Cloning and functional characterization of Phaeodactylum tricornutumfront-end desaturases involved in eicosapentaenoic acid biosynthesis Fre´de´ric Domergue1,*, Jens Lerchl2, Ulrich Za¨h
Trang 1Cloning and functional characterization of Phaeodactylum tricornutum
front-end desaturases involved in eicosapentaenoic acid biosynthesis
Fre´de´ric Domergue1,*, Jens Lerchl2, Ulrich Za¨hringer3and Ernst Heinz1
1
Institut fu¨r Allgemeine Botanik, Universita¨t Hamburg, Hamburg, Germany;2BASF Plant Science GmbH, BPS-A30,
Ludwigshafen, Germany;3Forschungszentrum Borstel, Borstel, Germany
Phaeodactylum tricornutum is an unicellular silica-less
diatom in which eicosapentaenoic acid accumulates up to
30% of the total fatty acids This marine diatom was used for
cloning genes encoding fatty acid desaturases involved in
eicosapentaenoic acid biosynthesis Using a combination of
PCR, mass sequencing and library screening, the coding
sequences of two desaturases were identified Both protein
sequences contained a cytochrome b5domain fused to the
N-terminus and the three histidine clusters common to all
front-end fatty acid desaturases The full length clones were
expressed in Saccharomyces cerevisiae and characterized as
D5- and D6-fatty acid desaturases The substrate specificity
of each enzyme was determined and confirmed their
involvement in eicosapentaenoic acid biosynthesis Using
both desaturases in combination with the D6-specific
elongase from Physcomitrella patens, the biosynthetic path-ways of arachidonic and eicosapentaenoic acid were recon-stituted in yeast These reconstitutions indicated that these two desaturases functioned in the x3- and x6-pathways, in good agreement with both routes coexisting in Phaeodacty-lum tricornutum Interestingly, when the substrate selectivity
of each enzyme was determined, both desaturases converted the x3- and x6-fatty acids with similar efficiencies, indicating that none of them was specific for either the x3- or the x6-pathway To our knowledge, this is the first report describing the isolation and biochemical characterization of fatty acid desaturases from diatoms
Keywords: front-end desaturase; diatom; polyunsaturated fatty acid; eicosapentaenoic acid
Diatoms are unicellular photosynthetic microalgae of the
phytoplankton that are particularly important in ocean
ecosystems; they are thought to be responsible for as much
as 25% of global primary productivity and for an
accord-ingly significant O2production [1] Because most of diatoms
are surrounded by a highly structured silica cell wall, they
also play a key role in the biogeochemical cycling of silica
[2] Diatoms are used commercially for various purposes
such as feeds in aquaculture, sources of polyunsaturated
fatty acids (PUFAs) or pharmaceutical drugs [3]
Phaeo-dactylum tricornutum, a silica-less diatom, is one of the most
widely utilized model systems for studying the ecology,
physiology, biochemistry and molecular biology of diatoms
[4] This organism became even more attractive with the
recent establishment of a procedure for its stable
transfor-mation [4–6], which enabled the demonstration of its
sensing system [7] and its conversion into a heterotrophic
organism, opening the possibility to grow it by large-scale
fermentation for commercial exploitation [8]
As its fatty acid composition contains up to 30% eicosapentaenoic acid (EPA, 20:5D5,8,11,14,17) and only traces of docosahexaenoic acid (DHA, 22:6D4,7,10,13,16,19),
P tricornutumrepresents an interesting alternative source for the industrial production of EPA, and gram-scale purification of this particular PUFA has already been achieved [9] Very long chain PUFAs like EPA, DHA and arachidonic acid (ARA, 20:4D5,8,11,14) have received great interest as such fatty acids are required in the human diet for normal health and development, particularly in the case
of new-borns and infants [10,11] Polyunsaturated fatty acids are important constituents of membranes and precursors of several biologically active eicosanoids, like prostaglandins and leukotrienes, which regulate many physiological functions in mammals [12] For mammals lacking D12- and D15-fatty acid desaturases, 18:2D9,12and 18:3D9,12,15 are considered to be essential fatty acids that must be supplied in the diet Although humans can synthesize very-long chain PUFAs from these precursors, dietary changes over the last decades resulted in very high x6- to x3-fatty acid ratios that have negative impacts on both health and development [13,14] In order to circum-vent this deficit in x3-fatty acids and to preserve marine reserves, alternative sources have been searched, among others the production of PUFAs in transgenic oilseed crops [15] Such a goal has led to the identification of most
of the genes coding for the enzymes involved in PUFA biosynthesis (fatty acid desaturases and elongases) in several organisms, such as the nematode Caenorhabditis elegans[16], the fungus Mortierella alpina [17,18] and the moss Physcomitrella patens [19,20], but so far not in any diatom, even though they represent an important group of primary x3-fatty acid producers
Correspondence to F Domergue, Institut fu¨r Allgemeine Botanik,
Universita¨t Hamburg, Ohnhorststrasse 18, 22609 Hamburg,
Germany Fax: +49 40 42816 254, Tel.: + 49 40 42816 373,
E-mail: fredDo@botanik.uni-hamburg.de
Abbreviations: PUFAs, polyunsaturated fatty acids; GLC-MS, gas
liquid chromatography-mass spectrometry; ARA, arachidonic acid;
EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; FAMEs,
fatty acid methyl esters; FID, flame-ionization detector.
