synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B.. napus cultivar
Trang 1Restoring enzyme activity in nonfunctional low erucic acid Brassica
Vesna Katavic1, Elzbieta Mietkiewska2,3, Dennis L Barton1, E Michael Giblin2, Darwin W Reed2
and David C Taylor2
1
Saskatchewan Wheat Pool Agricultural Research and Development, Saskatoon, Canada;2National Research
Council of Canada, Plant BiotechnologyInstitute, Saskatoon, Canada,3Plant Breeding and Acclimatization Institute,
Mlochow Research Center, Poland
Genomic fattyacid elongation 1 (FAE1) clones from high
erucic acid (HEA) Brassica napus, Brassica rapa and
Bras-sica oleracea, and low erucic acid (LEA) B napus cv Westar,
were amplified by PCR and expressed in yeast cells under the
control of the strong galactose-inducible promoter As
expected, yeast cells expressing the FAE1 genes from HEA
Brassicaspp synthesized very long chain monounsaturated
fatty acids that are not normally found in yeast, while fatty
acid profiles of yeast cells expressing the FAE1 gene from
LEA B napuswere identical to control yeast samples In
agreement with published findings regarding different HEA
and LEA B napus cultivars, comparison of FAE1 protein
sequences from HEA and LEA Brassicaceae revealed one
crucial amino acid difference: the serine residue at position
282 of the HEA FAE1 sequences is substituted by
phenyl-alanine in LEA B napus cv Westar Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B napus
cv Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain mono-unsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution Thus, for the first time, the low erucic acid trait in canola B napus can be attributed
to a single amino acid substitution which prevents the bio-synthesis of the eicosenoic and erucic acids
Keywords: Brassica; Brassicaceae; FattyAcid Elongation 1; 3-ketoacyl-CoA synthase; site directed mutagenesis
While de novo fatty acid synthesis occurs in plastids, the
synthesis of very long chain monounsaturated fatty acids
(VLCMFAs) is located in the cytosol and catalyzed by a
membrane-bound fatty acid elongation (FAE) complex on
the endoplasmic reticulum The initial substrate for the
elongation is oleic acid (18:1) The elongation of 18:1
involves the sequential addition of C2units from
malonyl-CoA to a long chain acyl-malonyl-CoA primer Each round of
elongation involves four enzymatic reactions catalyzed by
the FAE complex The FAE reactions are condensation
of malonyl-CoA with a long chain acyl-CoA to give a
3-ketoacyl-CoA, reduction to 3-hydroxyacyl-CoA,
dehy-dration to enoyl-CoA and final reduction of the enoyl-CoA resulting in an elongated acyl-CoA [1]
In high erucic acid (HEA) Brassicaceae, a seed-specific fatty acid elongase 1 (FAE1) is the condensing enzyme (3-ketoacyl-CoA synthase) that catalyzes the first of four enzymatic reactions of the FAE complex, resulting in the synthesis of VLCMFAs which are the major constituents of their seed oil It is assumed that the activities of the three subsequent enzymes crucial for VLCMFA biosynthesis, namely a 3-ketoacyl-CoA reductase, a 3-hydroxyacyl-CoA dehydrase and enoyl-CoA reductase, are present ubiqui-tously in plants and are common to all microsomal FAE systems In contrast, the condensing enzymes seem to be differentially expressed and are probably unique to each system Furthermore, it appears that FAE1 is a rate limiting enzyme for VLCMFA accumulation in seeds [2,3] while the other three enzymes of the elongase complex do not appear
to play a role in controlling VLCMFA formation [2] Millar & Kunst [2] demonstrated that the Arabidopsis FAE1gene is able to direct the synthesis of VLCFA in yeast Upon the expression of the Arabidopsis FAE1 coding region under the control of strong galactose-inducible (GAL1) promoter, the transformed yeast cells accumulated 20:1, 22:1 and 24:1 that are not normally present in nontrans-formed yeast cultures
Han et al [4–6] expressed Arabidopsis and B napus FAE1genes in yeast cells and concluded that in addition to 18:1 D9, both elongases are able to elongate the 16:1 D9 acyl chain However, the Arabidopsis FAE1 prefers to use 18:1 D9 and 18:1 D11 to produce 20:1 D11 and 20:1 D13, respectively, while the Brassica napus FAE1 more efficiently
Correspondence to V Katavic, NRC/PBI, 110 Gymnasium Place,
Saskatoon, SK, S7N 0W9, Canada.
