Fatty acid desaturation and elongation reactions of trichoderma sp. 1-OH-2-3

Article abstract of DOI:10.1007/s11746-008-1339-2

The fatty acid desaturation and elongation reactions catalyzed by Trichoderma sp. 1-OH-2-3 were investigated. This strain converted palmitic acid (16:0) mainly to stearic acid (18:0), and further to oleic acid (c9-18:1), linoleic acid (c9,c12-18:2), and α-linolenic acid (c9,c12,c15-18:3) through elongation, and Δ9, Δ12, and Δ15 desaturation reactions, respectively. Palmitoleic acid (c9-16:1) and cis-9,cis-12- hexadecadienoic acid were also produced from 16:0 by the strain. This strain converted n-tridecanoic acid (13:0) to cis-9-heptadecenoic acid and further to cis-9,cis-12-heptadecadienoic acid through elongation, and Δ9 and Δ12 desaturation reactions, respectively. trans-Vaccenic acid (t11-18:1) and trans-12-octadecenoic acid (t12-18:1) were desaturated by the strain through Δ9 desaturation. The products derived from t11-18:1 were identified as the conjugated linoleic acids (CLAs) of cis-9,trans-11-octadecadienoic acid and trans-9,trans-11-octadecadienoic acid. The product derived from t12-18:1 was identified as cis-9,trans-12-octadecadienoic acid. cis-6,cis-9-Octadecadienoic acid was desaturated to cis-6,cis-9,cis-12-octadecatrienoic acid by this strain through Δ12 desaturation. The broad substrate specificity of the elongation, and Δ9 and Δ12 desaturation reactions of the strain is useful for fatty acid biotransformation.

Full text of DOI:10.1007/s11746-008-1339-2

J Am Oil Chem Soc (2009) 86:227–233
DOI 10.1007/s11746-008-1339-2
ORIGINAL PAPER
Fatty Acid Desaturation and Elongation Reactions of Trichoderma
sp. 1-OH-2-3
Akinori Ando Æ Jun Ogawa Æ Shigenobu Kishino Æ
Taiyo Ito Æ Norifumi Shirasaka Æ Eiji Sakuradani Æ
Kenzo Yokozeki Æ Sakayu Shimizu
Received: 25 August 2008 / Revised: 14 October 2008 / Accepted: 26 November 2008 / Published online: 25 December 2008
Ó AOCS 2008
Abstract The fatty acid desaturation and elongation
reactions catalyzed by Trichoderma sp. 1-OH-2-3 were
investigated. This strain converted palmitic acid (16:0)
mainly to stearic acid (18:0), and further to oleic acid (c9-
18:1), linoleic acid (c9,c12-18:2), and a-linolenic acid
(c9,c12,c15-18:3) through elongation, and D9, D12, and
D15 desaturation reactions, respectively. Palmitoleic acid
(c9-16:1) and cis-9,cis-12-hexadecadienoic acid were also
produced from 16:0 by the strain. This strain converted
n-tridecanoic acid (13:0) to cis-9-heptadecenoic acid
and further to cis-9,cis-12-heptadecadienoic acid through
elongation, and D9 and D12 desaturation reactions,
respectively. trans-Vaccenic acid (t11-18:1) and trans-12-
octadecenoic acid (t12-18:1) were desaturated by the strain
through D9 desaturation. The products derived from t11-
18:1 were identified as the conjugated linoleic acids
(CLAs) of cis-9,trans-11-octadecadienoic acid and trans-
9,trans-11-octadecadienoic acid. The product derived from
t12-18:1 was identified as cis-9,trans-12-octadecadienoic
acid. cis-6,cis-9-Octadecadienoic acid was desaturated
to cis-6,cis-9,cis-12-octadecatrienoic acid by this strain
through D12 desaturation. The broad substrate specificity
of the elongation, and D9 and D12 desaturation reactions of
the strain is useful for fatty acid biotransformation.
Keywords Desaturation Á Elongation Á Polyunsaturated
fatty acid Á Conjugated linoleic acid Á Trichoderma sp.