*Present address: Plant Science Sweden AB, SE-26831 Svalo¨v, Sweden.
(Received 18 April 2002, revised 24 June 2002,
accepted 10 July 2002)
Trang 2Using various14C-labelled precursors, Moreno et al [21]
showed that P tricornutum synthesized EPA de novo by
elongation and aerobic desaturation of fatty acids As
shown in Fig 1, EPA can be synthesized by the classical
x6-pathway, the classical x3-pathway, a pathway relying on
intermediates of both of these pathways and by an
alter-native x3-pathway involving D9-elongation and
D8-desat-uration [22] Pulse-chase experiments with [14C]linoleic acid
suggested that the third route was the most active one,
invol-ving successively D6-desaturation (18:2D9,12to 18:3D6,9,12),
x3-desaturation (18:3D6,9,12to 18:4D6,9,12,15), D6-elongation
(18:4D6,9,12,15 to 20:4D8,11,14,17) and a final D5-desaturation
(20:4D8,11,14,17 to 20:5D5,8,11,14,17 [22]) P tricornutum is a
particularly interesting model for studies on PUFA
biosyn-thesis because EPA represents up to 30% of the total fatty
acids whereas all the intermediates of its pathway are only
present in traces This accumulation of only EPA may
indicate that the elongase and desaturases responsible for its
biosynthesis are very effective in channelling the different
intermediates towards the final product We therefore
decided to use this organism for cloning the genes encoding
the different fatty acid desaturases involved in EPA
biosynthesis and identified two sequences coding for fatty
acid desaturases with D5- and D6-regioselectivities Using
these two genes together with that of the D6-specific
elongase from P patens [20], the biosynthetic pathways of
ARA and EPA were successfully reconstituted in yeast,
showing that these desaturases were responsible for the
D5-and D6-activities of both the x3- D5-and x6-pathways
M A T E R I A L S A N D M E T H O D S
Materials
Restriction enzymes, polymerases and DNA modifying
enzymes were obtained from New England Bioloabs
(Frankfurt, Germany) unless indicated otherwise All other chemicals were from Sigma (St Louis, MO, USA) Phaeodactylum tricornutum culture
P tricornutumUTEX 646 was grown in f/2 culture medium supplemented with 10% organic medium [23] at 22Cwith aeration and photoperiods of 16 h of light (35 lEÆm)2Æs)1) cDNA library construction
Frozen cells of P tricornutum were ground in the presence
of liquid nitrogen and the resulting fine powder was resuspended in 2 mL of homogenization buffer (0.33M
sorbitol, 0.3M NaCl, 10 mM EDTA, 10 mM EGTA, 2% SDS, 2% 2-mercaptoethanol in 0.2M Tris/HCl pH 8.5) Phenol (4 mL) and chloroform (2 mL) were added succes-sively and the samples were shaken vigorously for 15 min at 40–50C After centrifugation (10 min · 10 000 g), the aqueous phase was successively extracted with 2 mL of phenol/chloroform (1 : 1, v/v) and 2 mL of chloroform 1/20 vol of 4MNaHCO3and 1 vol of ice-cold isopropanol were added and the sample stored overnight at)20 C Nucleic acids were precipitated (30 min· 10 000 g), washed with 70% ethanol and resuspended in 80 mM
Tris/borate pH 7.0 containing 1 mM EDTA 1/3 vol of
8M LiCl was added and the sample incubated 30 min at
4C After centrifugation (30 min · 10 000 g), the pellet was washed with ethanol 70% (v/v) and resuspended in RNase-free water Poly(A)+ RNA was isolated with Dynabeads (Dynal, Oslo, Norway) and cDNA first strand synthesis was achieved using the murine leukemia virus reverse transcriptase (Roche, Mannheim, Germany) The second strand synthesis was carried out by incubation with DNA polymerase I and Klenow enzyme, followed by RNaseH digestion cDNA was blunted with the T4 DNA
Fig 1 Possible biosynthetic routes leading to EPA biosynthesis in Phaeodactylum tricornu-tum The classical x6- and x3-pathways are framed and the alternative x3-pathway (involving D9-elongation and D8-desatura-tion) is shown with broken arrows The most active route according to Arao and Yamada [22] is shown with wide arrows and with text in bold.
