Formation of conjugated D
11
D
13
-double bonds by D
12
-linoleic acid
(1,4)-acyl-lipid-desaturase in pomegranate seeds
Ellen Hornung
1
, Christian Pernstich
1
and Ivo Feussner
1,2
1
Institut fu
¨
r Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben, Germany;
2
Albrecht-von-Haller-Institut
fu
¨
r Pflanzenwissenschaften, Georg-August Universita
¨
t Goettingen, D-37077 Goettingen, Germany
For the biosynthesis of punicic acid (18:3
D9Z,11E,13Z
)a
(11,14)-linoleoyl desaturase activity has been proposed. To
isolate this acyl-lipid-desaturase, PCR-based cloning was
used. This approach resulted in the isolation of two complete

(Z,E,Z) geometries [2], providing an easily accessible source
of these fatty acids. At least five different out of the six
theoretical invisible regio-isomers have been reported within
plant seed oils with double bond systems in the following
positions: (Z,E,Z)- and (E,E,Z)-8,10,12–18:3 and (Z,E,Z)-
(Z,E,E)- and (E,E,Z)-9,11,13–18:3. One of these, punicic
acid (18:3
D9Z,11E,13Z
) is the major constituent of the seed oil
of Punica granatum [3]. Seed oils harboring conjugated fatty
acids are of industrial interest, because the oil is used as
drying oil in paints and may be used for cosmetic purposes.
A number of enzymatic mechanisms have been published
to describe the biosynthesis of conjugated octadecatrienoic
acids in plants. These include an oxidase type reaction [4]
and the direct isomerization of linolenic acid [5,6] at the level
of free fatty acids in algae. In recent publications on the
biosynthesis of a-eleostearic acid (18:3
D9Z,11E,13E
) and of
calendic acid (18:3
D8E,10E,12Z
) [7–10], it became clear that the
responsible enzymes in higher plants belong to the growing
family of special acyl-lipid-desaturases (Fig. 1) [11,12].
Besides introducing conjugated double bonds by so-called
(1,4)-acyl-lipid-desaturases (FADX) this class of enzymes
catalyzes the formation of hydroxy, epoxy, and acetylenic
groups, respectively, within a fatty acid backbone [13,14].
Furthermore the reaction takes place while the acyl moiety

¨
, Sweden), methanol,
hexane and 2-propanol (all HPLC grade) were from Baker
(Deventer, the Netherlands).
Correspondence to I. Feussner, Biochemie der Pflanze, AvH,
Justus-von-Liebig-Weg 11, D-37077 Goettingen, Germany.
Fax: 49 551 395749, Tel.: + 49 551 395743,
E-mail:
Abbreviations: FAD12, D
12
-fatty acid desaturase; FADX, (1,4)-acyl-
lipid desaturase; FAD-OH, fatty acid hydroxylase; GC-FID, gas
chromatography-free induction decay; PCI, phenol/chloroform/
isoamyl alcohol; PVP, polyvinylpyrrolidone.
Note: The nucleotide sequences reported in this paper have been
submitted to the GenBank/EMBL data bank with accession numbers
PuFAD12 AJ437139, PuFADX AJ437140.
(Received 28 May 2002, revised 3 August 2002,
accepted 15 August 2002)
Eur. J. Biochem. 269, 4852–4859 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03184.x
Isolation of cDNAs
P. granatum seeds were harvested from fruits obtained
from a local market. For RNA isolation, 20 g of seeds
were ground in liquid nitrogen, 200 mL of extraction
buffer I [100 m
M
Tris/HCl, pH 7.5, 25 m
M
EDTA, 2%
(w/v) laurylsarcosyl, 4

