Multistep enzymatic synthesis of long-chain α,ω-Dicarboxylic and ω-hydroxycarboxylic acids from renewable fatty acids and plant oils

Article abstract of DOI:10.1002/anie.201209187

A multistep enzyme catalysis was successfully implemented to produce long-chain α,ω-dicarboxylic and ω-hydroxycarboxylic acids from renewable fatty acids and plant oils. Sebacic acid as well as ω-hydroxynonanoic acid and ω-hydroxytridec-11-enoic acid were produced from oleic and ricinoleic acid. Copyright

Full text of DOI:10.1002/anie.201209187

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DOI: 10.1002/anie.201209187
Enzyme Catalysis
Multistep Enzymatic Synthesis of Long-Chain a,w-Dicarboxylic and
w-Hydroxycarboxylic Acids from Renewable Fatty Acids and Plant
Oils**
Ji-Won Song, Eun-Yeong Jeon, Da-Hyun Song, Hyun-Young Jang, Uwe T. Bornscheuer, Deok-
Kun Oh, and Jin-Byung Park*
Global climate change and oil depletion have stimulated the
development of routes to produce renewable biofuels and
chemicals in an environmentally friendly way. Tremendous
scientific and technological developments in biocatalysis have
enabled sustainable processes for the production of biofuels,
chemicals, and polymers from carbohydrate-based biomass.[1]
However, when using the current technology it is sometimes
difficult to obtain high productivity and product yield; this is
particularly true for the production of energy-rich molecules
from plant-derived carbohydrates. For instance, oxygenated
long-chain carboxyl synthons (e.g., a,w-dicarboxylic acids, w-
hydroxycarboxylic acids, alcohols with chain length ꢀ C8), are
difficult to produce in high yields from plant carbohydrates
through biocatalysis. This difficulty is due to problems in the
tight regulation of carbon chain length, especially for
carboxylic acids having chains with an odd number of
carbon atoms, in the deregulation of cellular regulatory
systems, in the efficient regeneration of costly nicotine amide
cofactors, and in the regiospecific oxygenation of the fatty-
acid moiety involved in the biosynthesis.[2]
leic acid, are used annually for the preparation of nylon-6,10.
The majority of dicarboxylic acids are produced from
petrochemical feedstocks or fatty acids by chemical routes
under harsh reaction conditions that require high temper-
ature and pressure, strong acids (e.g., H2SO4, HNO3), and/or
toxic oxidants (e.g., ozone), which cause serious problems and
harm our environment.[3a–c] However, in the past few years
milder methods based on, for example, ruthenium catalysis in
combination with peracetic acid to replace ozonolysis, or the
metathesis reaction of unsaturated fatty acids to yield linear
diacids, have been developed.[4] Biotransformation routes that
use whole cell microorganisms for the preparation of
dicarboxylic acids and their precursors w-hydroxycarboxylic
acids have been also reported.[5] Sebacic acid and azelaic acid
can be produced by fermentation of Candida tropicalis.[3c]
However, in this case, petrochemical feedstocks, such as
decane and nonane, are used as starting materials. Dicarbox-
ylic acids and w-hydroxyfatty acids can be produced from
vegetable oil fatty acids by C. tropicalis.[6] However, the
diversity of the products is significantly constrained by the
limitations in the availability of the reactants used and the
range of intermediates accepted by the metabolic pathways in
C. tropicalis.
Herein, a biocatalytic process was designed and evaluated
for the production of long-chain a,w-dicarboxylic acids (e.g.,
C10) and w-hydroxycarboxylic acids (e.g., C9, C11, C13) from
renewable fatty acids of vegetable and animal origin (e.g.,
oleic acid, ricinoleic acid), which are currently the most
important renewable feedstock of the chemical industry.[4]
Hydration of internal carbon atoms of fatty acids by
a hydratase, oxidation of the hydroxy group to the ketone
by an alcohol dehydrogenase (ADH), Baeyer–Villiger mono-
oxygenase (BVMO) catalyzed oxidation to the ester, and
hydrolysis of this ester, yielded a,w-dicarboxylic acids or w-
hydroxy fatty acids.