Trang 3polymerase (Roche), and EcoRI/XhoI adapters (Pharmacia,
Freiburg, Germany) were ligated with the T4 DNA ligase
After XhoI digestion, phosphorylation with the
polynucleo-tide kinase (Roche) and gel separation, DNA molecules
larger than 300 bp were ligated into vector arms and
packaged into lambda ZAP Express phages using the
Gigapack Gold Kit (Stratagene, Amsterdam, Netherlands)
Random sequencing of the cDNA library
After in vivo mass excision of the cDNA library, plasmid
recovery and transformation of Escherichia coli DH10B
(Stratagene), plasmid DNA was prepared on a Qiagen
DNA preparation robot (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions and submitted
to random sequencing by the chain termination method
using the ABI PRISM Big Dye Termination Cycle
Sequencing Ready Reaction Kit (Perkin-Elmer,
Weiters-tadt, Germany) About 8400 clones were processed and
annotated using the standard software package EST-MAX
(Bio-Max, Munich, Germany), resulting in 3860
nonredun-dant sequences
PCR amplification
In parallel, a PCR-based cloning strategy was followed
using primer mixtures corresponding to the highly
con-served histidine clusters present in membrane-bound
desat-urases [24] After in vivo mass excision of the P tricornutum
cDNA library, the resulting plasmid bank was used as
template for PCR with different combinations of degenerate
primers [19] PCR amplifications were carried out using the
Pfu DNA polymerase (Stratagene) and the following
program: denaturation for 3 min at 96C; 5 cycles of 30 s
at 96C, 30 s at 55 C(decreasing by 3 Cin each
successive cycle), 1 min at 72C; 30 cycles of 30 s at 96 C,
30 s at 40C , 1 min at 72 C; and a final extension step of
10 min at 72C
cDNA library screening
Both methods yielded several sequences which were
anno-tated to putative desaturases For full length cloning via
library screening, the sequence of interest was cloned into
the pGEM-T vector (Promega, Madison, WI, USA) and a
digoxigenin-labelled DNA probe of this fragment was
synthesized by PCR The cDNA library described above
(about 5· 105 plaques) was screened according to the
manufacturer’s instructions (Boehringer, Mannheim,
Ger-many, Stratagene) After two rounds of screening, several
clones were isolated and the longest ones sequenced on both
strands
Functional characterization inS cerevisiae
For functional characterization, the P tricornutum cDNA
clones were cloned in different yeast expression vectors For
this purpose, the different open reading frames (ORF) were
modified by PCR to create appropriate restriction sites
adjacent to the start and stop codons and to insert the yeast
consensus sequence for enhanced translation [25] in front of
the start codon The amplified DNAs were first cloned into
pUC18 using the SureClone Ligation Kit (Pharmacia) The
ORFs were then released using the restriction sites created
by PCR and cloned into the same sites of different yeast expression vectors Using SpeI and SacI sites, the ORF of PtD5 was cloned behind the galactose-inducible promoter
PGAL10of the yeast expression vector pESC-LEU (Strata-gene), yielding pESC-LEU-PtD5 The ORF of PtD6 was cloned behind the constitutive promoter PADHof the yeast expression vector pVT102-U [26] using BamHI and XhoI sites, yielding pVT102-U-PtD6 To obtain pESC-LEU-PSE1-PtD5, the ORF of pse1 was released from pY2PSE1 [20] using BamHI, cloned behind the galactose-inducible promoter PGAL1of pESC-LEU before inserting the ORF of PtD5 as indicated above
C13ABYS86 Saccharomyces cerevisiae strain (leu2, ura3, his, pra1, prb1, prc1, cps [27]) was transformed with plasmid DNA by a modified PEG/lithium acetate protocol [28] After selection on minimal medium agar plates without uracil or leucine, cells harbouring the yeast plasmid were cultivated in minimal medium lacking uracil or leucine but containing 2% (w/v) raffinose and 1% (v/v) Tergitol NP-40 The expression was induced by supplementing galactose to 2% (w/v) when the cultures had reached a D600of 0.2–0.3
At that time, the appropriate fatty acids were added to a final concentration of 500 lM, unless indicated otherwise All cultures were then grown for a further 48 h at 20C, unless indicated otherwise, and used for fatty acid analysis Fatty acid analysis
Fatty acid methyl esters (FAMEs) were obtained by transmethylation of yeast cell sediments with 0.5M sulphu-ric acid in methanol containing 2% (v/v) dimethoxypropane
