The other one codes for a new type of 1,4-acyl-lipid-desaturase that converts a cis double bond located in the D12-position of linoleic acid or c-linolenic acid, but not in a-linolenic a
Trang 1Formation of conjugated D11 D13-double bonds by D12-linoleic acid (1,4)-acyl-lipid-desaturase in pomegranate seeds
Ellen Hornung1, Christian Pernstich1and Ivo Feussner1,2
1
Institut fu¨r Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben, Germany;2Albrecht-von-Haller-Institut fu¨r Pflanzenwissenschaften, Georg-August Universita¨t Goettingen, D-37077 Goettingen, Germany
For the biosynthesis of punicic acid (18:3D9Z,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
cDNAs The first isolated full-length cDNA harbors a
sequence of 1350 bp encoding a protein of 395 amino acids
The second cDNA was 1415 bp long encoding a protein of
387 amino acids For functional identification proteins
encoded by the cDNAs were expressed in Saccharomyces
cerevisiae, and formation of newly formed fatty acids was
analyzed by gas chromatography-free induction decay
(GC-FID) and GC/MS The expression of the heterologous
enzymes resulted in the first case in a significant amount of linoleic acid and in the second case, after linoleic acid sup-plementation, in formation of punicic acid The results presented here identify one cDNA coding for a classical
D12-acyl-lipid-desaturase The other one codes for a new type
of (1,4)-acyl-lipid-desaturase that converts a cis double bond located in the D12-position of linoleic acid or c-linolenic acid, but not in a-linolenic acid, into a conjugated cis–trans double bond system
Keywords: acyl-group desaturase; conjugase; Punica granatum; Saccharomyces cerevisiae; seed oil
The most common octadecatrienoic fatty acid in plants is
a-linoleic acid (18:3D9Z,12Z,15Z), which is the main
constitu-ent of chloroplastic membranes [1] Beside this,
triacylglyce-rols from seeds are sometimes composed of additional
conjugated octadecatrienoic acids having (Z,E,E) or
(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: 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 (18:3D9Z,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 (18:3D9Z,11E,13E) and of calendic acid (18:3D8E,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
is esterified to PtdCho as has been shown first for the classical acyl-lipid-desaturases [15] and then for the forma-tion of a-eleostearic acid as well [16] However, all (1,4)-acyl-lipid-desaturases isolated so far from plants convert a cis double bond either at position D9or D12, respectively, of the fatty acid backbone into a conjugated trans–trans double bond system To obtain additional information on the biosynthesis of conjugated octadecatrienoic acids we deci-ded to expand the analysis on the biosynthesis of punicic acid (18:3D9Z,11E,13Z) in the seeds of P granatum, since the biosynthesis of this conjutrienoic fatty acid involves the conversion of a cis double bond at position D12into a conjugated trans–cis double bond system Here, we describe the cloning of this new type of (1,4)-acyl-lipid-desaturase that catalyzes the formation of a conjugated triene fatty acid that harbors a (Z,E,Z)-9,11,13–18:3 double bond system
M A T E R I A L S A N D M E T H O D S Chemicals
Standards of fatty acids as well as all other chemicals were from Sigma (Deisenhofen, Germany) Conjugated fatty acids were from Larodan (Malmo¨, 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: ifeussn@gwdg.de
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)
Trang 2Isolation 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 mM Tris/HCl, pH 7.5, 25 mM EDTA, 2%
(w/v) laurylsarcosyl, 4M guanidinium thiocyanate, 5%
(w/v) polyvinylpyrrolidone (PVP), 1% (v/v)
a-mercapto-ethanol] was added, homogenized further with an
Ultra-turrax (IKA Labortechnik, Staufen, Germany) and the
homogenate was shaken for 10 min After centrifugation at
