-
drolyzed into 9-hydroxynonanoic acid and n-nonanoic
Transesterification of the ester intermediate (4) with acid with a high yield. The ester (4) was also convert-
methanol into 9-hydroxynonanoic acid (6) and n-no- ed into 9-hydroxynonanoic acid and the n-nonanoic
ACHTUNGTRENNUNGnanoic acid methyl ester (8) was investigated because acid methyl ester (8), which can be oxygenated into
the methyl ester (8) can be oxygenated into the 9-hy- the 9-hydroxynonanoic acid methyl ester (9) by the
droxynonanoic acid methyl ester (9) with a high yield AlkBGT from P. putida GPo1. Finally, 9-hydroxyno-
by the AlkBGT from P. putida GPo1 (Scheme 3).[7,14] nanoic acid was chemically oxidized to azelaic acid
The methyl ester (8) can also be converted into the 9- (7) with a high yield under mild reaction conditions.
aminononanoic acid methyl ester by serial combina- This study demonstrates that industrially relevant C9
tions of the AlkBGT from P. putida GPo1 and w- chemicals can be prepared from oleic acid with great
transaminase from Chromobacterium violaceum.[15]
efficiency, in terms of carbon economy and environ-
The treatment of the ester (4) with sodium methox- mental friendliness compared to the ozonolysis-based
ide in methanol did not work well, generating only chemical processes. This study will contribute to de-
the hydrolysis products (5, 6). By thorough screening velopment of sustainable processes for the production
of several reaction conditions, dibutyltin oxide-cata- of medium-chain fatty acids from renewable long-
lyzed methanolysis was found to be very successful. chain fatty acids and plant oils.
When the ester (4) was treated with a catalytic
amount of dibutyltin oxide in methanol at reflux con-
ditions for 12 h, the desired products, methyl nonan- Experimental Section
ACHTUNGTRENNUNGoate (8) and 9-hydroxynonanoic acid (6) were pro-
duced with rather high yields (80%). This indicates Microbial Strains and Culture Media
that the industrially relevant chemicals may be pro-
duced via combined whole-cell biotransformation of
oleic acid and methanolysis of the ester (4).
Recombinant E. coli BL21(DE3):pACYC-ADH, pJOE-
BVMO and recombinant E. coli BL21(DE3):pET-OhyA
were grown in a LB medium (5 gLÀ1 yeast extract, 10 gLÀ1
tryptone, and 10 gLÀ1 NaCl) supplemented with the appro-
priate antibiotics (i.e., chloramphenicol and ampicillin) for
seed cultivation. The Riesenberg medium,[16] supplemented
with 10 gLÀ1 glucose and the appropriate antibiotics, was
used for the main cultivation and biotransformation. The
Riesenberg medium consisted of 4 gLÀ1 (NH4)2HPO4,
13.5 gLÀ1 KH2PO4, 1.7 gLÀ1 citric acid, 1.4 gLÀ1 MgSO4, and
10 mLLÀ1 trace metal solution [10 gLÀ1 FeSO4, 2.25 gLÀ1
ZnSO4, 1.0 gLÀ1 CuSO4, 0.5 gLÀ1 MnSO4, 0.23 gLÀ1
Na2B4O7, 2.0 gLÀ1 CaCl2, and 0.1 gLÀ1 (NH4)6Mo7O24]. Re-
combinant gene expression was induced by adding 0.1 mM
isopropyl b-d-thiogalactopyranoside (IPTG) and/or 2 gLÀ1
rhamnose to the culture broth.
Preparation of the Recombinant Plasmid
The pCOLA-ADH-OhyA was constructed by transferring
the alcohol dehydrogenase gene from M. luteus from
pACYC-ADH[4b] and the hydratase gene from S. maltophilia
from pET28a-OhyA[17] into the pCOLA-Duet vector (Sup-
porting Information, Table S1). The ADH gene was ampli-
fied by a polymerase chain reaction (PCR) with P1 and P2
Scheme 3. Chemical conversion of the ester intermediate (4)
to methyl nonanoate (8) and 9-hydroxynonanoic acid (6).
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as the primers (see the primer sequences in the Supporting
Information, Table S2). The hydratase gene was amplified
with P3 and P4 primers. The vector and PCR product were
digested using appropriate restriction enzymes. The digested
vector and PCR product were mixed at a ratio of 1 to 3 and
ligated using T4 DNA ligase (ELPIS-Biotech, Daejeon, Re-
public of Korea).
ester, as reported previously by us.[4a,5a] Low melting white
solid; mp 38–408C; H NMR (300 MHz, CDCl3): d=4.05 (t,
J=6.0 Hz, 2H), 2.40–2.26 (m, 4H), 1.63–1.59 (m, 6H), 1.32–
1.26 (m, 18H), 0.88 (t, J=6.6 Hz, 3H); 13C NMR (75 MHz,
CDCl3): d=179.17, 174.09, 64.34, 34.41, 33.85, 31.85, 31.81,
29.23, 29.14, 29.11, 29.05, 29.03, 28.95, 28.61, 25.86, 25.02,
22.65, 14.10.
