For biotechno-logical applications, these organisms are regarded as Keywords desaturases; long chain polyunsaturated fatty acids; sphingolipids; Thalassiosira pseudonana; yeast expressio
Trang 1Thierry Tonon1,*, Olga Sayanova2, Louise V Michaelson2, Renwei Qing1,†, David Harvey1,
Tony R Larson1, Yi Li1, Johnathan A Napier2and Ian A Graham1
1 CNAP, Department of Biology, University of York, Heslington, York, UK
2 Rothamsted Research Institute, Harpenden, UK
The algae, as a group, represent the third largest
aqua-culture crop (after freshwater fish and molluscs) in the
world today [1,2] In recent years, considerable
atten-tion has been directed at marine microalgae for the
production of oils and fatty acids, in particular the use
of algal oils containing long chain polyunsaturated
fatty acids (LCPUFAs) The most prominent of these
are the health beneficial omega-3 eicosapentaenoic acid (EPA, 20:5D5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6D4,7,10,13,16,19) Among the alga groups iden-tified as producers of high levels of LCPUFAs, di-atoms are able to produce and accumulate EPA and DHA in triacylglycerols (TAGs) [3] For biotechno-logical applications, these organisms are regarded as
Keywords
desaturases; long chain polyunsaturated
fatty acids; sphingolipids; Thalassiosira
pseudonana; yeast expression
Correspondence
I A Graham, Department of Biology
(area 7), University of York, PO Box373, UK
Fax: +44 1904 328762
Tel: +44 1904 328750
E-mail: iag1@york.ac.uk
Present addresses
*UMR 7139, CNRS-GOEMAR-UPMC,
Station Biologique, BP 74, 29682 Roscoff
cedex, France
College of Life Science, Sichuan University,
Chengdu, China
Note
The sequences reported in this paper have
been submitted to GenBank database under
the accession number AY817152 (TpdesO),
AY817153 (TpdesA), AY817154 (TpdesB),
AY817155 (TpdesI) and AY817156 (TpdesK)
(Received 13 March 2005, revised 22 April
2005, accepted 9 May 2005)
doi:10.1111/j.1742-4658.2005.04755.x
Analysis of a draft nuclear genome sequence of the diatom Thalassiosira pseudonana revealed the presence of 11 open reading frames showing significant similarity to functionally characterized fatty acid front-end desaturases The corresponding genes occupy discrete chromosomal loca-tions as determined by comparison with the recently published genome sequence Phylogenetic analysis showed that two of the T pseudonana desaturase (Tpdes) sequences grouped with proteobacterial desaturases that lack a fused cytochrome b5 domain Among the nine remaining gene sequences, temporal expression analysis revealed that seven were expressed
in T pseudonana cells One of these, TpdesN, was previously characterized
as encoding aD11-desaturase active on palmitic acid From the six remain-ing putative desaturase genes, we report here that three, TpdesI, TpdesO and TpdesK, respectively encode D6-, D5- and D4-desaturases involved
in production of the health beneficial polyunsaturated fatty acid DHA (docosahexaenoic acid) Furthermore, we show that one of the remaining genes, TpdesB, encodes aD8-sphingolipid desaturase with strong preference for dihydroxylated substrates
Abbreviations
ARA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; EST, expressed sequence tag; FAME, fatty acid methyl ester; gDNA, genomic DNA; LCB, long chain base; LCPUFA, long chain polyunsaturated fatty acid; PUFAs, polyunsaturated fatty acids; RACE, rapid amplification of cDNA ends; TAG, triacyglycerol; Tpdes, Thalassiosira pseudonana desaturase.