3000 g for 15 min the floating solid lipid phase and the
pellet were discarded, and the remaining liquid phase was
extracted with an equal volume of PCI (phenol/chloroform/
isoamyl alcohol, 20 : 20 : 1, v/v/v) After centrifugation at
3000 g for 10 min the hydrophilic phase was reextracted
with an equal volume of chloroform and the centrifugation
step performed as before The hydrophilic phase was loaded
on a CsCl cushion (5MCsCl) of 8 mL and centrifuged at
18C and 100 000 g for 18 h The RNA precipitate was
dried, resuspended and extracted for 15 min in a mixture
consisting of 7.5 mL extraction buffer II (100 mMTris/HCl,
pH 8.8, 100 mMNaCl, 5 mMEDTA, 2% (w/v) SDS and
10 mL PCI) Centrifugation and washing of the hydrophilic
phase with chloroform followed and the RNA was
preci-pitated from the hydrophilic phase by an equal volume of
5MLiCl overnight at 4C After centrifugation for 30 min
at 120 000 g at 4C 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-KitTM(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-scriptIITM(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-PolymeraseTM
(Biozym, Hess Oldendorf, Germany) using an amplification
program of 2 min denaturation at 94C, followed by 10 cycles of 30 s at 94C, 45 s at 50 C, 1 min at 72 C, followed by 20 cycles of 30 s at 94C, 45 s at 50 C, 1 min
at 72C (time increment 5 s) and terminated by 2 min extension at 72C PCR products of the expected length were cloned in pGEM-TTM (Promega, Mannheim, Germany) and sequenced The fragments Pun1 and Pun2 were chosen for the isolation of full-length cDNA clones using the marathonTMcDNA amplification kit (Clontech, Heidelberg, Germany) To amplify the 5¢- and 3¢-ends
of Pun1 and Pun2 by PCR, specific primers were used 5¢-RACE primer C: 5¢-GGG ACG AGG AGC GAT GTG TGG AG-3¢, Pun1 3¢-RACE primer D: 5¢-AGT CCT CAT ATT AAA TGC ATT CGT GG-3¢, Pun2 5¢-RACE primer F: 5¢-ACG GAA CGA GGA GCG CTG AGTG-3¢, 3¢-RACE primer G: 5¢-CTG ATC GTG AAC GCA TTC CTG G-3¢ Amplification was carried out by using the Advantage cDNA PCR-KitTM (Clontech, Hei-delberg, Germany) according to the manufacturer’s instruc-tions The fragments were cloned in pGEM-TTM and sequenced To obtain the full-length cDNA clones by PCR and for expression in S cerevisiae, specific primers of the 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
J 5¢-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 ExpandTM High Fidelity-System (Roche Diagnostics, Mannheim, Germany) was used, with
an amplification program of 2 min denaturation at 94C, followed by 10 cycles of 30 s at 94C, 30 s at 58 C, 1 min
at 72C, followed by 15 cycles of 30 s at 94 C, 30 s at
58C, 1 min at 72 C (time increment 5 s) and terminated
by 5 min extension at 72C The fragments were cloned into pGEM-TTMand 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 GAL1into the shuttle vector pYES2TM(Invitrogen, Carls-bad, USA) or pESC-LEUTM(Stratagene, Amsterdam, the Netherlands) to yield the plasmids pYES-PuFADX and pESC-LEU-PuFAD12 The plasmids were transformed into the yeast strain INVSc1TM(Invitrogen, Carlsbad, USA) by lithium acetate [17] Individual colonies of cell were then grown overnight at 30C in SD media lacking uracil (pYES2TM) or leucin (pESC-LEUTM), supplemented with glucose Cells were then washed twice in SD media, before being diluted to A600¼ 0.2–0.4 in SD media supplemented with galactose If fatty acids were added, at a concentration of 0.02% (w/v), the media was also supplemented with tergitol type NP-40 at a concentration of 0.2% (w/v) Cultures were maintained either for 3 days at 30C or for 10 days at 16 C with shaking (150 r.p.m) to densities of A600¼ 2–3 Twenty millilitres of cell cultures were harvested by centrifugation and lyophilized
Lipid analysis For analysis of punicic acid content of transformed yeast cells, lyophilized cell pellets were homogenized by adding
Fig 1 Reactions catalyzed by members of the acyl-lipid-desaturase
family.