M
LiCl overnight at 4 °C. After centrifugation for 30 min
at 120 000 g at 4 °C the precipitate was washed with 70%
ethanol, dried and dissolved in 1 mL water. From this total
RNA fraction poly(A)
+
RNA was enriched using the Poly
Attract-Kit
TM
(Promega, Mannheim, Germany) according
to the manual provided, and used for all further experi-
ments. ss-cDNA was synthesized from poly(A)
+
RNA of
pomegranate seeds by reversed transcription with Super-
scriptII
TM
(Gibco BRL, Eggenstein, Germany).
Construction of expression plasmids and recombinant
protein synthesis
This ss-cDNA was used as template for PCR-based cloning.
PCR fragments of about 560 bp were amplified with the
degenerate sense primer A 5¢-TGGGTIAWHGCHCAYG
ARTGBGG-3¢ and antisense primer B 5¢-CCARTYCCAY
TCIGWBGARTCRTARTG-3¢, derived from the amino
acid sequences WVIAHEC and HYDS(S/T)EW(D/N)W,
respectively which in acyl-lipid-desaturases are highly con-
served. PCR was carried out with TfI-DNA-Polymerase
TM
(Biozym, Hess. Oldendorf, Germany) using an amplification

expected open reading frame of the entire cDNA with
suitable recognition sites were used for amplification. Pun1
sense primer H 5¢-ATG GGA GCT GAT GGA ACA
ATG TCT C-3¢, antisense primer I 5¢-ATT CAG AAC
TTG CTC TTG AAC CAT AG-3¢ and Pun2 sense primer
J5¢-ATG GGA GCC GGT GGA AGA ATG AC-3¢ anti-
sense primer K 5¢-TGA TCA GAG GTT CTT CTT
GTA CCA G-3¢. The Expand
TM
High Fidelity-System
(Roche Diagnostics, Mannheim, Germany) was used, with
an amplification program of 2 min denaturation at 94 °C,
followed by 10 cycles of 30 s at 94 °C, 30 s at 58 °C, 1 min
at 72 °C, followed by 15 cycles of 30 s at 94 °C, 30 s at
58 °C, 1 min at 72 °C (time increment 5 s) and terminated
by 5 min extension at 72 °C. The fragments were cloned
into pGEM-T
TM
and the resulting plasmids PuFADX and
PuFAD12 were sequenced. For expression in S. cerevisiae
the open reading frames of PuFADX and PuFAD12 were
cloned as a HindIII/BamHI or SalI/HindIII fragment,
respectively, behind the galactose-inducible promotor
GAL1 into the shuttle vector pYES2
TM
(Invitrogen, Carls-
bad, USA) or pESC-LEU
TM
(Stratagene, Amsterdam, the
Netherlands) to yield the plasmids pYES-PuFADX and

1.35 mL of a mixture of toluene and methanol (1 : 2, v/v)
and 0.5 mL sodium methoxide, using a glass rod. After
shaking the samples for 20 min at room temperature,
1.8 mL of 1
M
NaCl and 4 mL heptane were added and
fatty acid methyl esters were extracted by shaking vigor-
ously for 10 min. The organic phase was evaporated to
dryness under a nitrogen stream and the corresponding
fatty acid methyl esters were reconstituted in 40 lLof
acetonitrile. Then 1 lL of each sample was analyzed by GC,
performed with an Agilent GC 6890 system (Agilent,
Waldbronn, Germany) coupled with an FID detector
equipped with a capillary HP INNOWAX column
(30 m · 0.32 mm, 0.5 lm coating thickness, Agilent, Wald-
bronn, Germany). Helium was used as the carrier gas
(30 cm · s
)1
). The samples were measured with a split of
20 : 1 with an injector temperature of 220 °C. The tem-
perature gradient was 150 °C for 1 min, 150–200 °Cat
15 °Cmin
)1
, 200–250 °Cat2°Cmin
)1
,and250°Cfor
10 min. Fatty acids were identified by authentic standards.
Alternatively, the corresponding fatty acid methyl esters
were analyzed by GC/MS, performed with an Agilent GC
6890 system coupled with an Agilent 5973 N MS detector