Trang 2potentially good sources for the cloning of genes
enco-ding the enzymes required for LCPUFA biosynthesis
Different routes of LCPUFA synthesis have developed
in nature The production of EPA and DHA in marine
bacteria and some marine fungi relies on anaerobic
polyketide synthase systems, which are encoded by
sev-eral large polypeptides [4] More gensev-erally,
biosynthe-sis of these fatty acids requires aerobic desaturases and
elongases that catalyse a consecutive series of
desatura-tion and elongadesatura-tions of the fatty acyl chain to generate
EPA and DHA from a-linolenic acid (18:3D9,12,15)
Besides the main routes leading to DHA viaD6
-desatu-ration, some algae display an alternate pathway for
C20-PUFA production involving aD9-elongase [5] and
aD8-desaturase [6]
To date, at least one enzyme corresponding to each
of the front-end desaturases and elongases necessary
to convert a-linolenic acid to EPA has been isolated
from diverse origins [7] ‘Front-end’ desaturation can
be defined as desaturation between a pre-existing
dou-ble bond and the C-terminal end of a fatty acid, as
opposed to the much more prevalent (in plants)
methyl-directed desaturation Reconstitution of EPA
biosynthesis has been achieved in yeast [8,9] and in
plants [10,11], with encouraging levels of
C20-LCPUFA production Moreover, the D4-desaturase
gene encoding the last step in DHA biosynthesis has
recently been isolated from a number of marine
organ-isms [12–14] The final elongation step [of C20
polyun-saturated fatty acids (PUFAs) to C22] catalysed by a
D5-elongase was the last outstanding step remaining to
be functionally characterized at the molecular level
Very recently, characterization of such an activity has
been described in the microalgae Pavlova lutheri [15],
Ostreococcus tauri and Thalassiosira pseudonana [16]
These novel fatty acid elongases were used to
success-fully reconstitute DHA synthesis in yeast Therefore,
all the activities are now available to engineer plants to
produce this nutritionally important fatty acid
How-ever, all these enzymes have been isolated from a
diverse array of organisms, for instance several marine
microalgal species For the purposes of metabolic
engineering and in order to achieve optimal synthesis
in a heterologous host, it may be advantageous to use
a complementary set of desaturase and elongase
enzymes from the same organism This may be
partic-ularly relevant for the metabolic engineering of a
com-pound such as DHA in a heterologous host such as
linseed which would require the introduction of three
desaturase and two elongase steps in order to convert
the endogenous fatty acid a-linolenic acid to DHA
Gene discovery-based strategies such as targeted
expressed sequence tag (EST) databases or PCR
amplification using degenerate primers typically do not provide sufficient coverage to enable identification of complete sets of genes for a particular process from a single organism Bioinformatics-based analysis of com-plete genome sequences potentially allow an exhaustive approach to gene discovery from a single organism if the process in question is sufficiently understood in terms of enzymes and other proteins involved at the biochemical level Such a situation now exists for dis-covery of genes involved in PUFA biosynthesis follow-ing the completion of the genome sequence of the EPA and DHA producing diatom T pseudonana [17] Levels
of EPA and DHA in this organism are in the range of
17 and 5%, respectively, when in the exponential phase
of growth [3] Two elongases involved in LCPUFA biosynthesis from T pseudonana have been recently characterized [16] and analysis of a draft genome sequence of this organism prior to publication of the complete genome sequence revealed the presence of a family of putative front-end desaturases that are obvi-ous candidates for enzymes involved in the synthesis of EPA and DHA [18] However, rather surprisingly, the first of these genes to be functionally characterized was found to encode a cytochrome b5 fusion desaturase exhibiting D11-desaturase activity Here we report the cloning and characterization of the three desaturases involved in DHA synthesis, i.e a D6-, a D5- and a
D4-desaturase Moreover, heterologous expression of
an additional cytochrome b5 fusion desaturase has allowed the identification of a new D8-sphingolipid desaturase from Thalassiosira
Results
Phylogenetic and expression analysis of
T pseudonana genes with similarity to front-end desaturases
A recent phylogenetic analysis of the draft genome sequence of T pseudonana [18] reported the presence
of 12 sequences showing significant similarity to func-tionally characterized front-end desaturases, i.e a fused cytochrome b5 binding domain at their N-termi-nus and three histidine boxes [19] Now that contigs have been built for the entire genome and most assigned to 24 nuclear chromosomes [17], we re-exam-ined these 12 putative desaturase sequences based on cDNA characterization and genome analysis In our previous analyses, two partial desaturase coding sequences present near the ends of two distinct contigs were designated as unique genes, TpdesB and TpdesD TpdesB encoded an N-terminus region and TpdesD encoded a C-terminus region Our subsequent cDNA