Introduction
A. Ando Á J. Ogawa Á S. Kishino Á T. Ito Á E. Sakuradani Á
S. Shimizu (&)
Polyunsaturated fatty acids (PUFA) have various physio-
logical effects. For example, those of the C-20 series such as
dihomo-c-linolenic acid (DGLA, c8,c11,c14-20:3), arachi-
donic acid (AA, c5,c8,c11,c14-20:4), and eicosapentaenoic
acid (EPA, c5,c8,c11,c14,c17-20:5) are of interest as they
are precursors for prostaglandins, thromboxanes, leukotri-
enes, and prostacyclins [1]. In addition, conjugated PUFAs
(CPUFAs) have also attracted much attention as a novel
type of biologically beneficial functional lipid in the last
two decades. In particular, conjugated linoleic acid (CLA,
18:2 with conjugated double bonds) has been the most
intensively investigated and was revealed to have beneficial
effects on human and animal health [2].
Laboratory of Fermentation Physiology and Applied
Microbiology, Division of Applied Life Sciences,
Graduate School of Agriculture, Kyoto University,
Kitashirakawa-oiwakecho, Sakyo-ku,
Kyoto 606-8502, Japan
e-mail: sim@kais.kyoto-u.ac.jp
A. Ando Á J. Ogawa
Research Division of Microbial Sciences,
Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
e-mail: aando@kais.kyoto-u.ac.jp
S. Kishino Á K. Yokozeki
Laboratory of Industrial Microbiology,
Graduate School of Agriculture, Kyoto University,
Kitashirakawa-oiwakecho, Sakyo-ku,
Kyoto 606-8502, Japan
Since natural sources rich in these PUFAs and CPUFAs
are limited, recent investigations have been focused on
microorganisms as alternative sources of them. Several
groups have started to screen for microorganisms capable
of accumulating lipids containing PUFAs and CPUFAs in
N. Shirasaka
Department of Applied Biological Chemistry,
Faculty of Agriculture, Kinki University,
3327-204 Nakamachi, Nara 631-8505, Japan
123

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J Am Oil Chem Soc (2009) 86:227–233
order to obtain more suitable sources for large-scale
preparation of important nutritional components [3–5].
Previously, we found that a filamentous fungus, Morti-
erella alpina 1S-4 isolated from soil, is a potent producer
of triacylglycerols containing AA, DGLA and EPA [6–8].
Mutants that are considered to be defective in (or to have
low activity of) D5, D6, D12, D9 or x3 desaturase have
been derived from M. alpina 1S-4 [9–13], and many kinds
of PUFA have been produced by means of these mutants.
In fact, we have succeeded in their application to the
industrial production of a triacylglycerol with a high con-
tent of AA [14]. Other than Mortierella sp., some groups
investigated fatty acid compositions of Trichoderma sp.
and reported that they accumulate unusual fatty acids such
as hydroxylated fatty acids and conjugated fatty acids
although the chemical structures of these fatty acids are
still ambiguous[5, 15, 16]. We also isolated Trichoderma
sp. AM076 from fresh water and revealed that the strain
accumulated rarely occurring cis-9,cis-12-hexadecadienoic
acid (c9,c12-16:2), when grown with palmitoleic acid
(16:1) [17]. Moreover, the D9, D12, and D15 desaturases of
the strain seemed to act on a variety of fatty acids [18].
In this study, we investigated the specificity of the
desaturation and elongation reactions catalyzed by a
mutant Trichoderma strain for its application for the pro-
duction of PUFAs and CPUFAs. In particular, we focused
on mutant strain 1-OH-2-3, which was derived from
Trichoderma sp. AM076 exhibiting a high level of lipid
accumulation, and evaluated its potential as a catalyst for
fatty acid biotransformation.
University). Strains TU42 and 1-OH-2-3 were PUFA-
accumulating mutants derived from Trichoderma sp.
AM076 by nitrosoguanidine mutagenesis [17, 18]. All
strains were inoculated into 4 ml of GY medium (1%
glucose and 0.5% yeast extract; Oriental, Osaka, Japan),
pH 6.0, with or without each fatty acid substrate, 0.80% (w/
v), in a 20-ml Erlenmeyer flask, and then incubated with
reciprocal shaking (120 rpm) at 28 °C for 7 days. All
experiments were carried out in triplicate, and the averages
of three separate experiments, which were reproducible
within 10%, are presented in the figures and tables.