sequence exhibited highest identities to D
12
-fatty acid
desaturases from Gossypium hirsutum (accession number
Y10112) and Solanum commersonii (accession number
X92847). The corresponding amino acid sequence of
Pun2, a fragment of 567 bp, showed highest identities to
D
12
-fatty acid desaturases from Sesamum indicum (accession
number AF192486) and again to a D
12
-fatty acid desaturase
from S. commersonii. To isolate the full-length cDNA
clones, RACE with specific primers was used to amplify the
5¢-and3¢-ends of Pun1 and Pun2. The fragments were
cloned and sequenced. With specific primers for the
expected open reading frames containing specific restric-
tion sites the entire cDNAs of about 1.2 kb were amplified
by PCR and subcloned into pGEM-T
TM
. The resulting
fragments were sequenced. The full-length cDNA of Pun1
had a length of 1185 bp coding for a protein of 395 amino
acids with a calculated molecular mass of 45.8 kDa. The
amino acid sequence of this putative fatty acid desaturase
showed highest identities to the D
12
-fatty acid desaturases
from G. hirsutum (58%), from S. commersonii (59%) and

9
-(1,4)-acyl-lipid-desaturases form another
subgroup within this phylogenetic tree.
Functional expression in
S. cerevisiae
and fatty acid
analysis
To investigate the product and substrate specificity of
PuFAD12 and PuFADX, respectively, the full-length
cDNAs were cloned into yeast expression vectors under
the control of the inducible GAL1 promoter and the
encoded proteins were expressed in S. cerevisiae strain
INVSc1. In induced cultures of cells harboring the cDNA of
PuFAD12 accumulation of linoleic acid and to a much
lower extent of hexadecadienoic acid was observed (Fig. 4,
upper panel vs. middle panel). The accumulation was
dependent on the growth temperature of the cultures as little
or no linoleic acid and hexadecadienoic acid were detected
in cells maintained at 30 °C. Whereas linoleic acid and
hexadecadienoic acid accumulated up to 5% and 1% (w/w),
respectively, of the total fatty acids, if cells were grown at
16 °C.
Since PuFADX was expected to code for a (1,4)-acyl-
lipid-desaturase, cultures transformed with PuFADX were
supplemented with linoleic acid as putative substrate.
However in induced yeast cultures transformed with
PuFADX and without the addition of linoleic acid to the
growth medium, accumulation of linoleic acid up to 1.2%
(w/w) has been observed, if the cells were maintained at
30 °C (data not shown). Punicic acid could only be detected

homo-c-linolenic acid no formation of a conjugated fatty
acid was found (data not shown and Table 1). However
with c-linolenic acid the formation of a presumably
conjugated octatetraenoic fatty acid was found (Table 1).
In order to confirm the structure of this newly formed fatty
acid, mass spectrometry was used and in the lower panel of
Fig. 6 the resulting mass spectrum is shown. Again the fatty
acid methyl ester was characterized by an abundant
molecular ion that time of m/z ¼ 290, thus confirming the
Fig. 2. Sequence alignment of the D
12
-acyl-lipid-desaturases. The pro-
tein alignment was generated with the
CLUSTAL
-
X
program and was
performed with sequences from S. indicum (SiFAD12, accession
number AF192486), S. commersonii (ScFAD12, accession number
X92847), P. granatum (PuFAD12, accession number AJ437139),
G.hirsutum (GhFAD12, accession number Y10112), Crepis alpina
(CaFAD12, accession number Y16285), and the D
12
-acyl-lipid-
desaturase from P. granatum (PuFADX, accession number AJ437140).
Boxes indicate the three characteristic and highly conserved histidine
regions and identical amino acids are marked as bold letters. For the
alignment D
12
-acyl lipid desaturases were selected which displayed the