Long-chain a,w-dicarboxylic acids and w-hydroxycarbox-
ylic acids are used in the production of a variety of chemical
products and intermediates, such as, nylons and other
polyamides, polyesters, resins, hot-melt adhesives, powder
coatings, corrosion inhibitors, lubricants, plasticizers, greases,
and perfumes.[3] For instance, several 10000 tons of sebacic
acid (1,10-decanedioic acid), which is produced from ricino-
[*] M. Sc. J.-W. Song,[+] M. Sc. E.-Y. Jeon,[+] M. Sc. D.-H. Song,
M. Sc. H.-Y. Jang, Prof. J.-B. Park
Department of Food Science and Engineering
Ewha Womans University
Seoul 120-750 (South Korea)
E-mail: jbpark06@ewha.ac.kr
Prof. U. T. Bornscheuer
Institute of Biochemistry
Department of Biotechnology & Enzyme Catalysis
Greifswald University, 17487 Greifswald (Germany)
The first example was the cleavage of ricinoleic acid (1)
into n-heptanoic acid (4) and w-hydroxyundec-9-enoic acid
(5; Scheme 1). The recombinant Escherichia coli BL21(DE3)
expressing the ADH of Micrococcus luteus NCTC2665 and
the BVMO of Pseudomonas putida KT2440, which were
reported to catalyze oxidation of long-chain secondary
alcohols[7] and long-chain keto hydrocarbons (e.g., 4-dec-
anone),[8] respectively, catalyzed the transformation of rici-
noleic acid (1) into 3 via 12-ketooleic acid (2; Figure 1 and
Supporting Information, Figure S1B). The ester (3) was
produced in a final concentration of 0.76 mm from 1.0 mm
ricinoleic acid. Addition of the cell extract of E. coli BL21,
Prof. D.-K. Oh
Department of Bioscience and Biotechnology, Konkuk University
Seoul 143-701 (South Korea)
[+] These authors contributed equally to this work.
[**] This study was supported by the Marine Biomaterials Research
Center grant from Marine Biotechnology Program funded by the
Ministry of Land, Transport, and Maritime Affairs, Korea.
Supporting information (including full experimental details) for this
article is available on the WWW under http://dx.doi.org/10.1002/
anie.201209187.
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the cleavage pathway (Scheme 2). In addition, some BVMOs
were reported to catalyze abnormal-ester formation, which is
driven by migration of the less-substituted carbon center
during catalysis.[10] As the formation of abnormal esters may
generate different products, another source of BVMO was
also adopted in the synthetic pathway. To evaluate the
performance of the extended pathway, recombinant E. coli
cells that expressed the gene encoding oleate hydratase
(OhyA) from Stenotrophomonas maltophilia[11] in addition to
the ADH of M. luteus and the BVMO of P. putida KT2440,
were constructed and used for the biotransformation of oleic
acid (Figure 2a). Addition of oleic acid (6) to 1.0 mm in the
reaction medium allowed the recombinant biocatalyst to
Scheme 1. Designed biotransformation pathway. Ricinoleic acid (1) is
converted into n-heptanoic acid (4) and w-hydroxyundec-9-enoic acid
(5) by multistep enzyme-catalyzed reactions.
Figure 1. Time course of the biotransformation of ricinoleic acid (1) by
the recombinant E. coli BL21(DE3), expressing the ADH from M. luteus
and the BVMO from P. putida KT2440. The experiments were carried
out in triplicate and the standard deviation was less than 10%. The
Figure 2. Time course of the biotransformation of oleic acid by the
recombinant E. coli BL21(DE3), expressing the oleate hydratase from
S. maltophilia, the ADH from M. luteus and the BVMO from P. putida
KT2440 (a) or the BVMO from P. fluorescens (b). The experiments were
carried out in triplicate and the standard deviation was less than 10%.