Extraction, Esterification and Purification
The mycelia were harvested by filtration washed with
0.85% NaCl, and then dried at 100 °C overnight. The dried
cells were directly transmethylated with 10% methanolic
HCl, and the resultant fatty acid methyl esters were
extracted with n-hexane and then analyzed by gas–liquid
chromatography (GLC) [19]. The esters were quantitated
using a Shimadzu GC-17A equipped with a flame ioniza-
tion detector and a split injection system (split ratio, 1/50),
and fitted with
a
capillary column (HR-SS-10,
50 m 9 0.25 mm I.D.; Shinwa Kako, Kyoto, Japan). The
column temperature was initially 180 °C, and then was
raised to 220 °C at the rate of 2 °C/min and maintained at
that temperature for 5 min. The injector and detector were
operated at 250 °C. Helium was used as the carrier gas at
225 kPa/cm2.
After conversion of the reaction products to their methyl
esters, they were separated by reverse-phase high-perfor-
mance liquid chromatography (HPLC, LC-10A; Shimadzu
Co., Kyoto, Japan) on a Cosmosil 5C18 AR column
(20 9 250 mm; Nacalai Tesque, Kyoto, Japan). The
mobile phase was acetonitrile-H2O (8:2 v/v) at the flow
rate of 3.0 ml/min, and the effluent was monitored by
means of ultraviolet detection (205 nm). The chemical
structures of the fatty acid derivatives were determined by
mass spectroscopy (MS), proton nuclear magnetic reso-
nance (1H–NMR) spectroscopy, 1H–1H chemical shift
correlation spectroscopy (1H–1H COSY), 1H clean-total
correlation spectroscopy (TOCSY), and two-dimensional
nuclear Overhauser effect spectroscopy (NOESY).
Experimental Procedures
Chemicals
n-Tridecanoic acid (13:0), n-pentadecanoic acid (15:0),
n-heptadecanoic acid (17:0), and a mixture of trans-
vaccenic acid (t11-18:1) and trans-12-octadecenoic acid
(t12-18:1) were purchased from Tokyo Chemical Industry
Co., Ltd. (Tokyo, Japan). Mead acid (MA, c7,c10,c13-20:3),
AA, cis-6,cis-9,cis-12-octadecatrienoic acid ethyl ester
(c6,c9,c12-18:3EE), and vaccenic acid (c11-18:1) were
obtained from Funakoshi (Tokyo, Japan). cis-6,cis-9-Octa-
decadienoic acid ethyl ester (c6,c9-18:2EE) was obtained
from Suntory Ltd. (Osaka, Japan). All other reagents were of
analytical grade.
Preparation of Free Fatty Acids
Free fatty acids were prepared by heating the fatty acid
methyl esters (50 mg) in a mixture of 5.0 ml of 7.0 N
sodium hydroxide and 5.0 ml of methanol in a capped tube.
After being heated in a boiling water bath for 1 h, the
solution was acidified to pH 2.0 with 10% (w/v) sulfuric
acid in water. The free fatty acids were extracted with
diethylether. The organic extract was washed with water
Microorganisms and Cultivation
The Trichoderma species (FA607, FA608, FA610, FA611,
AM076, TU42 and 1-OH-2-3) were obtained from
the AKU culture collection (Div. Appli. Life Sci., Kyoto
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J Am Oil Chem Soc (2009) 86:227–233
229
1
1H-NMR, H-1H COSY, NOESY and TOCSY
and dried over anhydrous Na2SO4, and then the solvent was
removed under vacuum with a rotary evaporator.
Analyses
All NMR experiments were performed on a JEOL EX-400
(400 MHz at 1H; Jeol Ltd.), and chemical shifts were
assigned relative to the solvent signal. The fatty acid
methyl esters were dissolved in dichloromethane-d2 and the
diameter of the tube was 5 mm.
Preparation of Pyrrolidide Fatty Acids
Pyrrolidide derivatives were prepared by direct treatment
of the isolated methyl esters with pyrrolidine-acetic acid
(10:1 v/v) in a capped tube for 1 h at 100 °C, followed by
extraction from the acidified solution according to the
method of Andersson and Holman [20]. The dichloro-
methane extract was washed with water and dried over
anhydrous Na2SO4, and then the solvent was removed
under vacuum with a rotary evaporator.