hydroxylase.
Ó FEBS 2002 Punicic acid producing (1,4)-acyl-lipid-desaturase (Eur. J. Biochem. 269) 4855
formation of a conjugated fatty acid. To compare the
substrate specificity directly between linoleic acid and
c-linolenic acid, yeast cells harboring PuFADX were grown
in the presence of an equimolar mixture of linoleic acid,
a-linolenic acid and c-linolenic acid. The resulting fatty acid
profile is shown in the middle panel of Fig. 7. Punicic acid
and the conjugated octadecatetraenoic fatty acid derived
from c-linolenic acid but not from a-linolenic acid were
detected in a ratio of 2 to 0.6% (w/w) indicating a three- to
fourfold preference of PuFADX against linoleic acid under
these conditions.
DISCUSSION
Over the last five years more and more data have
accumulated which show that the growing family of special
acyl-lipid-desaturases catalyzes the formation of a wide
array of functional groups within unusual fatty acids
predominantly found in plant seed oils [18]. To this family
belong now besides the classical D
12
and D
15
-acyl-lipid-
desaturases [12], desaturases directly fused to their elec-
tron donor such as D
5
and D
6
-acyl-lipid-desaturases [19],

-
desaturases and D
12
-desaturase-related nonclassical acyl-
lipid-desaturases. Expression of PuFAD12 in yeast cells
indicated that it is a classical D
12
-acyl-lipid-desaturase
(Fig. 4). Expression of PuFADX in yeast cells revealed
that the enzyme produced fatty acid derivatives with
conjugated double bond systems from linoleic and
c-linolenic acid substrates, respectively (Fig. 5, Table 1).
Two names, ÔconjugaseÕ and Ô(1,4)-acyl-lipid-desaturaseÕ
were previously suggested to refer to enzymes that are
responsible for introducing conjugated double bonds into
acyl chains [7,9]. Both names were proposed, because they
describe the catalytic mechanism of these enzymes: ÔConju-
gaseÕ, since the enzyme forms two conjugated double bonds
out of one isolated double bond and Ô(1,4)-acyl lipid
desaturaseÕ, since this class of enzymes seem to catalyze an
(1,4)-elimination of hydrogen atoms bound to the aliphatic
carbon chain instead of an 1,2-syn elimination in case of the
classical acyl-lipid-desaturases [21]. Two (1,4)-acyl-lipid-
desaturases from Impatiens balsamina and Momordica cha-
rantia were found to be able to convert the D
12
-double bond
of linoleic acid into two conjugated and trans configurated
double bonds at the 11 and 13 positions, resulting in the
production of the conjugated linolenic acid 18:3

This seed oil must contain significant amounts of the envis-
aged product in a chemically pure manner. The amount of
an unusual fatty acid is determined by (a) the complex
Fig. 6. Mass spectra of conjugated fatty acid methyl esters. Conjugated
fatty acid methyl esters were isolated from yeast cells transformed with
PuFADX and supplemented either with linoleic acid (upper panel) or
c-linolenic acid (lower panel), respectively. The lipids were extracted
from lyophilized yeast cells, esterified fatty acids were transmethylated
and analyzed by GC/MS as described under materials and methods.
All fatty acids were characterized by coelution of authentic standards.
The mass spectra of the substances eluting at the retention times of the
conjugated fatty acid methyl esters were recorded.
Table 1. Substrate specificity of PuFADX. Yeast cells transformed with PuFADX were grown in the presence of different fatty acids. The lipids
were extracted from lyophilized yeast cells, esterified fatty acids were transmethylated and GC/FID analysis of fatty acid methyl esters isolated from
these yeast cultures was performed as described under materials and methods. All fatty acids were characterized by coelution of authentic standards.
Fatty acid detected
Supplemented fatty acid (%)
a
18:2
D9Z,12Z
18:3
D6Z,9Z,12Z
18:3
D9Z,12Z,15Z
20:3
D8Z,11Z,14Z
16:0 15.0 19.0 12.9 16.0
16:1
D9Z
14.9 9.2 28.9 30.0