at 80Cfor 1 h FAMEs were extracted in petroleum ether and analysed by gas-liquid chromatography (GLC) using a Hewlett-Packard 6850 gas chromatograph (Hewlett-Pac-kard, Palo Alto, CA, USA) equipped with a polar capillary column (ZB-Wax, 30 m· 0.32 mm internal diameter, 0.25 lm film, Torrance, CA, USA) and a flame-ionization detector (FID) Data were processed using the HP Chem Station Rev A06.03 FAMEs were identified by compar-ison with appropriate reference subtances or by GLC-MS of FAMEs and 4,4-dimethyloxazoline derivatives as described previously [29]
R E S U L T S Isolation of two fatty acid desaturase-like cDNA clones Two full length clones encoding putative fatty acid desat-urases were isolated from the P tricornutum cDNA library using a combination of PCR amplification, mass sequencing and library screening as described in the Materials and methods The first clone was 1689 bp and contained an ORF of 1434 bp coding for a polypeptide of 477 amino acids This polypeptide showed high sequence homologies
to various D6-desaturases It was, for example, 43% identical (59% similar) to the D6-desaturase from Pythium irregulare (GenBank accession number AAL13310) and 37% identical (53% similar) to the D6-desaturase from Mortierella alpina[30] Accordingly, this clone was anno-tated PtD6 (for P tricornutum delta 6-desaturase) The second clone was 1672 bp and contained an ORF of
1410 bp coding for a polypeptide of 469 amino acids; it had
Trang 4highest sequence similarities to the two D5-fatty acid
desaturases recently cloned from Dictyostelium discoideum
For example, it was 21% identical (39% similar) to the
second D5-desaturase from D discoideum [31] This clone
was consequently annotated PtD5
Protein domains
Figure 2 shows the amino acid sequences of the proteins
encoded by PtD5 and PtD6 (PtD5p and PtD6p,
respect-ively) Both proteins contain the three conserved histidine
clusters characteristic for all membrane-bound desaturases
[24] They also contain a cytochrome b5domain fused at the
N-terminus and an H to Q substitution in the third
histidine-box, both of these features being typical of
front-end desaturases [32] Hydrophobic plots have indicated the
presence of several long hydrophobic stretches and the four
potential transmembrane helices fitting to the topological
model developed for membrane-bound desaturases by
Shanklin et al [33] are shown In good agreement with this
model, the program developed by Nakai & Horton for
prediction of cellular localization [34] indicated that both
proteins were localized in the endoplasmic reticulum, although no di-lysine retention signal was present in the sequences
Figure 2 also shows the amino acid sequence of the free cytochrome b5(PtCytb5), which was obtained in full length from the mass sequencing Whereas the cytochrome b5 domain of PtD6p contains the eight invariant amino acids
of the cytochrome b5superfamily (Fig 2, dots; [35]), PtD5p has two substitutions (W to E and Y to F), with only the last one being conserved Such substitutions have in fact already been found for four of the eight conserved amino acids as only the HPGG cluster is systematically present in all the cytochrome b5domains of front-end desaturases cloned so far Another characteristic feature of the cytochrome b5 domain of fused desaturase was observed in P tricornutum The sequence of the free microsomal cytochrome b5 between the two highly conserved W and G (marked with arrows) contains 12 acidic amino acids (aspartate and glutamate), whereas the corresponding domain sequences in PtD5p and PtD6p have only eight and five acidic residues, respectively Such a decrease in the number of acidic amino acids in fused cytochrome b5domains has been observed in
Fig 2 Amino acid sequences of PtD5p and PtD6p For alignment the CLUSTAL X program was used (gap opening 12, gap extension 0.05) and the five highest BLAST scores of each desaturase (not shown) in order to obtain correct alignment of the histidine clusters The conserved amino acids are on black back-ground The eight amino acids which are usually conserved in the cytochrome b 5
superfamily are marked with dots, the three histidine boxes framed and the potential transmembrane domains underlined The sequence data published here have been submitted to the DDBJ/EMBL/GenBank sequence data bank with the accession number AY082392, AY082393 and AF503284 for PtD5p, PtD6p and PtCytb5, respectively.