Results and Discussion
Profiles of Fatty Acids Synthesized de novo
by Trichoderma sp.
GC–MS Analysis
Table 1 shows the amounts and compositions of fatty acids
synthesized de novo by the Trichoderma species. In strain
1-OH-2-3, c9-18:1 was a major unsaturated fatty acid
accumulated due to a leaky defect in D12 desaturation.
The strain 1-OH-2-3, however, accumulated much larger
amounts of fatty acids compared with the others. Tricho-
derma sp. 1-OH-2-3 was selected for the following
experiments to investigate the substrate specificity of
desaturation and elongation reactions.
GC–MS QP5050 with GC-17A (Shimadzu Co.) was used
for mass spectral analyses. The GLC separation of fatty
acid methyl esters was performed on a HR-SS-10 column
as described above at the same temperature. MS was per-
formed in the electron impact mode at 70 eV with a source
temperature of 250 °C. Split injection (split ratio, 1/50)
was performed with the injector port at 250 °C. The GLC
separation of fatty acid pyrrolidide derivatives was per-
formed on a HR-1 column (25 m 9 0.5 mm I.D., Shinwa
Kako) at 300 °C.
Fatty Acid Transformation by Trichoderma
sp. 1-OH-2-3
MS–MS Analysis
13:0 Transformation
MS–MS analyses were performed on the free acids of the
fatty acids with a Tandem Mass Spectrometer, JEOL
HX110A/HX110A (Jeol Ltd, Tokyo, Japan). The ioniza-
tion method comprised fast atom bombardment (FAB) and
the accelerating voltage was 3 kV. Glycerol was used as
the matrix.
As shown in (Fig. 1b), four unknown peaks were detected
on GLC when 13:0 was added to the medium. Based on
comparison of their retention times on GLC and HPLC
with those for standards, and on the results of mass spectral
analysis, these unknown fatty acids were identified as 15:0,
Table 1 Fatty acid production and composition of Trichoderma species and mutants
Strains
Total FA (mg g-1 dry cell)
FA composition (%)a
16:0
16:1
18:0
c9-18:1
c9,c12-18:2
c9,c12,c15-18:3
FA607
FA608
FA610
FA611
TU42
44.7
50.4
16.7
41.9
31.7
18.4
102.9
36.4
58.4
41.7
49.1
54.7
72.3
47.3
2.7
6.5
1.5
1.9
ND
ND
4.1
3.3
4.2
2.0
4.6
3.4
5.4
3.7
9.9
9.7
38.4
18.4
38.5
27.5
23.7
15.0
18.9
9.3
2.8
9.3
4.3
6.0
ND
3.7
7.0
12.6
12.2
7.3
AM076
1-OH-2-3
22.3
All strains were grown in GY medium without fatty acid substrate at 28 °C for 1 week as described under ‘‘Experimental Procedures’’
ND not detected
a
FA fatty acid, 16:0 palmitic acid, 16:1 palmitoleic acid, 18:0 stearic acid, c9-18:1 oleic acid, c9,c12-18:2 linoleic acid, c9,c12,c15-18:3 a-
linolenic acid
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J Am Oil Chem Soc (2009) 86:227–233
Fig. 1 GLC chromatogram of
methyl esters of standard fatty
acids (a) and those of fatty acids
produced from 13:0 (b),
c6,c9-18:2 (c), a mixture of
t11-18:1 and t12-18:1 (d), and
c11-18:1 (e) by Trichoderma sp.
1-OH-2-3
(a) Control
(b) 13:0
13:0
15:0
16:0
17:0
c9-17:1
c9-18:1
18:0
c9,c12-17:2
c9,c12-18:2
c9-18:1
c9,c12-18:2
c9,c12,c15-18:3
(c) c6,c9-18:2
(d) t11-18:1+t12-18:1
(e) c11-18:1
c9-18:1
+t11-18:1
+t12-18:1
c6,c9-18:2
c9-16:1
c9-18:1
+c11-18:1
a
c6,c9,c12-18:3
b
c
17:0, c9-17:1, and c9,c12-17:2. But the D15 desaturation
product, c9,c12,c15-17:3, was not detected. These results
suggest that 13:0 is elongated to 15:0, and further to 17:0.