Trang 31.35 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 1MNaCl 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 lL of
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 220C The
tem-perature gradient was 150C for 1 min, 150–200 C at
15C min)1, 200–250C at 2 C min)1, and 250C for
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
(Agilent, Waldbronn, Germany) The GC was equipped
with a capillary HP-5 column (5% diphenyl : 95%
polydi-methyl siloxane, 30 m· 0.25 mm, 0.25 lm coating
thick-ness, Agilent, Waldbronn, Germany) and helium was used
as the carrier gas (40 cm· s)1) An electron energy of
70 eV, an ion source temperature of 230C, and a
temperature of 275C for the transfer line were used The
samples were measured in the EI mode, and the splitless
injection mode (opened after 1 min) with an injector
temperature of 250C The temperature gradient was 60–
110C at 25 CÆmin)1, 110C for 1 min, 110–270 C at
10CÆmin)1, and 270C for 10 min
R E S U L T S
PCR-based cloning and isolation of full-length cDNAs
coding for acyl-lipid-desaturases
For PCR-based cloning degenerated primers, deduced from
conserved regions of acyl-lipid-desaturases, were
synthes-ized [9] The template used was ss-cDNA from P granatum,
which was reverse-transcribed from mRNA of seeds from
fruits PCR products of the expected length were cloned and
sequenced Database searches and alignments with these
fragments indicated two different fragments (Pun1 and
Pun2) with similarities to plant acyl-lipid-desaturases Pun1
was a fragment of 586 bp The corresponding amino acid
sequence exhibited highest identities to D12-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
D12-fatty acid desaturases from Sesamum indicum (accession
number AF192486) and again to a D12-fatty acid desaturase
from S commersonii To isolate the full-length cDNA
clones, RACE with specific primers was used to amplify the
5¢- and 3¢-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-TTM 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 D12-fatty acid desaturases from G hirsutum (58%), from S commersonii (59%) and from Corylus avellana (61%, accession number A65100), respectively A more detailed comparison of these sequences
is shown in Fig 2 Due the low sequence identity of the encoded protein against classical D12-fatty acid desaturases
it was expected that this clone may code for a (1,4)-acyl-lipid-desaturase It was therefore named PuFADX The full-length cDNA of Pun2 had a length of 1161 bp coding for a protein of 387 amino acids with a calculated molecular mass of 44.3 kDa The corresponding amino acid sequence showed higher identities against those of the classical D12 -fatty acid desaturases from S indicum (78%), S commerso-nii (77%) and C avellana (78%), respectively, and was therefore named PuFAD12 (Fig 2) These findings were substantiated further by phylogenetic tree analysis Fig 3 This analysis indicates that PuFADX, similar to all other (1,4)-acyl-lipid-desaturases isolated so far which modify a double bond at position D12of linoleic acid, groups into one subgroup of the acyl-lipid-desaturase family There is only one exception that is the (1,4)-acyl-lipid-desaturase from Impatiens, which in contrast modifies a-linolenic acid preferentially D12-acyl-lipid-acetylenases and epoxygenases
as well as D9-(1,4)-acyl-lipid-desaturases form another subgroup within this phylogenetic tree
Functional expression inS 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 30C 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
16C
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
30C (data not shown) Punicic acid could only be detected after supplementation of the growth media with linoleic acid thus confirming again this fatty acid as the precursor of plant (1,4)-acyl-lipid-desaturases producing trienoic fatty acids (Fig 5, upper panel vs middle panel) The accumu-lation of punicic acid was reduced at lower temperatures This was in contrast to the increased accumulation of linoleic acid and hexadecadienoic acid in cells expressing
Trang 4PuFAD12 at this temperature In cells maintained at 30C
and supplemented with linoleic acid, punicic acid accounted
for up to 1.6% (w/w) of the total fatty acids The identity of
the conjutrienoic fatty acid methyl ester punicic acid in yeast
cell cultures was established by gas chromatographic
retention times of the methyl esters of three different
positional isomers as authentic standards In the lower panel
of Fig 5 it is shown that the different positional isomers of conjutrienoic fatty acids could be clearly separated under the conditions used In addition, mass spectrometry was performed to confirm the identity of the substance assigned
as punicic acid The mass spectrum of this fatty acid methyl ester, shown in the upper panel of Fig 6, was identical to that of methyl punicic acid and was characterized by an abundant molecular ion of m/z¼ 292 which has been shown before to be characteristic for conjugated fatty acids [7]
To investigate further the substrate specificity of PuFADX, induced yeast cultures were supplemented either with cis- or trans-vaccenic acid, a- or c-linolenic acid or with homo-c-linolenic acid, respectively, and grown at 30C With cis- and trans-vaccenic acid, a-linolenic acid and 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/z¼ 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 D12-acyl lipid desaturases were selected which displayed the
highest amino acid identities towards the two newly described D12
-acyl-lipid-desaturases from pomegranate.