Trang 5most front-end desaturases cloned so far and may be
correlated with the necessary close interaction between this
domain and the desaturase part [32]
Functional characterization inSaccharomyces cerevisiae
The activities of the proteins encoded by these two cDNAs
were confirmed by heterologous expression in S cerevisiae
The open reading frames of PtD6 and PtD5 were cloned in
the yeast expression vectors pVT102-U [26] and pESC-LEU
(Strategene), respectively, and these constructs and the
empty vectors were transformed into the S cerevisiae strain
C13ABYS86 [27] Expression were carried out for 48 h at
20Cin the presence of the potential substrates for D6- or
D5-fatty acid desaturases, 18:2D9,12and 20:3D8,11,14,
respect-ively In the presence of the empty vector pVT102-U and
500 lMlinoleic acid, only the endogenous yeast fatty acids
and the additional substrate could be detected indicating
that yeast did not metabolize the exogenously added fatty
acid (Fig 3A) In contrast, in the yeast containing
pVT102-U-PtD6, a new fatty acid corresponding to c-linolenic acid
(18:3D6,9,12) was detected The structure of this fatty acid was
confirmed by GLC-MS analyses demonstrating that PtD6p
was a D6-desaturase In pVT102-U, the expression of PtD6
was under the control of a constitutive promoter (PADH)
which resulted in a high desaturation of linoleic acid (25–
30%) When PtD5p was expressed in the presence of
20:3D8,11,14, a peak corresponding to arachidonic acid
(ARA, 20:4D5,8,11,14) was detected (Fig 3B) The structure
was confirmed by GLC-MS analyses, demonstrating that
PtD5p was a D5-desaturase
Specificity ofPhaeodactylum tricornutum front-end
desaturases
The substrate specificity of each desaturase was
subse-quently determined in the same transgenic yeast using
different potential substrates (Table 1) The favourite
sub-strate of PtD6p was linoleic acid, but a-linolenic acid
(18:3D9,12,15) was converted to stearidonic acid (18:4D6,9,12,15)
with similar efficiency In the absence of exogenously fed
fatty acids, 5–6% of the endogenous monounsaturated fatty
acids of yeast were desaturated (16:1D9to 16:2D6,9and 18:1D9
to 18:2D6,9), similar to the D6-desaturases from P patens
[19] and M alpina [17] In order to check if PtD6p could
also be responsible for the D8-desaturation activity involved
in the alternative x3-pathway (see Fig 1), similar to the
bifunctional (D5/D6) fatty acid desaturase recently cloned
from zebrafish [36], 20:3D11,14,17was assayed as a potential
substrate No conversion of this substrate could be detected indicating that PtD6p did not display any D8-desaturase activity (Table 1) The highest activity of PtD5p was obtained with 20:3D8,11,14as substrate (24,7% conversion), but PtD5p was also active with both 20:2D11,14 and 20:3D11,14,17, as it has already been reported for the D5-desaturase from C elegans [37] PtD5p also showed low activities with 20:1D11and vaccenic acid (18:1D11), but
no activity was detected with 20:1D8(Table 1) or with oleic, linoleic and a-linolenic acid (data not shown)
Reconstitution of PUFA biosynthetic pathways
inSaccharomyces cerevisiae Using the two front-end desaturases from P tricornutum and Pse1p, the D6-specific elongase from P patens [20], the possibility of reconstituting the biosynthetic pathways of arachidonic and eicosapentaenoic acid in yeast was inves-tigated Pse1p was used as it allows the synthesis of 20:3D8,11,14 and 20:4D8,11,14,17 from 18:3D6,9,12 and 18:4D6,9,12,15, respectively [20] When PtD5p was coexpressed with Pse1p in the presence of c-linolenic acid, 20:3D8,11,14
and ARA were synthesized (Fig 4A) About 50% of 18:3D6,9,12was elongated by Pse1p and 15% of the resulting 20:3D8,11,14was converted to 20:4D5,8,11,14by PtD5p Simi-larly, when 18:4D6,9,12,15was exogenously fed, 20:4D8,11,14,17
Fig 3 Fatty acid profiles of yeast transformed
with pVT102-U-PtD6 (A) or pESC-LEU-PtD5
(B) C13ABYS86 yeast strain transformed
with either the empty vector (dashed line) or
the different constructs (full line) was
supple-mented with different fatty acids (A, 500 l M
18:2D9,12; B, 500 l M 20:3D8,11,14) and grown for
48 h at 20 C FAMEs from the whole cells
were prepared and analysed by GLCas
indi-cated under Material and methods.
Table 1 Substrate specificity of PtD5p and PtD6p expressed in Sac-charomyces cerevisiae Yeast strain C13ABYS86 was transformed with the different constructs (pESC-LEU-PtD5 or pVT102-U-PtD6) and grown for 48 h at 20 Cin the presence of different fatty acid substrates (500 l M or as indicated) FAMEs from the whole cells were prepared and analysed by GLCas indicated under Material and methods Desaturation (%) was calculated as (product · 100)/(educt + prod-uct) using values corresponding to percent of total fatty acids Each value is the mean ± SD from three to five independent experiments.
Substrate Desaturation Substrate Desaturation 18:1D11 2.0 ± 0.2 16:1D9 a 5.7 ± 0.6
20:1 D11 b 3.7 ± 0.7 18:2 D9,12 27.8 ± 3.6 20:2D11,14 b 11.8 ± 1.0 18:3D9,12,15 26.7 ± 3.3 20:3D11,14,17 10.8 ± 0.3
20:3 D8,11,14 24.7 ± 3.3 20:3 D8,11,14 0
a
In the absence of exogenously fed fatty acid.bIn the presence of
1 m M exogenously fed fatty acid.