The resulting 17:0 is desaturated to c9-17:1 through D9
desaturation, and further to c9,c12-17:2 through D12
desaturation.
desaturases in microorganisms usually convert C–C single
bonds to C=C double bonds in the cis conformation,
compound (a) was deduced to be c9,t12-18:2.
The pyrrolidide derivative of compound (b) showed a
molecular weight of 333 (Fig. 2b). This result suggested
that compound (b) is a C18 fatty acid containing two
double bonds. The FAB-MS data for the free fatty acids
of compound (b) revealed a molecular weight of m/z 280
([M-H]?, 279). Typical fragments (m/z) for compound (b)
were 127, 141, 167, 193, 207 and 208 (data not shown).
The m/z 141, 167, and 193 fragments were derived
through cleavage of single bonds between C8–C9, C10–
C11, and C12–C13, numbered from the carboxyl group.
The m/z 127 and 207 fragments, derived through cleavage
of the single bond between the a and b positions from the
double bond, were clearly detected. Hence, compound (b)
was identified as a 9, 11 positional isomer of octadeca-
dienoic acid. Furthermore, 1H–NMR, 1H–1H COSY,
NOESY, and TOCSY analyses were carried out to
determine the geometric configuration of compound (b)
(data not shown). Consequently, the data showed that
compound (b) was c9,t11-18:2, a CLA isomer, which we
reported previously to be produced by lactic acid bacteria
from linoleic acid [21].
c6,c9-18:2 Transformation
An unknown peak was detected on GLC, when c6,c9-18:2
was added (Fig. 1c). Based on comparison of its retention
times on GLC and HPLC to those for standards, and on the
result of mass spectral analysis, this unknown fatty acid
was identified as c6,c9,c12-18:3. This suggests that c6,c9-
18:2 is desaturated to c6,c9,c12-18:3 through D12 desatu-
ration. The D15 desaturation product, c6,c9,c12,c15-18:4,
was not detected.
t11-18:1 and t12-18:1 Transformation
Unknown peaks, a, b, and c, were detected on GLC, when a
mixture of t11-18:1 and t12-18:1 was added to the medium
(Fig. 1d). These unknown fatty acid methyl esters were
purified by HPLC and then subjected to structural deter-
mination by MS and NMR analysis. Pyrrolidide derivative
of compound (a) showed a molecular weight of 333
(Fig. 2a). This result suggested that compound (a) is a C18
fatty acid containing two double bonds. Intervals of 12
atomic mass units (amu) were found between m/z 196 (C8)
and 208 (C9), and m/z 236 (C11) and m/z 248 (C12),
indicating double bonds at D9 and D12, respectively. Based
on these results, and considering that fatty substrates
comprise a mixture of t11-18:1 and t12-18:1, and that
The pyrrolidide derivative of compound (c) likewise
showed a molecular weight of 333 (Fig. 2c). This result
again suggests that compound (c) is a C18 fatty acid con-
taining two double bonds. Based on the composition
determined from mass spectra, and the retention time on
GLC relative to those of standard CLA (Sigma, USA) and
CLA prepared by Lactobacillus plantarum AKU 1009a
[21], compound (c) was strongly suggested to be t9,t11-
18:2.
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J Am Oil Chem Soc (2009) 86:227–233
231
Fig. 2 GC–MS spectra of
pyrrolidide derivatives of
compounds (a), (b), and (c)
(a) Compound a
182
O
C
70
112
N
70
112 140 168
262
333
333
c9,t12-18:2
140
168
262
50
100
150
200
250
300
(b) Compound b
112
182
O
C
126
N
70
70
112 140 168
262
333
c9,t11-18:2
333
140
150
168 182
50
100
200
250
300
(c) Compound c
182
O
C
70
112
N
70
112 140 168
248 276 304 333
98
t9,t11-18:2
333
140
150
168 182
304
300
248
250
276
50
100
200
c11-18:1 Transformation
15 lg/ml of c6,c9-17:2 were produced, the proportions as
to the total fatty acids being 0.67 and 0.26%, respectively.