~
symbols indicate concentration of ricinoleic acid (1; ), 10-ketooleic
!
&
acid (2; ), 3 ( ), and n-heptanoic acid (4) and w-hydroxyundec-9-
~
enoic acid (5; ).
expressing the esterase gene of P. fluorescens SIK WI, which
has been reported to hydrolyze long-chain esters (e.g., n-
octylacetate),[9] resulted in the formation of n-heptanoic acid
(4) and w-hydroxyundec-9-enoic acid (5) from 3 (Supporting
Information, Figure S1C). According to GC–MS analysis, the
final products (4 and 5) were generated from ricinoleic acid in
a greater than 70% yield. Compound 5 was isolated from the
reaction broth in a yield of over 70% with a purity of
approximately 90% and identified by NMR analysis (Sup-
porting Information, Figures S1D and S6B).
*
The symbols indicate concentration of oleic acid (6; ), 10-hydroxy-
~
!
&
stearic acid (7; ), 10-ketostearic acid (8; ), 9 or 12 ( ), and a) n-
~
nonanoic acid (10) and w-hydroxynonanoic acid (11; ) or b) n-octanol
~
(13) and 1,10-decanedioic acid (14; ).
produce ester 9 via 10-hydroxystearic acid (7) and 10-
ketostearic acid (8; Supporting Information, Figure S2C).
The ester (9) was produced to a concentration of 0.67 mm and
was completely converted into n-nonanoic acid (10) and w-
hydroxynonanoic acid (11) by the esterase from P. fluorescens
(Supporting Information, Figure S2D). Remarkably, the
replacement of the gene encoding BVMO of P. putida
Most fatty acids that are available from plant oils occur as
nonhydroxylated forms.[4] Therefore, an enzyme to catalyze
the formation of hydroxylated fatty acids was introduced into
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w-hydroxytridec-11-enoic acid were pro-
duced from lesquerolic acid (Supporting
Information, Figure S4). Linoleic acid was
converted in a similar fashion into oleic acid
(Supporting Information, Figure S5). This
result indicated that versatile carboxyl syn-
thons can be produced from renewable fatty
acids through multistep biocatalysis involv-
ing fatty acid hydroxylases and BVMOs.
Fatty acids are produced from plant oils.
To further extend the applicability of the
biocatalytic cascade reaction, we finally
developed a biotransformation process that
directly uses a plant oil (i.e., olive oil) as
starting material. Olive oil was hydrolyzed
with lipase and the resulting hydrolyzate
was subjected to the oxidative cleavage
reactions. Lipase treatment of 5 gLÀ1 olive
oil resulted in the production of 11.3 mm
oleic acid and 1.2 mm palmitic acid. The
subsequent biotransformation was con-
ducted by the recombinant E. coli BL21
expressing hydratase, ADH, and BVMO
from P. putida KT2440. The ester 9 was
produced to 6.9 mm in the medium from
oleic acid (6; Figure 3). Hydrolysis of ester 9
Scheme 2. Extended biotransformation pathway. Oleic acid (6) is converted into either n-
nonanoic acid (10) and w-hydroxynonanoic acid (11) or n-octanol (13) and 1,10-decanedioic
acid (14) by multistep enzyme-catalyzed reactions.
Table 1: Products accessible through the multistep biocatalysis.
KT2440 with that of P. fluorescens
DSM 50106[12] enabled the recombi-
nant cells to transform 10-ketostea-
ric acid (8) into 12, which is differs
from 9 in the position of the ester
oxygen atoms in the fatty acid
skeleton, as confirmed by MS anal-
ysis (Supporting Information, Fig-
ure S2E). This change in the reac-
tion pathway resulted in the pro-
duction of n-octanol (13) and 1,10-
decanedioic acid (i.e., sebacic acid;
14) instead of n-nonanoic acid (10)
and w-hydroxynonanoic acid (11;
Figure 2b and Supporting Informa-
tion, Figure S2F). This result indi-
cates that the diversity of the prod-
ucts of the synthetic pathway could
be broadened by employing fatty
acid hydratases and different types
of BVMOs.