Fax: + 1 306 975 4839, Tel.: + 1 306 975 5273,
E-mail: Vesna.Katavic@nrc.ca
Abbreviations: FAE1, fattyacid elongation 1; FAE1, fatty acid
elongase 1; FAMEs, fatty acid methyl esters; HEA, high erucic acid;
LEA, low erucic acid; LPAT, lyso-phosphatidic acid acyltransferase;
3-KCS, 3-ketoacyl-CoA synthase; MDE, microspore derived embryo;
SC-ura, synthetic complete medium lacking uracil; SDM, site directed
mutagenesis; VLCMFAs, very long chain monounsaturated fatty
acids; VLCFAs, very long chain fatty acids; WS-SDM, Westar
culti-var site directed mutated; WS-wt, Westar culticulti-var wild-type.
Note: The nucleotide sequence data reported are deposited in the
GenBank database under accession numbers AF490459, AF490460,
AF490461 and AF490462.
(Received 3 July 2002, revised 21 August 2002,
accepted 18 September 2002)
Trang 2utilizes 20:1 D11 and 20:1 D13 to make 22:1 D13 and 22:1
D15, respectively
As a part of our effort to increase the amounts of
industrially valuable VLCFAs, particularly erucic acid
(22:1) in Canadian HEA cultivars, we focused our research
on manipulating genes/enzymes which are involved in the
accumulation of VLCFAs in seed oil: an
erucoyl-CoA-utilizing lyso-phosphatidic acid acyltransferase (LPAT)
crucial for trierucin bioassembly, and the seed-specific
FAE1, crucial for erucic acid biosynthesis Earlier, we
reported on the expression of yeast LPAT gene SLC1-1 and
Arabidopsis FAE1coding regions in target HEA B napus
germplasm, and the performance of transgenic progeny in
the field [3,7,8]
Although the cloning of genes encoding 3-ketoacyl-CoA
synthase (3-KCS) from different plant species has been
achieved [4,9–21], knowledge about the mechanism of
action and properties of FAE1 and other elongase
conden-sing enzymes is limited due to the fact that these enzymes are
membrane-bound, and as such are inherently more difficult
to characterize biochemically than soluble condensing
enzymes
Recently, Ghanevati & Jaworski [22] generated and
analyzed a number of Arabidopsis FAE1 mutants to decipher
the importance of cysteine and histidine residues as possible
catalytic residues of FAE1 condensing enzymes Their
results have shown that cysteine 223 is essential for FAE1
KCS activity, and that it seems to have a similar role to
the active-site cysteines present in other condensing enzymes
To get more insight into how the coding region of the
FAE1 condensing enzymes in Brassicas determines the
proportions and amounts of VLCFAs in their seed oils, we
have expressed FAE1 coding regions from HEA B napus,
Brassica rapa(formerly Brassica campestris) and Brassica
oleraceain yeast
Intense research is ongoing by several groups to elucidate
the mutations involved in the loss of FAE1 condensing
enzyme activity in LEA B napus cultivars Han et al [6]
speculated that the presence of serine at position 282 in all
functional proteins instead of phenylalanine in
nonfunc-tional LEA B napus FAE1 could be important for the
activity of the condensing enzyme Roscoe et al [23]
hypothesized that the LEA phenotype could be the result
of one or more lesions in the genes that encode or regulate
FAE1 activity To clarify this controversy, we decided to
examine the role of the amino acid serine at position 282 in
the FAE1 protein sequence to determine if this apparent
mutation from serine to phenylalanine led to the LEA
B napusphenotype We introduced a point mutation into
the LEA B napus cv Westar FAE1 coding region to
substitute phenylalanine with serine at position 282 in an