From 0.80% of c6,c9-18:2EE, 18 lg/ml c6,c9,c12-18:3
was produced, the proportion as to the total fatty acids
being 2.49%. As to the production of CLA isomers, 45 lg/
ml of c9,t11-18:2 and 19 lg/ml of t9,t11-18:2 were pro-
duced from 0.80% of t11-18:1 added to the medium, the
proportions as to the total fatty acids being 0.70 and 0.30%,
respectively. These results indicated that Trichoderma sp.
1-OH-2-3 exhibits desaturation activity with flexible sub-
strate specificity, especially for D9 and D12 desaturation.
Previously, we reported that Trichoderma sp. AM076
produced c9,c12-16:2 and c9,c12,c15-16:3 from c9-16:1
and c9,c12-16:2 added to the culture medium, respectively
[17, 18]. These results indicated that Trichoderma sp. have
unique fatty acid transformation activities. In this report,
we investigated the substrate specificity of the desaturation
and elongation reactions catalyzed by a mutant strain
An unknown peak was detected on GLC when c11-18:1
was added (Fig. 1e). Based on the retention times on GLC
and HPLC relative to those of standards, and the results of
mass spectra analysis, this unusual fatty acid was identified
as c9-16:1 (data not shown). The amount of c9-16:1
derived from c11-18:1 was much higher than that of
de novo synthesized c9-16:1 (Fig. 1a, e). These results
suggest that c11-18:1 is converted to c9-16:1 through
b-oxidation. The D9 desaturation product, c9,c11-18:2, was
not detected.
Production of Various Fatty Acids
Table 2 shows the amounts of typical fatty acids produced
by strain 1-OH-2-3 for 7 days cultivation. When 0.80% of
17:0 was added to the medium, 40 lg/ml of c9-17:1 and
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Table 2 Transformation of fatty acids by Trichoderma sp. 1-OH-2-3
Additional substrate
Newly generated fatty acids from added substrate (lg ml-1
)
c9-17:1
c9,c12-17:2
c6,c9,c12-18:3
c9,t11-18:2
t9,t11-18:2
Others
t11-18:1
c6,c9-18:2EE
17:0
ND
ND
40
ND
ND
15
ND
18
45
19
35
ND
ND
ND
ND
ND
ND
ND
The fungus was grown in GY medium supplemented with 0.8% of each substrate for 7 days
ND not detected
COOH
13:0
EL
EL
COOH
15:0
17:0
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
16:0
18:0
t11-18:1
t12-18:1
EL
∆9
∆9
∆9
∆9
∆9
COOH
c6,c9-18:2
COOH
COOH
COOH
c9-16:1
c9-18:1
c9-17:1
c9,t11-18:2
c9,t12-18:2
c11-18:1
-OX
β
12
12
∆
∆
12
12
∆
∆
COOH
t9,t11-18:2
COOH
c6,c9,c12-18:3
COOH
c9,c12-16:2
COOH
c9,c12-18:2
c9,c12-17:2
15
15
∆
∆
COOH
COOH
c9,c12,c15-16:3
c9,c12,c15-18:3
Fig. 3 Proposed pathway for FA transformation by Trichoderma sp.
1-OH-2-3. EL elongation, D15 D15 desaturation, D12 D12 desatura-
tion, D9 D9 desaturation, b-OX b-oxidation. The gray arrows indicate
the de novo biosynthetic pathway. The black arrows indicate our
previous observations [18]
1-OH-2-3 accumulating much higher amounts of fatty acid
than parental strain.
In conclusion, we revealed that the substrate specificity
of elongation, and D9 and D12 desaturation by mutant
strain 1-OH-2-3 are broad and that the D9 desaturase of this
strain showed an ability to produce specific CLA isomers
(c9,t11-18:2 and t9,t11-18:2).