Trang 6and EPA were detected (Fig 4B) In the presence of
a-linolenic acid (18:3D9,12,15), the yeast containing the three
heterologous genes synthesized 18:4D6,9,12,15, 20:3D11,14,17,
20:4D8,11,14,17and 20:5D5,811,14,17(EPA, Table 2) The
pres-ence of 20:3D11,14,17 resulted from the elongation of
18:3D9,12,15by Pse1p Although the activity of Pse1p upon
18:3D9,12,15 is usually low, the high proportion of the
exogenously fed a-linolenic acid in the medium lead to a
significant production of 20:3D11,14,17(Table 2) In addition
to the unexpected synthesis of this fatty acid, traces of
16:2D6,9, 18:2D6,9and 20:4D5,11,14,17were detected (data not
shown), in agreement with the specificities of both
desatu-rases (Table 1) EPA was present and accumulated up to
0.23% of the total fatty acids, confirming that its complete
biosynthetic pathway from a-linolenic acid, involving
D6-desaturation, D6-elongation and D5-desaturation
(x3-pathway), had been successfully reconstituted About
16% of 18:3D9,12,15was converted by PtD6p, 24% of the
resulting 18:4D6,9,12,15 was then elongated by Pse1p and
about 14% of the elongated product was finally desaturated
to EPA by PtD5p When 18:2 was the exogenously fed
substrate, the synthesis of arachidonic acid (20:4D5,8,11,14)
was achieved, confirming that the three proteins were also
active in the x6-pathway (Table 2) PtD6p converted about 23% of 18:2D9,12to c-linolenic acid, Pse1p elongated 14% of 18:3D6,9,12 to 20:3D8,11,14 and finally, PtD5p desaturated about 10% of the elongated product to ARA Arachidonic acid represented 0.16% of the total fatty acids (Table 2) and, similar to the results obtained with the reconstitution of the EPA biosynthetic pathway, several side-products (20:2D11,14 and traces of 16:2D6,9, 18:2D6,9and 20:3D5,11,14) were present in the fatty acid profiles (data not shown)
Selectivity ofPhaeodactylum tricornutum front-end desaturases
In order to evaluate if EPA was preferentially synthesized in
P tricornutumthrough the x6- or x3-pathway, the select-ivity of both front-end desaturases was investigated In the x6-pathway, the D6-fatty acid desaturase acts on 18:2D9,12 before the action of the D6-elongase followed by D5- and x3-desaturases, whereas in the x3-pathway, the D6-desat-urase converts the product of the x3-desatD6-desat-urase, 18:3D9,12,15,
to 18:4D6,9,12,15 (see Fig 1) The selectivity of PtD6p was assayed in the presence of 125 lM of both 18:2D9,12and 18:3D9,12,15 so that the four potential substrates of PtD6p
Fig 4 Fatty acid profiles of yeast transformed with pESC-LEU-PSE1-PtD5 C13ABYS86 yeast strain transformed with either the empty vector (dashed line) or pESC-LEU-PSE1-PtD5 (full line) was supplemented with
500 l M 18:3D6,9,12(A) or 18:4D6,9,12,15(B) and grown for 48 h at 20 C FAMEs from the whole cells were prepared and analysed
by GLCas indicated under Material and methods.
Table 2 Reconstitution of pathways for EPA and ARA biosynthesis in yeast C13ABYS86 yeast strain was transformed with pVT102-U-PtD6 and pESC-LEU-PSE1-PtD5 or the corresponding empty vectors The transformants were grown for 96 h at 20 Cin the presence of 250 l M 18:3 D9,12,15
or 18:2D9,12 FAMEs from the whole cells were prepared and analysed by GLCas indicated under Material and methods Each value corresponds
to the percent of total fatty acids and is the mean ± SD from three independent experiments.
18:3 D9,12,15 exogenously supplied 18:2 D9,12 exogenously supplied Fatty
acid
Empty vectors
PtD6 + PSE1 + PtD5
Empty vectors
PtD6 + PSE1 + PtD5
18:3 D9,12,15 42.6 ± 1.2 36.7 ± 1.9 – –
Trang 7(16:1D9, 18:1D9, 18:2D9,12 and 18:3D9,12,15, Table 1) were
present in similar proportions (Fig 5A) PtD6p desaturated
the intermediates of the x3- and x6-pathway, 18:3D9,12,15
and 18:2D9,12, respectively, with similar efficiency At the
same time, it was also active on the monounsaturated fatty
acids (16:1D9and 18:1D9) Interestingly, the conversions of
the four potential substrates by PtD6p observed in this
experiment (6.3, 5.8, 33.5 and 25.6% for 16:1D9, 18:1D9,
18:2D9,12 and 18:3D9,12,15, respectively) were very close to
those obtained when feeding a single substrate (see Table 1)
Therefore, PtD6p showed much higher activities towards
the dienoic and trienoic intermediates of PUFA biosynthesis
than towards the monounsaturated fatty acids, but it was
not confined to either the classical x6- or x3-pathway
The D5-fatty acid desaturase is the last enzyme involved
in the x3-pathway, converting 20:4D8,11,14,17 to EPA,
whereas in the x6-pathway, it converts 20:3D8,11,14 to
20:4D5,8,11,14 before an x3-desaturase synthesizes