attempt to restore the FAE1 condensing enzyme activity
Here we report and discuss the results of analyses of
heterologous expression in yeast and site-directed mutation
of Brassica FAE1 condensing enzymes
M A T E R I A L S A N D M E T H O D S
Plant materials
HEA B napus cv Hero [24], B rapa microspore-derived
embryo line, MDE R500 [25], B oleracea
microspore-derived embryo line, MDE 103 [26], and LEA B napus
canola cv Westar were used in this study for the cloning of FAE1 coding regions
Cloning FAE1 coding regions and heterologous expression in yeast
Based on known FAE1 sequences from Arabidopsis and
B napus, the forward primer VBE4 (5¢-ACCATG ACGTCCATTAACGTAAAGCTCC-3¢) and the reverse primer VBE3 (5¢-GGACCGACCGTTTTGGGCACG-3¢) were designed, synthesized and used to amplify FAE1 coding regions from target species by PCR Genomic DNA was isolated according to Edwards et al [27] from seed at mid-development from B napus cv Hero and cv Westar, and from MDEs at mid-development from B rapa and B oler-acea This was used as template DNA for PCR, carried out using Vent DNA polymerase (New England Biolabs) Amplified products without stop codons were cloned into the yeast expression vector pYES2.1/V5-His-TOPO (Invi-trogen) downstream of the galactose-inducible promoter (GAL1) Omitting a stopcodon allows for the PCR product
to be expressed as a fusion to the C-terminal V5 epitope and polyhistidine tag for protein detection and purification All products were confirmed by sequence analyses using external primer Gal1 Forward primer (Invitrogen) and V5 C-terminus Reverse primer (Invitrogen), and primers VBE3 and VBE4 Yeast cells (line Inv Sc1, Invitrogen), were transformed with pYES2.1/V5-His-TOPO constructs bear-ing different FAE1 genes, usbear-ing the S.c EasyCompTM Transformation Kit (Invitrogen) Yeast cells transformed with pYES2.1/V5-His-TOPO plasmid only were used as a control Transformants were selected by growth on synthetic complete medium lacking uracil (SC-ura), supplemented with 2% (w/v) glucose The colonies were transferred into liquid SC-ura with 2% (w/v) glucose and grown at 28C overnight For expression studies the overnight cultures were used to inoculate 25 mL of SC-ura supplemented with 2% (w/v) galactose to give an initial D600of 0.2 The cultures were subsequently grown overnight at 20C or 28 C to D600of 1.4 and used for biochemical analyses
Fatty acid analyses and enzyme assays The yeast cultures were grown overnight and cells were pelleted Cell pellets were saponified in methanolic-KOH [10% (w/v) KOH, 5% (v/v) H2O in methanol] for 2 h at
80C After saponification, samples were cooled on ice and then washed with hexane to remove nonsaponifiable material The remaining aqueous phase was then acidified with 6MHCl Free fatty acids were extracted in hexane, the solvent removed under a stream of N2, and the free fatty acids were transmethylated in 3Mmethanolic HCl for 2 h
at 80C Fatty acid methyl esters (FAMEs) were extracted
in hexane, the solvent removed under a N2stream and the residue was dissolved in hexane for GC under the conditions described previously [28]
Fatty acid elongase activity of the yeast microsomal membrane preparation was assayed essentially as described
by Katavic et al [3] The assay mixture consisted of
80 mMHepes-NaOH, pH 7.2; 1 mMATP, 1 mMCoA-SH, 0.5 mM NADH, 0.5 mM NADPH, 2 mM MgCl2, 1 mM malonyl-CoA, 18lM[1–14C]oleoyl-CoA (0.37 GBqÆmol)1)
in a final volume of 500 lL The reaction was started by
Trang 3the addition of 0.5 mg of microsomal protein and incubated