1
9-Hydroxynonanoic acid (6): The ester intermediate (4)
(260 mg, 0.83 mmol) was treated with NaOH (1N) in
MeOH-H2O (4/1, 10 mL) at 608C for 3 h. The reaction mix-
ture was cooled down and its pH was adjusted to 2 by slow
addition of aqueous HCl (6N) under ice-cooled conditions.
The reaction mixture was evaporated to reduce the volume
to approximately 2 mL. Water (5 mL) was added, and the
residual solvent was saturated with NaCl and extracted with
ethyl acetate (3ꢁ15 mL). The combined organic extract was
washed with brine (10 mL) and water (10 mL), and dried
over anhydrous Na2SO4, filtered, and evaporated to obtain
a yellow semi-solid material. The crude product was purified
by silica gel column chromatography using 50% ethyl ace-
tate-hexane as an eluent to afford 9-hydroxynonanoic acid
(6) as a low melting white solid; yield: 122 mg (85%); mp
50–528C (lit.[18] mp 49.5–51.08C). 1H NMR (300 MHz,
CDCl3): d=3.64 (t, J=6.0 Hz, 2H), 2.35 (t, J=7.5 Hz, 2H),
1.66–1.52 (m, 4H), 1.38–1.32 (m, 8H); 13C NMR (75 MHz,
CDCl3): d=178.85, 63.03, 33.83, 32.68, 29.14 (2C), 28.93,
25.61, 24.63.
Biotransformation in a Flask and Bioreactor
The biotransformation was carried out as previously repor-
ted.[12a] In brief, the biotransformation was initiated at the
stationary growth phase, usually 8 h after the induction of
gene expression with 0.1 mM IPTG and/or 2 gLÀ1 rhamnose.
After changing the pH of the culture broth to 8.0 and in-
creasing the temperature to 358C, 5–30 mM oleic acid or 10-
hydroxystearic acid and 0.5 gLÀ1 Tween80 were added into
the culture broth containing a cell concentration of 3 to 10 g
dry cells/L. Cultivation and biotransformation was per-
formed in a 250-mL flask (working volume: 20 mL) in
a shaking incubator (200 rpm). The bioreactor experiment
was conducted in a 1-L scale reactor (Biotron, Bucheon,
Korea). The agitation speed and aeration rate were set at
400–1000 rpm and 1 vvm, respectively, to avoid any oxygen
limitation during cultivation and biotransformation.
Product Analysis by Gas Chromatography/Mass
Spectrometry (GC/MS)
Azelaic acid (7), Method A:[8] A stock solution of H5IO6/
CrO3 was prepared by dissolving periodic acid (H5IO6)
(11.4 g, 50 mmol) and CrO3 (23 mg, 0.23 mmol) in wet ace-
tonitrile (0.75% water) to a volume of 114 mL (complete
dissolution typically required 1–2 h). The H5IO6/CrO3 stock
solution (3.25 mL, 2.5 equiv. of H5IO6 and 0.01 equiv. of
CrO3) was then added to a solution of compound 6 (100 mg,
0.57 mmol) in wet acetonitrile (4 mL, 0.75% water) while
maintaining the reaction temperature at 0–58C for 30 min.
The mixture was aged at 0–58C for 45 min. The reaction
was quenched by adding an aqueous solution of Na2HPO4
(0.2 g in 2 mL H2O). Ethyl acetate (10 mL) was added and
stirred. The organic layer was separated. The aqueous part
was saturated with NaCl and extracted further with 10%
MeOH-ethyl acetate (4ꢁ10 mL). The combined organic
layer was washed with brine (10 mL), 5% NaHSO3 (10 mL),
and brine (10 mL). The organic layer was dried over anhy-
drous Na2SO4 and then concentrated to give the crude dicar-
boxylic acid, which was further purified by recrystallization
from ethyl acetate-hexane to yield pure azelaic acid (7) as
a white solid; yield: 85 mg (79%).
Concentrations of the remaining fatty acids and accumulat-
ing carboxylic acids in the medium [e.g., oleic acid (1), 10-
hydroxystearic acid (2), 10-ketostearic acid (3), and the
ester (4)] were determined as described previously.[4a] The
reaction medium was mixed with an equal volume of ethyl
acetate containing 0.1 or 0.5 gLÀ1 methyl palmitate as an in-
ternal standard. The organic phase was harvested after vigo-
rous vortexing and then subjected to derivatization with N-
methyl-N-(trimethylsilyl)trifluoroacetamide (TMS). The
TMS derivatives were analyzed using a Thermo Ultra Trace
GC system connected to an ion trap mass detector (Thermo
ITQ1100 GC-ion Trap MS, Thermo Scientific, Indianapolis,
IN, USA). The derivatives were separated on a non-polar
capillary column (30 m length, 0.25 mm film thickness, HP-
5MS, Agilent Technologies, Palo Alto, CA, USA). A linear
temperature gradient was programmed as 908C, 58CminÀ1
to 2808C. The injection port temperature 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 was used for the detection
and fragmentation analysis of the reaction products.