Starting material Origin of
BVMO
Final products
Yield
[%][a]
P. fluorescens
>60
>60
5-hydroxydeca-
noic acid
P. putida
P. fluorescens
P. putida
lesquerolic acid
ricinoleic acid
>50
>70
P. fluorescens
P. putida
P. fluorescens
P. putida
>60
>60
oleic acid[b]
P. fluorescens
P. putida
>50
>50
linoleic acid[b]
In addition to ricinoleic acid and
oleic acid, 5-hydroxydecanoic acid,
lesquerolic acid ((11Z, 14R)-14-
hydroxyicos-11-enoic acid), and
linoleic acid were subjected to the
oxidative cleavage by the enzyme
[a] The product yield was calculated based on the substrate depletion and the product concentration
determined by GC–MS 2 h after biotransformation. The substrate was added to 1.0 mm in 50 mm Tris-
HCl buffer (pH 8.0), containing 3.6 g dry cellsLÀ1 of recombinant E. coli biocatalysts.[15] [b] With
coexpression of the oleate hydratase from S. maltophilia.
cascade (Table 1). 5-Hydroxydecanoic acid was degraded into
either 1,5-dicarboxylic acid and n-heptanol or g-hydroxybu-
tyric acid and n-hexanoic acid depending on the BVMO used
(Supporting Information, Figure S3). n-Heptanoic acid and
was mediated by cell extract of E. coli BL21 expressing the
esterase gene from P. fluorescens, because the esterase
reaction products (i.e., nonanoic acid and w-hydroxynonanoic
acid) were toxic to viable E. coli cells. At the end, w-
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biocatalysis can be further broadened by adopting new fatty
acid hydroxylases.
Received: November 16, 2012
Published online: January 30, 2013
Keywords: biotransformations · carboxylic acids ·
.
enzyme catalysis · fatty acids · oxygenation
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Figure 3. Biotransformation of olive oil with lipase and the recombi-
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ADH from M. luteus, and BVMO from P. putida KT2440. Olive oil was
hydrolyzed with lipase in 50 mm Tris-HCl buffer (pH 7.5; data not
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*
symbols indicate concentration of oleic acid (6; ), 10-hydroxystearic
~
!
&
acid (7; ), 10-ketostearic acid (8; ), 9 ( ), and n-nonanoic acid (10)
~
and w-hydroxynonanoic acid (11; ).
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In summary, we developed a synthetic biocatalytic cas-
cade reaction, which can be used to produce high value long-
chain a,w-dicarboxylic and w-hydroxycarboxylic acids from
cost-effective renewable fatty acids and/or plant oils. The
biocatalytic cascade reactions allowed the production of 1,10-
decanedioic acid as well as w-hydroxycarboxylic acids with
chain lengths of C9, C11, and C13; these carboxylic acids can be
converted into a,w-dicarboxylic acids by simple oxidation of
the hydroxy group. In addition, carboxylic acids with chain
lengths of C7 and C9, and primary alcohols with chain length
of C8 were produced from ricinoleic acid, lesquerolic acid,
oleic acid, and linoleic acid (Table 1). The diversity of the
carboxylic products can be extended by a number of ways.
One of them could be to engineer oleate hydratases to accept
other unsaturated fatty acids, such as petroselinic acid ((Z)-
octadec-6-enoate) and erucic acid ((Z)-docos-13-enoic acid),
which are commercially available.[4] Another way is to employ
new sources of fatty acid hydroxylases. For instance, cyto-
chrome P450 monooxygenases[13] and lipoxygenases[14] were
reported to catalyze regiospecific hydroxylation of internal
carbon atoms in the fatty acid skeleton. Therefore, we expect
that the applicability of our synthetic method for multistep
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