at 30C for 1 h Reactions were stopped by adding 3 mL of
100 gÆL)1KOH in methanol FAMEs were prepared and
quantified by radio-HPLC as described by Katavic et al [3]
Site-directed mutagenesis
To introduce the desired point mutation into the FAE1
coding region isolated from LEA B napus cv Westar, we
used a QuikChangeTMsite-directed mutagenesis kit
(Strat-agene) We have designed the oligonucleotide primers
SDF-3 (5¢-TGTTGGTGGGGCCGCTATTTTGCTCT
CCAACAAG-3¢) and SDF-4 (5¢-CTTGTTGGAGAGC
AAAATAGCGGCCCCACCAACA-3¢) containing the
desired mutation (bold) Primers were complementary to
opposite strands of pYES2.1/V5-His-TOPO containing the
FAE1gene During the PCR, primers were extended with
PfuTurbo DNA polymerase This polymerase replicated
both strands with high fidelity and without displacing the
mutated oligonucleotide primers PCR incubations were
run 30 sec at 95C (denaturation) followed by 16 cycles of
30 sec at 95C, 1 min at 55 C, 15 min at 68 C and
terminated by 15 min at 68C Following temperature
cycling, the product was treated with Dpn1 endonuclease
(target sequence 5¢-Gm6ATC-3¢) which is specific for
methylated and hemimethylated DNA, and is used to
digest parental DNA template and to select
mutation-containing synthesized DNA
Microsomal membrane preparation
Yeast microsomes were prepared essentially according to
Ghanevati & Jaworski [22] Briefly, cells were harvested and
washed with 10 mL of ice-cold isolation buffer (IB, 80 mM
Hepes-NaOH, pH 7.2, 5 mMEGTA, 5 mMEDTA, 10 mM
KCl, 320 mM sucrose, 2 mM dithiothreitol), pelleted and
resuspended in 500 lL of IB Cells were broken using three
60 s pulses with the Mini-BeadbeaterTM(BioSpec Products,
Inc., Bartlesville, OK, USA) using 0.5 mm glass beads The
supernatant was collected and centrifuged briefly to remove
unbroken cells The microsomal membrane pellet was
recovered after centrifugation at 100 000 g for 60 min and
resuspended in IB containing 20% (v/v) glycerol Protein
concentration was determined using the method according
to Bradford [29]
Immunoblot analysis
Microsomal proteins (100 lg) were separated on 15% SDS/
PAGE Ready Gel (Bio-Rad) After electrophoresis,
proteins were electro-transferred (1.5 h, 180 mA, 4C)
to poly(vinylidene difluoride) (PVDF) membrane (HybondTM-P, Amersham) using a Mini Trans-blot (Bio-Rad) apparatus and transfer buffer [10 mM CAPS, 10% (v/v) methanol, pH 11.0] An anti-(FAE1 3-KCS) Ig (gift from Dr L Kunst, Dep artment of Botany, University
of British Columbia, Canada) was used at a dilution of
1 : 5000 Secondary antibody (horseradish peroxidase-linked anti-rabbit IgG from sheep, Amersham) was diluted
1 : 10 000 and detected using Western blotting together with the ECL Plus system (Amersham) and Super RX film (Fujifilm)
R E S U L T S
Sequence alignment ofBrassica FAE1 proteins
We isolated by PCR clones corresponding to the coding regions of FAE1 genes from HEA B napus cv Hero, HEA B rapa line R500, HEA B oleracea line 103 and LEA B napus cv Westar Nucleotide sequences corres-ponding to open reading frames of 1523 bp were trans-lated and proteins of 506 amino acids were deduced Aligned FAE1 proteins from different HEA Brassica species showed high homology to published B napus FAE1 protein sequences (GenBank coding region acces-sion numbers AF006563, cv Golden, and AF274750, cv Ascari) However, several unique differences among FAE1 protein sequences were observed HEA B napus cv Hero FAE1 has two unique differences, one at position 118 with asparagine instead of aspartic acid, while at the position 484 in Hero, aspartic acid is substituted by a glutamic acid residue HEA B rapa FAE1 has a serine residue at position 179 while all other aligned FAE1 proteins have asparagine residues at this position When