Azelaic acid (7), Method B:[13a] A 50-mL two-necked,
round-bottomed flask, equipped with a condenser was
charged with compound 6 (0.13 g, 0.74 mmol), TEMPO
Step-by-Step Chemical Conversion of the Ester
Intermediate (4) to Azelaic Acid (7)
(2,2,6,6-tetramethyl-piperidin-1-yl
oxyl)
(4.6 mg,
0.029 mmol, 4 mol%), and 6 mL of acetonitrile. A solution
of sodium chlorite was prepared by dissolving 80% NaClO2
(81 mg, 0.89 mmol) in 1.0 mL of water, and a solution of
dilute sodium hypochlorite (NaOCl, 0.5 mL) was prepared
by diluting household bleach (5.25% NaOCl, 0.53 mL, ca.
2.0 mol% with 9.5 mL of water). Approximately 20% of the
NaClO2 solution was added from a syringe, followed by
20% of the diluted bleach solution. The remaining portions
of both reagents were then added simultaneously over
30 min. The resulting mixture was stirred at room tempera-
9-(Nonanoyloxy)nonanoic acid (4): The purification of the
crude ester, which was obtained by extraction, was done by
column chromatography (silica gel) with eluting by 20%
ethyl acetate-hexane. The isolation/recovery yield of the
ester (4) was approximately 85%, based on the measured
concentration of the ester by GC/MS analysis after the bio-
transformation reaction. In addition to the following NMR
analysis, the structure of the ester (4) was also thoroughly
characterized by GC/MS analysis after the silylation of the
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ture for 8 h. Water (7.5 mL) was added and the pH was ad-
justed to 8.0 by the addition of ca. 1.2 mL of 2.0N NaOH.
The reaction mixture was then poured into ice-cold sodium
sulfite solution (0.34 g in 4.4 mL of water), maintained
below 208C with an ice/water bath. After stirring for 15 min
at room temperature, 8 mL of methyl tert-butyl ether
(MTBE) was added and the resulting mixture was stirred
for 15 min. The organic layer was separated and discarded.
More MTBE (8 mL) was added and the rapidly stirred mix-
ture was acidified with 2.0N HCl to pH 3–4. The organic
layer was separated and the aqueous layer was extracted
with 2ꢁ8 mL portions of MTBE. The combined organic
phases were washed with two 6 mL portions of water and
6 mL of brine, and then concentrated to give almost pure
azelaic acid (7) as a white solid without further recrystalliza-
tion; yield: 131 mg (94%).
mixture was poured into a saturated sodium bicarbonate so-
lution (5.0 mL) and extracted three times with ethyl acetate.
The combined organic layers, which contained the dibutyltin
oxide as a fine white precipitate, were filtered through
celite, dried over sodium sulfate, and filtered. After removal
of the solvent under vacuum, methyl nonanoate (8) was pu-
rified by vacuum distillation as a colorless liquid; yield:
110 mg (80%); bp 728C (10 mmHg) (lit.[20] bp 1228C
1
(45 mmHg)); H NMR (300 MHz, CDCl3): d=3.62 (s, 3H),
2.26 (t, J=7.5 Hz, 2H), 1.60–1.56 (m, 2H), 1.29–1.20 (m,
10H), 0.84 (t, J=6.0 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d=173.84, 51.03, 33.83, 31.68, 29.10, 29.02, 29.00, 24.79,
22.49, 13.82.
Azelaic acid (7), Method C:[13b] A 10% aqueous solution
of NaHCO3 (2.25 mL) was added to a solution of compound
6 (0.13 g, 0.74 mmol) in acetone (8 mL) at 08C, followed by
solid NaBr (7.6 mg, 10 mol) and TEMPO (2,2,6,6-tetrameth-
yl-piperidin-1-yl oxyl) (2.3 mg, 0.015 mmol, 2 mol%). Tri-
chloroisocyanuric acid (TCCA) (0.258 g, 1.11 mmol) was
then slowly added within 20 min at 08C. The resulting mix-
ture was warmed to room temperature and stirred for the
required time (4 h) until the completion of the reaction, and
then 2-propanol (0.4 mL) was added. The mixture was fil-
tered on celite, concentrated under vacuum, and treated
with 2.7 mL of a saturated solution of Na2CO3. The aqueous
phase was washed with two portions of 8 mL of MTBE,
treated with 1N HCl, and extracted twice with 10 mL of
10% MeOH-ethyl acetate. The organic layers were dried
(Na2SO4), and the solvent was evaporated to yield almost
pure azelaic acid (7) as a white solid without further recrys-
tallization; yield: 130 mg (93%).
Acknowledgements
This study was supported by the R&D Program of MOTIE/
KEIT, Republic of Korea [10044604 and 10044647]. J.-H.
Seo was partially supported by Ewha Womans University
(RP-Grant 2015).
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10 Combined Biocatalytic and Chemical Transformations of
Oleic Acid to w-Hydroxynonanoic Acid and a,w-
Nonanedioic Acid
Adv. Synth. Catal. 2016, 358, 1 – 10
Satish Koppireddi, Joo-Hyun Seo, Eun-Yeong Jeon,
Partha Sarathi Chowdhury, Hyun-Young Jang,
Jin-Byung Park,* Yong-Uk Kwon*
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