c
This fatty acid may be
derived due to substrate impurities.
Ó FEBS 2002 Punicic acid producing (1,4)-acyl-lipid-desaturase (Eur. J. Biochem. 269) 4857
biosynthetic pathway of unusual fatty acids in seeds [18],
since many of these functional groups are introduced into
the fatty acid backbone while the fatty acid is esterified to a
molecule of PtdCho [15,16], and (b) by the specificity of the
respective enzyme which introduces this functional group.
Since this class of enzymes needs linoleic acid as substrate, it
needs a 18:2-platform to fulfill its function. With that respect
oil crop plants are needed which harbor high amounts of
linoleic acid within their seed oils such as soybean, flax or
sunflower [3]. However, their oils contain substantial
amounts of a-linolenic acid and all (1,4)-acyl-lipid-desatu-
rases reported so far showed no preference against linoleic
acid in the presence of a-linolenic acid. This problem may be
solved by using this new type of (1,4)-acyl-lipid-desaturase
that converts a double bond located only in the D
12
-position
of linoleic acid or c-linolenic acid, but not in a-linolenic acid,
into a conjugated double bond system. Therefore this
enzyme may have advantages over the previously known
enzymes, since c-linolenic acid is not found in the seed oils of
most crop plants.
ACKNOWLEDGEMENTS
The authors are grateful to M. Pu
¨
rschel for expert technical assistance.

Acad. Sci. USA 96, 12935–12940.
8. Cahoon, E.B., Ripp, K.G., Hall, S.E. & Kinney, A.J. (2001)
Formation of conjugated D
8
, D
10
double bonds by delta12-oleic
acid desaturase related enzymes. Biosynthetic origin of calendic
acid. J. Biol. Chem. 276, 2083–2087.
9. Fritsche,K.,Hornung,E.,Peitzsch,N.,Renz,A.&Feussner,I.
(1999) Isolation and characterization of a calendic acid producing
(8,11)-linoleoyl desaturase. FEBS Lett. 462, 249–253.
10. Qiu, X., Reed, D.W., Hong, H., MacKenzie, S.L. & Covello, P.S.
(2001) Identification and analysis of a gene from Calendula
officinalis encoding a fatty acid conjugase. Plant Physiol. 125,
847–855.
11. Shanklin, J. & Cahoon, E.B. (1998) Desaturation and related
modifications of fatty acids. Ann. Rev. Plant Physiol. Plant Mol.
Biol. 49, 611–641.
12. Heinz, E. (1993) Biosynthesis of polyunsaturated fatty acids. In
Lipid Metabolism in Plants (Moore, J.T.S., ed.), pp. 33–89. CRC
Press, London.
13. Lee, M., Lenman, M., Banas, A., Bafor, M., Singh, S., Schweizer,
M., Nilsson, R., Liljenberg, C., Dahlqvist, A., Gummeson, P O.,
Sjo
¨
dahl, S., Green, A. & Stymne, S. (1998) Identification of non-
heme diiron proteins that catalyze triple bond and epoxy group
formation. Science 280, 915–918.
14. Broun, P. & Somerville, C. (1997) Accumulation of ricinoleic,

donor. Eur. J. Lipid Sci. Technol. 103, 158–180.
20. Cahoon, R.E., Carlson, T.J., Hitz, W.D. & Ripp, K.G. (2000)
Genes for plant fatty acid modifying enzymes associated with
conjugated double bond formation in PCT WO 00/11176.
21. Svatos, A., Kalinova, B. & Boland, W. (1999) Stereochemistry
of lepidopteran sex pheromone biosynthesis: a comparison of
fatty acid CoA D11-(IX) -desaturases in Bombyx mori and
Manduca sexta female moths. Insect Biochem. Mol. Biol. 29, 225–
232.
Ó FEBS 2002 Punicic acid producing (1,4)-acyl-lipid-desaturase (Eur. J. Biochem. 269) 4859