we compared FAE1 protein sequences from different HEA Brassica spp with the LEA B napus cv Westar FAE1, we could detect two unique substitutions in Westar FAE1 At position 282 serine is substituted with phenyl-alanine, and at position 303 threonine is substituted with alanine However, the alignment of several microsomal 3-KCSs from Arabidopsis thaliana and different Brassic-aceae revealed that the only crucial difference among the protein sequences from functional microsomal 3-KCSs and the nonfunctional FAE1 condensing enzyme from LEA B napus cv Westar is at position 282 While all functional elongases have a serine amino acid residue at that position, in the catalytically inactive protein from LEA cv Westar serine 282 is substituted by phenylalanine (Fig 1)
Fig 1 Alignment and comparison of amino-acid sequences of several 3-ketoacyl-CoA synthases Protein sequences spanning the region of amino acids 266–325 from A thaliana CUT1 (accession number AF129511) and amino acids 264–323 from A thaliana FAE1 (accession number AF053345), HEA B napus cv.s Golden and Ascari (accession numbers AF00953 and AF274750), HEA B napus cv Hero, B oleracea MDE line
103, B rapa MDE line R500 and LEA B napus cv Westar were aligned Amino acid residues at position 282 are shaded in black and indicated by the black arrow The amino acid residues at position 303 are indicated by the white arrow.
Trang 4Expression of FAE1 genes in yeast and GC analyses
of FAMEs
For the functional expression of FAE1 clones in yeast,
DNA fragments corresponding to open-reading frames of
BrassicaFAE1 genes as well as Arabidopsis FAE1 (used as a
positive control for FAE1 expression) were linked to GAL1
in the expression vector pYES2.1/V5-His-TOPO Yeast
cells were transformed with FAE1 expression constructs or
with expression vector pYES2.1/V5-His-TOPO only (a
negative experimental control) Upon expression, the fatty
acid composition of induced yeast cell lysates was analyzed
by GC All HEA FAE1 genes were functionally expressed in
yeast, and heterologous 3-KCS enzymes together with the
endogenous dehydratase and two reductases catalyzed the
elongation of long chain fatty acid substrates into VLCFA
products All HEA Brassica FAE1-expressing yeast cells
were able to utilize both 18:1 isomers (D9 and D11) as
substrates for elongation reactions to produce 20:1 D11 and
D13 isomers, and 22:1 D13 and D15 isomers, respectively
The relative proportions of the different VLCFAs are
shown in Table 1 The A thaliana FAE1 more poorly
utilized 20:1 as a substrate in elongation process compared
to FAE1 from HEA Brassica species
The results of expression experiments at two different
temperatures (28C vs 20 C) showed that the overall
activity of the FAE1 condensing enzyme was reduced at the
lower temperature, but the trends in the relative proportions
of VLCMFAs produced were similar at both temperatures
Site directed mutagenesis of the LEAB napus cv
WestarFAE1 gene
In order to test the importance of serine 282–3-KCS
func-tion, we used a site-directed mutagenesis (SDM) approach
to change the phenylalanine 282 residue in LEA cv Westar
FAE1 to the highly conserved serine residue The sequence
analyses of five different clones revealed that two of
them (WS-SDM1 and WS-SDM18) had been successfully
mutated with a serine at position 282 (data not shown)