Trang 3analysis of these sequences has revealed that they
actu-ally represent a single gene present on chromosome 4
that is from now on referred to as TpdesB
Compar-ison of the full-length TpdesB cDNA with the genomic
sequence demonstrated that no introns are present in
the gene The TpdesB ORF gives a predicted protein
of 493 amino acids A further annotation update
relates to TpdesH which, based on EST sequence
ana-lysis, appears to contain three characteristic histidine
boxes but lacks the N-terminal cytochrome b5 domain
TpdesL is a close homolog of TpdesH that also lacks
the cytochrome b5 domain Both these genes share
higher overall sequence similarity with putative
proteo-bacterial desaturases that typically contain three
histi-dine boxes but also lack the cytochrome b5 fusion
domain; for this reason they were excluded from the functional analysis described in this present study Based on information contained in the GenBank data-base we have allocated 10 of the 11 T pseudonana Tpdes sequences to specific chromosomes (Fig 1A) These 10 Tpdes sequences are distributed among six of the 24 chromosomes, with three sequences on chromo-some 5, two on chromochromo-somes 4 and 6, and one each on chromosomes 3, 7 and 21 Material used to sequence the T pseudonana genome was derived from a single diploid founder and this revealed the presence of two haplotypes with on average 0.75% polymorphism at the nucleotide level [17] However, the Tpdes genes occupy distinct chromosomal positions and therefore even the pairings with highest sequence similarity such
Fig 1 Evolutionary relationship of T pseudonana putative desaturases (TpDES) with known front end desaturases and expression analysis
of corresponding genes T pseudonana sequences were arbitrarily designated TpDESA to TpDESO (A) The phylogenetic tree of TpDES and functionally characterized front-end desaturases was established using the PHYLIP 3.5c software package and based on 148 alignable amino acid residues [18] Percentage bootstrap values above 60 are indicated above the nodes Chromosome location and availability of cDNAs for each gene is shown after the gene name (B) For RT-PCR based gene expression analysis, cells were harvested at various time of incubation and growth stage monitored by measuring the percentage of nitrogen degraded (inset table) PCR analysis was performed with gene speci-fic primers on undiluted (lane 1) and five-fold serial dilutions (lanes 2–4) of cDNA Size of the expected cDNA amplified fragment is indicated
in brackets below the gene name Inset PCR products from genomic DNA are shown in order to validate the TpdesG and TpdesM primer pairs M, DNA molecular size ladder.
Trang 4as TpdesO-TpdesM, TpdesN-TpdesG and
TpdesH-TpdesLare not due to haplotype variation
The phylogenetic tree of TpDES sequences was
re-constructed with the updated Tpdes sequences noted
above in combination with front-end desaturases from
a range of organisms The resulting tree shows three
major groupings containing TpDES sequences
(Fig 1A) The first group, containing TpDESL,
TpDESH and four proteobacterial sequences, is
char-acterized by the lack of a fused cytochrome b5
domain None of these genes have been functionally
characterized and we did not include TpDESL or
TpDESH in our functional analysis The remaining
nine Tpdes genes show significant similarity to other
functionally characterized desaturases As a first step
to characterizing the Tpdes genes we analysed temporal
expression analysis throughout the growth phases of
the algal cell culture using semiquantitative RT-PCR
(Fig 1B) The second phylogenetic group contains
TpDESN together with previously characterized
D5- andD4-desaturases TpDESO and TpDESM group
with the diatom Phaeodactylum tricornutum D5
-desatu-rase PtDEL5 (Fig 1A) Comparison of genomic
sequences of TpDESO and TpDESM showed 72%
identity, and one intron could be detected in each
sequence RT-PCR analysis revealed that of these two
genes only TpdesO is transcribed (Fig 1B) The
TpdesOORF is 1425 bp long and encodes a protein of
474 amino acids Alignment with the corresponding
genomic sequence confirmed the presence of a 99 bp
intron in the TpdesO gene The amino acid sequence
of PtDEL5 and TpDESO exhibited 63% identity,
sug-gesting that TpDESO could catalyse the D5
-desatura-tion step in the PUFA biosynthetic pathway The
TpDESK gene sequence forms a subgroup with
func-tionally characterized D4-desaturases from
Thrausto-chytrium sp and Euglena gracilis (Fig 1A) TpDESK
is expressed at a low level relative to TpdesO during
the exponential phase of growth TpDESN and
TpDESG do not group with functionally characterized
front-end desaturases from other organisms TpDESN
has already been characterized as a 16:0 specific
D11-desaturase [18] Alignment of the TpdesG genomic
sequence with other desaturases showed that although
it contains a cytochrome b5 domain and three histidine
boxes a start methionine cannot be determined by