20:5D5,8,11,14,17(see Fig 1) To check the activity of PtD5p
on both substrates, PtD5p was coexpressed with the
elongase Pse1p in the presence of both c-linolenic acid
and stearidonic acid (Fig 5B) Pse1p activity led to the
synthesis of 20:3D8,11,14 and 20:4D8,11,14,17, both of them
being further desaturated by PtD5p In good agreement
with Zank et al [20], the elongase displayed no selectivity
when facing two of its favourite substrates Fifty percent of
18:3D6,9,12and 45% of 18:4D6,9,12,15were elongated by Pse1p,
which is nearly identical to the data obtained upon feeding
the substrates separately [20] Similarly, PtD5p did not show
any preference for 20:3D8,11,14 or 20:4D8,11,14,17, as about
15% of each elongation product was desaturated
Conse-quently, like PtD6p, the D5-fatty acid desaturase from
P tricornutum has no preference for the x6- or the
x3-pathway These experiments confirmed that both
front-end desaturases were acting simultaneously in the
x3- and x6-pathway because they accept every potential
substrate encountered
When experiments similar to those reported in Fig 5
were conducted with the D5-desaturases from M alpina or
other D6-desaturases (from M alpina, Homo sapiens,
Borago officinalis, Ceratodon purpureus and P patens), none
of these front-end desaturases was specific for the x3- or the
x6-fatty acids (data not shown) Although these desaturases
differed in their level of activity and in substrate preference,
all the D5- and D6-desaturases tested showed similar
activities towards the two substrates provided
simulta-neously (20:3D8,11,14 and 20:4D8,11,14,17 or 18:2D9,12 and
18:3D9,12,15, respectively) These results concerning the
selectivity of front-end desaturases suggest that such enzymes are in general not specifically restricted to one of the two classical x3- or x6-pathways as depicted in Fig 1 but that the same enzymes are involved in both routes
D I S C U S S I O N
In the present study, we report the cloning and functional characterization of a D5- and a D6-fatty acid desaturase from P tricornutum Both contain the typical features of membrane-bound desaturases and a cytochrome b5domain fused to their N-terminal extremity (Fig 2), similar to other front-end desaturases with the exception of those from cyanobacteria [32,33] Such fused domains are also found at the C-terminal end of some D9-desaturases and in several hydroxylases, sulfite oxidases and nitrate reductases, all these proteins being members of the cytochrome b5 super-family [38] It should be noted that in a phylogenetic tree with the D5- and D6-desaturases from various organisms, PtD5p and PtD6p fell into different branches like the front-end desaturases from M alpina, whereas in the case of
C elegans or man, each pair of front-end desaturases formed a separate branch (data not shown) This may indi-cate that the D5- and D6-desaturases from P tricornutum and M alpina have evolved separately a long time ago while
a common ancestor has been functioning for a longer time
in the case of man and C elegans
Heterologous expression in S cerevisiae was used to confirm the D5- and D6-regioselectivity of PtD5p and PtD6p, respectively (Fig 3), and to determine each sub-strate specificity (Table 1) PtD6p desaturated 25–30% of both 18:2D9,12and 18:3D9,12,15(Table 1), while PtD5p was as active on 20:4D8,11,14,17as on 20:3D8,11,14when coexpressed with the D6-elongase from P patens, Pse1p (Fig 4) These specificities fit perfectly with their involvement in EPA biosynthesis Because their coexpression with Pse1p in the presence of 18:2D9,12or 18:3D9,12,15led to the synthesis of ARA or EPA (Table 2), respectively, PtD5p and PtD6p can function in both the x3- and x6-pathway Concerning selectivity, it was shown that neither PtD5p nor PtD6p showed any preference for x3- or x6-fatty acids (Fig 5) Moreover, each desaturase displayed similar activities towards the different substrates, when they were provided
in a mixture or fed as a single substrate As corresponding results were obtained with several D5- and D6-desaturases from different organisms, indiscriminate use of x3- and x6-fatty acids may be a general feature of front-end desaturases
Fig 5 Fatty acid profiles of yeast transformed
with pVT102-U-PtD6 (A) or
pESC-LEU-PSE1-PtD5 (B) C13ABYS86 yeast strain
transformed with either the empty vector
(dashed line) or the different constructs (full
line) was supplemented with different fatty
acids (A, 100 l M 18:2D9,12and 100 l M
18:3D9,12,15; B, 250 l M 18:3D6,9,12and 250 l M
18:4 D6,9,12,15 ) and grown for 48 h at 20 C.
FAMEs from the whole cells were prepared
and analysed by GLCas indicated under
Material and methods.