We expressed wild-type FAE1 (WS-wt), two mutated WS
FAE1clones (WS-SDM1 and WS-SDM18) and the empty
plasmid control (pYES2.1/V5-His-TOPO) in yeast cells
The results of fatty acid analyses of transformed yeast cell
lysates by GC of the FAMEs revealed that condensing
enzyme activity was restored in both mutated WS clones; yeast cells expressing mutated WS-SDM clones produced 20:1 D11 and D13 isomers, and 22:1 D13 and D15 isomers
In contrast, yeast cells expressing WS-wt or plasmid only had fatty acid profiles typical of yeast, with no detectable monounsaturated VLCFAs present (Fig 2)
Immunoblot analyses of yeast microsomes
In order to detect FAE1 proteins in yeast cells expressing cv Westar wild-type FAE1 and two mutated cv Westar FAE1 clones, Western blot analyses were performed using micro-somes isolated from yeast cells after FAE1 heterologous expression and using anti-FAE1 Igs raised against the C-terminus domain of the Arabidopsis FAE1 protein Protein bands corresponding to FAE1/V5-His fusion were detected in all experimental samples except in the pYES2.1/ V5-His-TOPO-only control (Fig 3)
Elongase activity in microsomal fraction of yeast cells Microsomal fractions were isolated from lysates of yeast cells upon expression of WS-wt FAE1 clone and two mutated clones WS-SDM1 and WS-SDM18 As a control, the microsomal fraction from yeast cells containing only the empty plasmid (pYES2.1/V5-His-TOPO) was used To analyze the elongase activity, microsomal proteins were incubated with [1-14C]18:1-CoA and malonyl CoA The results of the elongase activity assays are summarized in Table 2 The elongase activity in WS-wt was low as expected, but the activity in the two mutated WS-SDM clones was comparatively very high
D I S C U S S I O N
We have isolated genomic clones corresponding to FAE1 coding regions from several HEA Brassica species and from LEA B napus No introns were present in the genomic clones, which seems to be a general characteristic of FAE1 genes in Brassicaceae Our earlier work [3] as well as findings from other groups [2,5,6] have shown that it is the FAE1 3-KCS coding region that determines the preference of its translated protein for either 18:1 moieties or 20:1 moieties for elongation The A thaliana FAE1 condensing enzyme used 20:1-CoA more poorly as an elongation substrate
Table 1 Fatty acid composition of yeast cells expressing FAE1 condensing enzymes from different HEA Brassica speciesa Lysates from yeast cells expressing B napus cv Hero, B oleracea MDE line 103, B rapa MDE line R500, LEA B napus cv Westar FAE1 condensing enzyme were analyzed Cells expressing FAE1 from A thaliana (ecotype Columbia) and pYES2.1/V5-His-TOPO (pYES2.1) were used as positive and negative controls, respectively FA (%), relative percent of total fatty acids.
pYES2.1/FAE1
FA (%) 16:0 16:1 18:0 18:1 D9 18:1 D11 20:1 D11 20:1 D13 22:1 D13 22:1 D15 24:1 VLCMFA
B n Hero 16.34 43.27 3.97 15.15 1.52 0.45 0.99 2.07 1.25 0.15 4.91
B o 103 15.77 44.84 3.62 13.90 1.56 0.49 1.10 2.19 1.07 0.17 5.02
B r R500 15.22 43.76 3.96 14.97 1.46 0.45 0.94 1.94 1.54 0.46 5.33
B n Westar 14.91 45.50 4.75 29.03 1.02 0.07 0.00 0.00 0.00 0.00 0.07
A t Col 16.12 40.64 4.69 18.63 1.68 1.67 3.17 0.85 0.37 0.07 6.13 pYES2.1 19.22 40.67 5.88 24.69 1.01 0.00 0.00 0.00 0.00 0.00 0.00 a
The data for 26:0 which is normally present in yeast at the amount of approximately 5%, and other fatty acids (12:0, 14:0, 14:1, 20:0, 22:0, 24:0) which were present in similarly minor percentages in all our samples are not shown.