in silicoanalyses alone, indicating that it may represent
a pseudogene RT-PCR based expression analysis
sug-gested that the gene is not expressed during the growth
phase and we have not investigated it further In the
third major group, TpDESE, TpDESI, TpDESB and
TpDESA are separated on four branches (Fig 1A)
The TpdesE ORF is incomplete in the draft genome sequence in comparison to other desaturases, and 5¢-RACE experiments failed to produce full-length cDNA for this gene despite the fact that it shows relat-ively high mRNA expression levels throughout the growth phases TpdesI is similarly highly expressed and the corresponding full length amino acid sequence has 70% sequence identity and groups with the Phaeod-actylum tricornutum PtDEL6 D6-desaturase TpdesI contains an intron of 149 bp, and the corresponding cDNA contains a 1455 bp ORF giving a predicted polypeptide of 484 amino acids The remaining two TpDES sequences, TpDESB and TpDESA, showed only 25% identity at the amino acid level Comparison
of TpdesA genomic and cDNA sequences revealed that the gene contains three introns of 84, 88 and 68 nucle-otides The 1548 bp cDNA of TpdesA gives a predicted polypeptide of 515 amino acids No function could be predicted for these enzymes from comparison of their primary structure with other front-end desaturases as they do not cluster with any functionally characterized desaturases with significant confidence
Incorporation of intron presence and positional information in genomic sequences does not shed any further light on the phylogenetic relationship of the cytochrome b5 containing T pseudonana desaturase genes TpdesB and TpdesN do not contain introns, TpdesK, TpdesO and TpdesI each have a single intron and TpdesA contains three introns Full-length cDNAs are not available for TpdesG and TpdesE therefore definite information on intron position is not available for these two genes Intron⁄ exon junction is not con-served between any pair of the Tpdes genes analyzed and our analysis suggests that introns appear to have evolved independently in each case
Characterization of PUFAs front-end desaturases
To establish the function of the different putative front-end desaturases, the full-length cDNAs of TpdesA, TpdesB, TpdesI, TpdesK and TpdesO were cloned into the vector pYES2, under the control of an inducible galactose promoter, to produce the constructs pYDESA, pYDESB, pYDESI, pYDESK and
pYDES-O They were first expressed in the Saccharomyces cere-visiae strain Invsc1 (or W303A1 for pYDESK) Transformants were incubated in the presence of a range of potential fatty acid substrates (18:2D9,12, 18:3D9,12,15, 20:2D11,14, 20:3D11,14,17, 20:3D8,11,14, 20:4D8,11,14,17, 22:4D7,10,13,16, 22:5D7,10,13,16,19) of desatu-rases involved in the PUFA biosynthesis pathway After addition of such substrates, no new peaks were detected for pYDESA and pYDESB, indicating that
Trang 5these desaturases were not involved in production of
LCPUFAs However, comparison of fatty acid profiles
derived from yeast transformed with pYES2 and
pYDESI showed two new peaks in the TpdesI
trans-formants even in the absence of exogenous fatty acids
in the medium These new peaks corresponded to
16:2D6,9and 18:2D6,9(Fig 2) These fatty acids are
pro-duced byD6-desaturation of 16:1D9and 18:1D9,
respect-ively, the two major fatty acids found in yeast When
linoleic (18:2D9,12) and a-linolenic (18:3D9,12,15) acids
were added to the culture medium, 18:3D6,9,12 and
18:4D6,9,12,15 were detected (Fig 2), confirming that
TpdesI encodes a D6-desaturase Analysis of the fatty
acid composition in the pYDESI transformants after
6 days of incubation showed the percentage conversion
of the 16:1D9 and 18:1D9 substrates were 29 and 38%,
respectively For the exogenous substrates 18:2D9,12 and 18:3D9,12,15, TpDESI exhibited a slight preference for the omega-3 fatty acid, as 68 and 80% of these substrates were converted to their corresponding
D6-products
Fatty acid profiling of extracts from TpDESO
S cerevisiae transformants detected new peaks when the culture medium was supplemented with 20:3D8,11,14 and 20:4D8,11,14,17 (Fig 3A) These new FAMEs were identified as 20:4D5,8,11,14 (arachidonic acid, ARA) and 20:5D5,8,11,14,17 (EPA), respectively These results dem-onstrate that pYDESO introduces a double bond at position 5 from the C-terminus of 20:3D8,11,14 and 20:4D8,11,14,17, indicating it is a D5-desaturase Analysis
of cells harvested after incubation for 6 days in the presence of both fatty acid substrates showed 16–19% conversion of each to their corresponding D5 fatty acids, suggesting that pYDESO does not have a
Fig 2 GC analysis of FAMEs from yeast transformed with the
empty plasmid pYES2 or the plasmid containing TpDESI Yeast
cells transformed with either pYES2 (bottom chromatogram) or
pYDESI (top chromatogram) were induced for six days in the
pres-ence of 18:2D9,12and 18:3D9,12,15exogenously fed before sampling
for fatty acid analysis New fatty acids are underlined I.S., internal
standard (17:0) The experiment was repeated twice and results of
a representative experiment are shown.