Trang 8The results presented in Fig 5B also show that the
D6-elongase converted equally well 18:3D6,9,12and 18:4D6,9,12,15
The fact that the presence or absence of an x3-double bond
has no effect on front-end desaturase and elongase activities
may be correlated with the regiochemistry of the reactions
catalysed Such enzymes are acting at the carboxyl end of
the molecule, whereas the structural difference between
x3-or x6-fatty acids resides in the methyl end Accx3-ordingly, it is
probable that neither the positioning of the substrate in the
reaction center nor the catalysis is affected by the presence
of an x3-double bond In this regard, the recent
demon-stration that the human D6-desaturase is not only active on
18:2D9,12and 18:3D9,12,15 but as well on 24:4D9,12,15,18 and
24:5D9,12,15,18,21[39] suggests that front-end desaturases may
tolerate some differences at the methyl end of their
substrates and only require an appropriate carboxylic end,
where the new double bond is to be inserted It should be
noted that the inverse situation was demonstrated in the
case of x3-desaturases Expression of the x3-desaturases
from Brassica napus and C elegans (FAT-1) in yeast clearly
showed that both were insensitive to the fatty acid chain
length and to the presence of double bonds proximal to the
carboxyl end [40,41] Similarly, a very low selectivity was
demonstrated for the x3-desaturase FAT-1 as this
desatur-ase expressed in mammalian cells converted almost all
x6-fatty acids to the corresponding x3-fatty acids
(18:2D9,12 to 18:3D9,12,15, 20:2D11,14 to 20:3D11,14,17,
20:3D8,11,14 to 20:4D8,11,14,17, ARA to EPA and even
22:4D7,10,13,16to 22:5D7,10,13,16,19[42])
In P tricornutum, the different intermediates of the EPA
biosynthetic pathway are only present in trace amounts,
suggesting that P tricornutum has developed highly efficient
mechanisms to achieve this specific fatty acid composition
Using in vivo labelling techniques, Arao & Yamada [22]
showed that four different routes led to the synthesis of EPA
in P tricornutum and that the preferred route relied on
intermediates of both the x6- and the x3-pathway (see
Fig 1) The results presented in Table 2 show that PtD5p
and PtD6p are involved in both pathways and they support
the preferred route, as the activity of PtD6p is higher with
18:2D9,12than with 18:3D9,12,15and that of PtD5p higher
with 20:4D8,11,14,17 than with 20:3D8,11,14 Nevertheless, the
substrate specificities and selectivities of PtD5p and PtD6p
cannot account for the nearly exclusive accumulation of
EPA observed in P tricornutum Moreover, and in good
agreement with Beaudoin et al [43], the synthesis of side
products suggests that all the enzymes involved in PUFAs
biosynthesis are rather unspecific and modify all the
different fatty acids they encounter (Table 2 and Fig 5)
Because of the relative insensitivity of front-end desaturases
and elongases to the presence of an x3-double bond and of
x3-desaturases to the structure of the carboxyl end of the
fatty acids, the existence of separate x3- and x6-pathways as
depicted in Fig 1 becomes questionable The results
presented here on front-end desaturases together with those
concerning x3-desaturase [42] and D6-elongase [20] are all in
favour of a scenario in which the same enzymes are
contributing to both pathways, which in fact do not exist
separately under these conditions As the activities of the
enzymes modifying the carboxyl end (D5- and
D6-desatu-rases and D6-elongase) and the methyl end of PUFAs
(x3-desaturase) do not depend on the structure of the opposite
end of the substrate, it is more likely that these enzymes act
simultaneously at several steps of interconnected pathways The resulting complex reaction network makes it impossible
to separate an x6- from an x3-pathway as the x3-desatur-ase is pulling all the intermediates into the x3-pathway when simultaneously, the front-end desaturases and elongase are pushing towards the end-product Accordingly, if these enzymes are expressed simultaneously in the same cellular compartment, it may not be appropriate to divide such a complex reaction network into separate x6- and x3-path-ways as depicted in Fig 1
To conclude, we have cloned and functionally charac-terized a D5- and a D6-fatty acid desaturase involved in EPA biosynthesis In addition, several lines of evidence presented in this study strongly suggest that front-end desaturases are not specific for the desaturation of a single fatty acid in a straight biosynthetic pathway but that they rather act simultaneously in both the x3- and x6-pathway and on other potential substrates This low specificity leads in yeast to the synthesis of undesirable side-products, which implies that the organisms accumulating a single PUFA like P tricornutum have developed highly effective strategies to channel all biosynthetic intermediates towards the accumulation of a single end-product In view of these results, elucidating and understanding the regulatory mechanisms leading to these highly selective accumula-tions becomes a clear prerequisite in order to implement PUFA biosynthesis in oilseed crops such as linseed or rapeseed
A C K N O W L E D G E M E N T S
This research has been supported by a Marie Curie Fellowship of the European Community programme Human Potential under the contract number HPMF-CT-1999-00148 We thank Dr T K Zank for providing the pY2PSE1 clone and BASF Plant Science GmbH (Ludwigshafen, Germany) for providing the k-ZAP Express library and for performing the random sequencing.
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