The transformation of fatty acids by the mutant strain
can be summarized as follows (Fig. 3): 13:0 is transformed
to 15:0 and further to 17:0 through elongation. 17:0 is
transformed to c9-17:1 and further to c9,c12-17:2 through
D9 and D12 desaturation, respectively. c9,c12-17:2 is not
further converted to c9,c12,c15-17:3 through D15 desatu-
ration. c6,c9-18:2 is transformed to c6,c9,c12-18:3 through
D12 desaturation. c6,c9,c12-18:3 was not converted to
c6,c9,c12,c15-18:4 through D15 desaturation. t11-18:1 was
transformed to c9,t11-18:2 and t9,t11-18:2 through D9
desaturation. t9,t11-18:2 might be produced from c9,t11-
18:2 through spontaneous chemical isomerization or by the
cis-trans isomerase of this strain. t12-18:2 was desaturated
to c9,t12-18:2 through D9 desaturation. t11-18:1 was
converted to CLA by strain 1-OH-2-3, while c11-18:1 was
scarcely transformed.
Recently, conjugated polyunsaturated fatty acids have
attracted considerable attention because of their potentially
beneficial effects. We reported that some lactic acid bac-
teria could convert linoleic acid to specific CLA isomers
c9,t11-18:2 and t9,t11-18:2 [3]. Among these isomers,
c9,t11-18:2 has been suggested to be one of the most
important isomers in terms of biological activity because it
is the major isomer in naturally occurring dairy products
and it is incorporated into the phospholipid fraction of
tissues of animals fed a mixture of CLA isomers [22].
Accordingly, methods for producing specific CLA isomers
are strongly desired to evaluate the function of CLA iso-
mers accurately. Though t11-18:1 (trans-vaccenic acid) is
123

J Am Oil Chem Soc (2009) 86:227–233
233
desaturase-defective mutant of Mortierella alpina 1S-4. J Am Oil
Chem Soc 69:939–944
10. Jareonkitmongkol S, Sakuradani E, Shimizu S (1993) A novel
D5-desaturase-defective mutant of Mortierella alpina 1S-4 and
its dihomo-c-linolenic acid productivity. Appl Environ Microbiol
59:4300–4304
11. Jareonkitmongkol S, Sakuradani E, Shimizu S (1994) Isolation
and characterization of an x3-desaturation-defective mutant of an
arachidonic acid-producing fungus, Mortierella alpina 1S-4.
Arch Microbiol 161:316–319
converted to c9,t11-18:2 by mammalian cells through D9
desaturation, there have not been any reports that fungi can
produce CLA isomers from t11-18:1 [23]. This is the
important report of the production of specific CLA isomer,
c9,t11-18:2, by fungi through D9 desaturation although
further studies to increase the productivity and the selec-
tivity are necessary.
Acknowledgments This work was partially supported by the Pro-
ject for the Development of a Technological Infrastructure for
Industrial Bioprocesses on R&D of New Industrial Science and
Technology Frontiers (to S.S.), the Industrial Technology Research
Grant Program (No. 05A07003d to E.S.) of the New Energy and
Industrial Technology Development Organization, Japan, Grants-in-
Aid for Scientific Research (No. 16688004 to J.O., No. 18208009 to
S.S., and No. 19688006 to E.S.), and COE for Microbial-Process
Development Pioneering Future Production Systems (to S.S.) from
the Ministry of Education, Science, Sports, and Culture, Japan.
12. Jareonkitmongkol S, Sakuradani E, Shimizu S (2002) Isolation
and characterization of a D9-desaturation-defective mutant of
an arachidonic acid-producing fungus, Mortierella alpina 1S-4.
J Am Oil Chem Soc 79:1021–1026
13. Jareonkitmongkol S, Shimizu S, Yamada H (1992) Fatty acid
desaturation-defective mutants of an arachidonic-acid-producing
fungus, Mortierella alpina 1S-4. J Gen Microbiol 138:997–1002
14. Shimizu S, Ogawa J, Kataoka M, Kobayashi M (1997) Screening
of novel microbial enzymes for the production of biologically and
chemically useful compounds. Adv Biochem Eng Biotechnol
58:45–87
15. Serrano-Carreon L, Hathout Y, Bensoussan M, Belin J-M (1992)
Production of 6-pentyl-a-pyrone by Trichoderma harzianum from
18:n fatty acid methyl esters. Biotechnol Lett 14:1019–1024
16. Suutari M (1995) Effect of growth temperature on lipid fatty
acids of four fungi (Aspergillus niger, Neurospora crassa, Pen-
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(1998) Microbial conversion of palmitoleic acid to 9,12-hex-
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(2005) The characteristics of desaturation of Trichoderma sp.
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