Fig 3 GC analysis of FAMEs from yeast transformed with the empty plasmid pYES2 or the plasmid containing TpDESO Yeast cells transformed with either pYDESO (top chromatograms) or pYES2 (bottom chromatograms) were induced for six days in the presence of 20:3 D8,11,14 and 20:4 D8,11,14,17 (A), 20:3 D11,14,17 (B), and 20:2 D11,14 (C) exogenously fed before sampling for fatty acid analy-sis New fatty acids are underlined The experiment was repeated twice and results of a representative experiment are shown.
Trang 6preference for one substrate over the other The
Cae-norabditis elegans D5-desaturase is capable of inserting
double bonds in a nonmethylene interrupted pattern
into 20:2D11,14 and 20:3D11,14,17 as well as in a
methy-lene interrupted pattern into fatty acids with aD8
dou-ble bond [20] TpdesO transformants were incubated in
the presence of 20:3D11,14,17 and 20:2D11,14 to establish
if the T pseudonana enzyme exhibited similar
charac-teristics Profiling of extracts of TpdesO transformants
fed with 20:3D11,14,17 and 20:2D11,14 revealed the
pres-ence of new peaks in both corresponding to
20:4D5,11,14,17 (juniperonic acid) and 20:3D5,11,14
(podo-carpic acid), respectively (Fig 3B,C) The percentage
conversion of 20:2D11,14and 20:3D11,14,17to theirD5
-de-saturated products was 4.7 and 8.4, respectively, which
was significantly less than the percentage conversion
determined for D5-desaturation of the D8-desaturated
fatty acids 20:3D8,11,14and 20:4D8,11,14,17
Heterologous expression of TpDESK in S cerevisiae
identified this enzyme as a D4-desaturase, as feeding of
transformants with 22:5D7,10,13,16,19 resulted in the
appearance a new peak identified as 22:6D4,7,10,13,16,19
(Fig 4) The quantities of both D4-desaturated FAs
detected were low but significant, with a conversion value of 3.0% for DHA These were the only unique peaks detected in the TpDESK transformants fed with the full range of fatty acids compared to the empty vector pYES2 controls
Characterization of sphingolipid related front-end desaturase(s)
Previous data have demonstrated that sphingolipid long chain base (LCB) desaturases have a paralogous relationship to front-end PUFA desaturases To deter-mine if any of the five T pseudonana cytochrome b5 fusion candidate desaturases (TpdesA, TpdesB, TpdesI, TpdesK and TpdesO) functioned as sphingolipid desaturases, LCBs were extracted from S cerevisiae cells after galactose-induced expression of the heterolo-gous gene Total LCBs (i.e both free LCBs and those deacylated from sphingolipids) were extracted and ana-lysed using previously reported methodology [21] LCB desaturation was determined by separation of deri-vatized LCBs by HPLC and LC-MS as previously described [22], with candidate T pseudonana enzymes tested for activity using both trihydroxy (i.e phyto-sphingosine) and dihydroxy (i.e sphinganine) sub-strates Of the five candidate desaturases tested by expression in S cerevisiae, only one, TpdesB, displayed any ability to desaturate sphingolipid LCBs, resulting
in the appearance of one additional (non-native) LCB (Fig 5) This activity was more pronounced when the pYDESB plasmid was expressed in the yeast sur2D mutant (which lacks the LCB C-4 hydroxylase Sur2p and hence trihydroxylated LCBs, Fig 5A), indicating