Trang 5compared to B napus and its ancestral species B oleracea
and B rapa
The alignment of FAE1 polypeptides revealed several
differences among FAE1 condensing enzymes from
differ-ent Brassica species Some of these changes indicate that the
B napuscv Hero allele could be more related to B oleracea
than to the B rapa FAE1 allele For example, both Hero
and B oleracea have arginine at position 286, lysine at
position 395 and glycine at position 406, while the other
FAE1s have glycine, arginine and alanine at these positions
respectively
The alignment of our Brassica FAE1 proteins with
3-ketoacyl-CoA synthases from A thaliana (CUT1 and
FAE1) showed that the only highly conserved amino acid
residue in all 3-KCSs was the serine 282 residue (Fig 1)
Similar findings were reported by Han et al [6] when they
compared the sequences of FAE1 condensing enzymes from
HEA B napus cv Ascari and LEA B napus cv Drakkar Furthermore, in all 3-KCS protein sequences available in the databases, the serine residue at position 282 is conserved
or conservatively substituted by threonine (e.g in Sorghum bicolor, broom corn) Indeed, the single-base change of nucleotide 845 from thymidine to cytosine, which resulted in the substitution of phenylalanine with serine in FAE1 condensing enzyme from LEA cv Westar at position 282, led for the first time to successful experimental restoration of elongase activity in a previously catalytically inactive enzyme (Fig 2)
The analyses of translation rates of LEA cv Westar FAE1 condensing enzyme by Western blots showed that translation is not impaired and the strong band corres-ponding to the FAE1 protein of the expected size was detected (Fig 3) Roscoe et al [23] reported that the mutations that eliminate 3-KCS activity in LEA rapeseed
Fig 2 GC chromatographs showing fatty acid profiles of transgenic yeast cells FAMEs were prepared from yeast cell lysates expressing FAE1 condensing enzymes from LEA B napus cv Westar wild-type and mutated Westar clones and analyzed by GC WS-wt, LEA B napus cv Westar FAE1, WS-SDM1, site directed mutated clone 1, WS-SDM18, site directed mutated clone 18; pYES2.1, pYES2.1/V5-His-TOPO experimental negative control.
Trang 6act post-transcriptionally, and that the loss of enzyme
activity is related to reduced quantity or stability of the
enzyme Although our results show that the loss of activity
is not due to the reduced quantity of protein, our
experiments were carried out in a heterologous system It
is possible that the regulation of elongase protein may be
quite different in yeast than it is in plants The immunoblot
results clearly indicate that the loss of FAE1 activity is not
due to a lower level of expression, since inactive wild-type
LEA FAE1 condensing enzyme was expressed in yeast at a
level similar to the mutant clones Recently, Ghanevati &
Jaworski [22] studied the role of conserved cysteine and
histidine residues in FAE condensing enzyme activity by
generating several different mutants, some of them showing
complete loss of enzyme activity Similar to our findings,
they concluded that the loss of activity was not related to
changes in protein expression level, since their mutant
proteins were expressed to the same extent as the FAE1
wild-type protein
It is not unusual that the substitution of a single amino acid results in loss of enzyme activity Bruner et al [30] reported that the mutation of aspartate at position 150 to an asparagine residue resulted in nearly complete loss of the peanut oleoyl-PC desaturase (D12 desaturase) activity when the mutated gene was expressed in yeast
Our study constitutes the first mutagenesis of catalytically inactive 3-KCS from a low erucic acid B napus (canola) cultivar and successful experimental restoration of conden-sing enzyme activity
As mentioned earlier, all our FAE1 expression experi-ments were performed in yeast cells We expect that the expression of restored FAE1 condensing enzyme from LEA
B napus in planta would result in similar increases in VLCFA content of seed oil as reported by Han et al [6] When they expressed FAE1 enzyme from HEA B napus cv Ascari in LEA cv Drakkar, the seed oil of certain transgenic lines contained upto 20% 20:1 and 30% 22:1 They concluded that nonfunctional FAE1 enzyme causes LEA phenotype at the E1 locus
We are now exploring several other FAE1 condensing enzymes from Brassicaceae to enhance our understanding
of the role of certain amino acid residues in determining substrate preference and specific activity of these condensing enzymes
A C K N O W L E D G E M E N T S
We thank Don Schwab, Barry Panchuk and Dr Larry Pelcher of the PBI DNA Technologies Groupfor p rimer synthesis and DNA sequencing We thank Arvind Kumar from Plant Biotechnology Institute (PBI) Seed Oil Modification Groupfor his technical helpwith immunoblot preparation, Dr Ljerka Kunst from the University of British Columbia for kindly supplying the anti-FAE1 3-KCS Igs and
Dr Jitao Zou from PBI for critical comments and suggestions during the preparation of the manuscript.