Fig 3 Phylogenetic tree analysis of plant acyl-lipid-desaturases The protein alignment was generated with the CLUSTAL - X program, and the phylogenetic tree was made with TREEVIEW Arabidopsis thaliana: AtFAD12 (ATD12aaa); Calendula officinalis: CoFAD12 (AF343065), CoFADX-1 (AF310155), CoFADX-2 (AF310156); Crepis alpina: CaFAD12ace (Y16285); Crepis palaestina: CpFAD12 (Y16284); CpFAD12epo (Y16283); Daucus carota: DcFAD12-OH (AF349965); Dimorphotheca sinuata: DmFAD12t (WO 01/128000), DmFADX-OH (WO 01/128000); G hirsutum: GhFAD12 (Y10112); Glycine max: GmFAD12 (L43921); Helianthus annuus: HaFAD12 (AF251842);
I balsamina: IbFADX (AF182520); Lesquerella fendleri:
LfFAD12-OH (AF016103); Licania michauxii: LmFADX (WO 00/11176); Momordica charantia; McFADX (AF182521); P granatum; PuFAD12 (AJ437139), PuFADX (AJ437140); Ricinus communis; RcFAD12-OH (U23378); S commersonii: ScFAD12 (X92847);
S indicum: SiFAD12 (AF192486); Vernonia galamensis: VgFAD12-1 (AF188263), VgFAD12-2 (AF188264) FAD12: D12-fatty acid desaturase, FADX: (1,4)-acyl-lipid-desaturase, FAD-OH: fatty acid hydroxylase.
Trang 5formation 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
D I S C U S S I O N
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 D12 and D15 -acyl-lipid-desaturases [12], -acyl-lipid-desaturases directly fused to their elec-tron donor such as D5 and D6-acyl-lipid-desaturases [19], hydroxylases [14], acetylenases and epoxygenases [13], (1,4)-acyl-lipid-desaturases [7,10], and recently desaturases which form hydroxy groups in conjugation to double bonds [20] Here, we report on the isolation of two diverse acyl-lipid-desaturases, PuFAD12 and PuFADX, respectively, from pomegranate seeds Both cDNAs have sequence similarity
to acyl-lipid-desaturases from plants PuFAD12 has higher amino acid identity to the classical D12-acyl-lipid-desaturases (approximately 80%), whereas PuFADX has equal
Fig 4 GC/FID analysis of fatty acid methyl esters isolated from yeast
cells transformed with pESC-LEU-PuFAD12 The lipids were extracted
from lyophilized yeast cells, esterified fatty acids were transmethylated
and analyzed by GC as described under materials and methods The
upper panel shows the fatty acid profile of nontransformed yeast cells
as controls All fatty acids were characterized by coelution of authentic
standards and the lower panel shows a linoleic acid standard.
Fig 5 GC/FID analysis of fatty acid methyl esters isolated from yeast cells supplemented with linoleic acid and transformed with pYES-PuFADX The lipids were extracted from lyophilized yeast cells, este-rified fatty acids were transmethylated and analyzed by GC as described in Materials and methods The upper panel shows the fatty acid profile of nontransformed yeast cells as controls All fatty acids were characterized by coelution of authentic standards and the lower panel shows a standard mixture of three different conjutrienoic fatty acids.
Trang 6sequence identity (approximately 60%) to both classical D12
-desaturases and D12-desaturase-related nonclassical
acyl-lipid-desaturases Expression of PuFAD12 in yeast cells
indicated that it is a classical D12-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, conjugase and (1,4)-acyl-lipid-desaturase 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: Conju-gase, since the enzyme forms two conjugated double bonds out of one isolated double bond and (1,4)-acyl lipid desaturase, 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-rantiawere found to be able to convert the D12-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 18:3D9Z,11E,13E [7] In addition (1,4)-acyl-lipid-desaturases isolated from
C officinalis have been shown to convert the D9-double bond of linoleic acid again into two conjugated and trans configurated double bonds at the 8 and 10 positions to produce another conjugated linolenic acid derivative 18:3D8E,10E,12Z [8–10] As shown here by heterologous expression in yeast cells the enzyme PuFADX converts linoleic acid into another conjugated linolenic acid deri-vative (Fig 5) In contrast to all other yet known (1,4)-acyl-lipid-desaturases, this enzymes converts the D12-double bond of linoleic acid into two conjugated and trans–cis configurated double bonds at the 11 and 13 positions, resulting in the production of the conjugated linolenic acid 18:3D9Z,11E,13Z It will be interesting to see by which mechani-stic parameters within the different enzymes the formation
of either a (Z,E,E) or (Z,E,Z)-configurated double bond system of the different linolenic acid isomers is determined Plant oils containing conjugated linolenic acid derivatives are of commercial interest, since they are used as drying oils
in paints Thus seed oils may be useful to be produced via transgenic approaches in a commercially important crop 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
16:2 D9Z,12Z b
a Amount of each fatty acid was expressed as relative ratio of all fatty acids found b New detected fatty acids c This fatty acid may be derived due to substrate impurities.