a preference for dihydroxylated substrates (Fig 5C) The molecular ion for the pYDESB-dependent LCB had an m⁄ z of 465, consistent with the identification of this product as a dihydroxylated long chain base of 18 carbons, containing one double bond (data not shown) The precise regiospecificity of the activity encoded by TpdesB was further investigated by comi-gration with authentic standards for desaturated dihydroxy-LCBS This indicated that the novel prod-uct present on expression of pYDESB in sur2D was not sphingosine (d18:1D4t) (Fig 5D), but instead comi-grated with the trans-isomer of d18:1D8 (Fig 5B) (as determined by coinjection with LCBs resulting from expression of the borage sphingolipid D8-desaturase in sur2D 23,24); Thus, TpdesB encodes a sphingolipidD8 -desaturase with strong preference for dihydroxylated substrates In addition, it appears that the TpDESB desaturase differs from higher plant orthologs, since it only synthesizes the trans stereoisomer of the D8 -dou-ble bond (Fig 5C, cf the stereo-unselective borage
Fig 4 GC analysis of FAMEs from yeast transformed with the
empty plasmid pYES2 or the plasmid containing TpDESK Yeast
cells transformed with either pYDESK (top chromatogram) or
pYES2 (bottom chromatogram) were induced for six days in the
presence of 22:5 D7,10,13,16,19 exogenously fed before sampling for
fatty acid analysis New fatty acid is underlined The experiment
was repeated twice and results of a representative experiment are
shown.
Trang 7sphingolipid D8-desaturase, Fig 5B) To provide
fur-ther evidence for the correct assignment of function of
TpdesB as a sphinganine D8t-desaturase, we examined
the sphingolipid LCB composition of T pseudonana
This indicated that the diatom sphingolipids are
com-posed predominantly of dihydroxylated C18-LCBs, of
which the majority are unsaturated: 76%
dienine-con-taining and 17% sphingenine-condienine-con-taining sphingolipids,
compared with 7% sphinganine-containing
sphingoli-pids Thus, 93% of T pseudonana LCBs contain one
or more double bonds
Discussion
Using a combination of molecular cloning and
bio-informatics analysis of the available T pseudonana
genome, we have been able to identify 11 putative
front-end desaturases Two lacked cytochrome b5
fusion domain and grouped with functionally
unchar-acterized putative proteobacterial desaturases Among
the remaining nine cytochrome b5 fusion domain
con-taining desaturase sequences, seven were shown to be
transcriptionally active in T pseudonana cells based on semiquantitative RT-PCR We were unable to obtain a full-length ORF for TpdesE due possibly to problems
of secondary structure in the mRNA We previously showed that despite having significant sequence similar-ity, TpDESN actually encodes aD11-desaturase specific for 16:0 and so cannot be considered as a member of the front-end desaturase functional class [18] Of the five remaining sequences we expected that at least some
if not all of these would encode desaturases involved in the biosynthesis of EPA and DHA from stearidonic acid (18:4D6,9,12,15) According to the phylogenetic ana-lysis, TpdesI, TpdesO and TpdesK were good candidates for genes encodingD6-,D5- andD4-desaturases, respect-ively Results of heterologous expression in yeast of these genes confirm these predictions TpDESI introdu-ces a double bond at position 6 from the C-terminus of endogenous 16:1D9, 18:1D9 and exogenous fatty acids 18:2D9,12 and 18:3D9,12,15 Production of 16:2D6,9 and 18:2D6,9 from yeast fatty acids has already been observed after transformation with D6-desaturase from the oleaginous fungus Pythium irregulare [25], the moss Physcomitrella patens [26], the diatoms P tricornutum [9] and higher plants [27] The fatty acid profile of
T pseudonana cells [18] suggests the existence of a
D6-desaturase that can act on 16:2D9,12 to produce the corresponding D6 fatty acid 16:3D6,9,12 TpDESI is a good candidate for this activity considering its broad substrate specificity However, we were unable to test this hypothesis by direct feeding experiments as, to our knowledge, 16:2D9,12is not commercially available TpDESO acts as aD5-desaturase on C20 fatty acids
to produce 20:4D5,8,11,14and 20:5D5,8,11,14,17as predicted from the clustering of the gene in the phylogenetic tree This enzyme is also able to introduce a double bond in