R E F E R E N C E S
1 Somerville, C., Browse, J., Jaworski, J.G & Ohlrogge, J.B (2000) Lipids In Biochemistryand Molecular Biologyof Plants (Bucha-nan, B.B., Gruissem, W & Jones, R.L., eds), p p 481–483 American Society of Plant Physiologists, Rockville, MD, USA.
2 Millar, A.A & Kunst, L (1997) Very-long-chain fatty acid bio-synthesis is controlled through the expression and specificity of the condensing enzyme Plant J 12, 121–131.
3 Katavic, V., Friesen, W., Barton, D.L., Gossen, K.K., Giblin, E.M., Luciw, T., An, J., Zou, J.-T., MacKenzie, S.L., Keller, W.A., Males, D & Taylor, D.C (2001) Improving erucic acid content in rapeseed through biotechnology: what can the Arabidopsis FAE1 and the yeast SLC1-1 genes contribute? Crop Sci 41, 739–747.
4 Han, J., Lu¨hs, W., Sonntag, K., Borchardt, D.S., Frentzen, M & Wolter, F.P (1998) Functional characterization of b-ketoacyl-CoA synthase genes from Brassica napus L In Advances in Plant Lipid Research (Sa´nchez, J., Cerda´-Olmedo, E & Martı´nez-Force, E., eds), pp 665–667 Universidad de Sevilla, Sevilla, Spain.
5 Han, J & Jaworski, J (2001) Analysis of isomers of very long chain unsaturated fatty acids in transgenic yeast by GC/MS Book
of Abstracts, p 29 Biochemistry and Molecular Biology of Plant Fatty Acids and Glycerolipids Symposium, June 6–10, South Lake Tahoe, CA, USA.
6 Han, J., Lu¨hs, W., Sonntag, K., Za¨hringer, U., Borchardt, D.S., Wolter, F.P., Heinz, E & Frentzen, M (2001) Functional char-acterization of b-ketoacyl–CoA synthase genes from Brassica napus L Plant Mol Biol 46, 229–239.
Fig 3 Immunoblot analysis of yeast microsomes expressing FAE1
condensing enzymes Proteins (100 lg per lane) from yeast expressing
wild-type LEA B napus cv Westar site-directed mutagenized clones,
A thaliana and empty plasmid pYES2.1 were probed with antibodies
raised against the C-terminus of A thaliana FAE1 Detected protein
bands correspond to the FAE1/V5-His fusion protein of
approxi-mately 61 kDa (56 kDa FAE1 + 5 kDa V5-His) WS-wt, LEA
B napus cv Westar FAE1, WS-SDM1, site directed mutated clone 1,
WS-SDM18, site directed mutated clone 18, A.t., A thaliana (positive
control), pYES2.1, pYES2.1/V5-His-TOPO experimental negative
control.
Table 2 Elongase activity assayed in microsomal preparation from
transgenic yeast cell lysates Microsomes were prepared from yeast cells
upon expression of LEA B napus cv Westar FAE1 (WS-wt), site
directed mutated cv Westar FAE1 clones SDM1 and
WS-SDM18 and plasmid pYES2.1/V5-His-TOPO only (pYES2.1) Protein
samples (0.5 mg) were incubated at 30 C with shaking at 100 r.p.m.
for 60 min with 18 l M [1– 14 C] oleoyl-CoA (0.37 GBqÆmol)1) and 1 m M
malonyl-CoA in the presence of 1 m M ATP, 1 m M CoA-SH, 0.5 m M
NADH, 0.5 m M NADPH and 2 m M MgCl 2 in a final volume of 500
lL After incubation, reaction mixtures were saponified,
transmeth-ylated and analyzed by radio-HPLC as described in Materials and
methods Results are the average of three determinations.
Transformant
Elongase Activity (20:1 + 22:1) (pmolÆmin)1Æmg protein)1)
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