Trang 7biosynthetic 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 D12-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
A C K N O W L E D G E M E N T S
The authors are grateful to M Pu¨rschel for expert technical assistance This work was supported by the BASF Plant Science GmbH.
R E F E R E N C E S
1 Somerville, C., Browse, J., Jaworski, J.G & Ohlrogge, J (2000) Lipids In Biochemistry and Molecular Biology of Plants (Bucha-nan, B.B., Gruissem, W & Jones, R.L., eds), pp 456–527 American Society of Plant Physiologists, Rockville, ML.
2 Tulloch, A.P (1982) 13 C Nuclear magnetic resonance spectro-scopic analysis of seed oils containing conjugated unsaturated acids Lipids 17, 544–550.
3 Gunstone, F.D., Harwood, J.L & Padley, F.B (1994) The Lipid Handbook, 2nd edn Chapman & Hall, London.
4 Hamberg, M (1992) Metabolism of 6,9,12-octadecatrienoic acids
in the red alga Lithothamnion corallioides: mechanism of forma-tion of a conjugated tetraene fatty acid Biochem Biophys Res Commun 188, 1220–1227.
5 Wise, M.L., Hamberg, M & Gerwick, W.H (1994) Biosynthesis
of conjugated triene-containing fatty acids by a novel isomerase from the red marine alga Ptilota filicina Biochemistry 33, 15223– 15232.
6 Zheng, W., Wise, M., Wyrick, A., Metz, J., Yuan, L & Gerwick, W (2002) Polyenoic fatty acid isomerase from the marine alga Ptilota filicina: protein characterization and functional expression of the cloned cDNA Arch Biochem Biophys 401, 11–20.
7 Cahoon, E.B., Carlson, T.J., Ripp, K.G., Schweiger, B.J., Cook, G.A., Hall, S.E & Kinney, A.J (1999) Biosynthetic origin of conjugated double bonds: Production of fatty acid components of high-value drying oils in transgenic soybean embryos Proc Natl 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, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean Plant Physiol 113, 933–942.
15 Sperling, P., Linscheid, M., Stocker, S., Muhlbach, H.P & Heinz,
E (1993) In vivo desaturation of cis-delta 9-monounsaturated to
Fig 7 GC/FID analysis of fatty acid methyl esters isolated from yeast
cells supplemented with equimolar amounts of linoleic acid, a-linolenic
acid and c-linolenic acid and transformed with pYES-PuFADX The
lipids were extracted from lyophilized yeast cells, esterified fatty acids
were transmethylated and analyzed by GC as described under
Mate-rials and methods The upper panel shows the fatty acid profile of
nontransformed yeast cells as controls All fatty acids were
charac-terized by coelution of authentic standards and the lower panel shows
a standard mixture of three different conjutrienoic fatty acids.
Trang 8cis-delta 9,12-diunsaturated alkenylether glycerolipids J Biol.
Chem 268, 26935–26940.
16 Liu, L., Hammond, E.G & Nikolau, B.J (1997) In vivo studies of
the biosynthesis of a-eleostearic acid in the seed of Momordica
charantia L Plant Physiol 113, 1343–1349.
17 von Pein, M (1992) PhD Thesis, Heinrich Heine-Universita¨t,
Du¨sseldorf.
18 Millar, A.A., Smith, M.A & Kunst, I (2000) All fatty acids are
not equal: discrimination in plant membrane lipids Trends Plant
Sci 5, 95–101.
19 Sperling, P & Heinz, E (2001) Desaturases fused to their electron 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.