a nonmethylene interrupted pattern at the D5-position
of 20:3D11,14,17 and 20:2D11,14 but at much lower effi-ciency than the methylene interrupted pattern of activ-ity with fatty acids containing a double bond at the
D8 position The C elegans D5-desaturase was shown
to exhibit similar activities as found with TpDESO [20] The significance of this nonmethylene interrupted pattern of activity in a biological context is not clear,
as the resulting fatty acids are considered to be ‘dead end’ metabolites since they do not appear to act as precursors for signalling molecules such as prostaglan-dins and they are not present as a major fatty acid component in T pseudonana
Of the three PUFA desaturases characterized in the heterologous system, TpDESK exhibited the lowest activity with only 3.0% of 22:5D7,10,13,16,19 being desaturated to DHA Due to the low D4-desaturase activity of TpDESK on what is likely to be the
Fig 5 HPLC analysis of sphingolipid LCB profiles of yeast
trans-formed with TpdesB Total LCBs were extracted from yeast sur2D
expressing pYDESB, derivatized and separated as described (A)
LCB profile from yeast mutant sur2D which synthesizes only
dihydroxylated LCBs (e.g d18:0 ¼ dihydroxylated 18 carbon LCB,
saturated) (B) LCB profile from sur2D yeast expressing the
stereo-unselective borage sphingolipid D 8 -desaturase: note the presence
of cis and trans D 8
-desaturated dihydroxylated LCBs (inset) (C) LCB profile from sur2D yeast expressing pYDESB: note the
pres-ence of only the trans isomer of the D 8 -desaturated dihydroxylated
LCB (D) The LCB profile of pYDESB was coinjected with a
deriva-tized authentic standard for sphingosine (d18:1 D4t ): note that
sphingosine does not coelute with the novel D 8t -LCB which arises
from pYDESB expression.
Trang 8preferred substrate it is not possible to reach a
conclu-sion on the substrate preference of this enzyme for
omega-3 vs omega-6 fatty acids with the current data
TpDESI and TpDESO displayed almost no selectivity
between the omega-3 and omega-6 fatty acid
sub-strates, being equally active on both substrates
Inter-estingly the D6-desaturase is more active than the
D5-desaturase which in turn is more active than the
D4-desaturase activity of TpDESK It is not possible to
conclude if this has any significance as regards rate of
flux through these different enzymes in vivo as the
heterologous expression system could introduce
arte-facts with respect to overall activity TpDESO and
TpDESI also exhibited additional activities with
TpDESO producing 16:2D6,9 and 18:2D6,9and TpDESI
producing 20:3D5,11,14 and 20:4D5,11,14,17 However,
none of these products were detected in extracts from
T pseudonanacells [18]
An alternate pathway for PUFA desaturation has
been demonstrated in several lower eukaryotes [28]
and a gene encoding this enzyme has been isolated
from Euglena gracilis [6] In this alternate pathway, a
front end D8-desaturase acts on 20:2D11,14
(eicosadi-enoic acid) and 20:3D11,14,17 (eicosatrienoic acid) to
produce 20:3D8,11,14 and 20:4D8,11,14,17 The E gracilis
D8-desaturase is in the same subgroup as TpDESI and
TpDESE in the phylogenetic tree (Fig 1) We have
functionally characterized TpDESI in the current work
but have been unable to characterize TpDESE The
fact that we did not detect eicosadienoic acid in
T pseudonana cells, and only very low level of
eicos-atrienoic acid were measured suggests the D8
-desatu-rase alternate pathway is not present in this organism
Furthermore, acyl-CoA profiling of T pseudonana
cells, did not detect 20:2D11,14 or 20:3D11,14,17 CoA
(Tonon et al unpublished data) Therefore it is
unli-kely that TpdesE or any of the other Tpdes genes
encode a PUFAD8-desaturase
As well as the three T pseudonana cytochrome b5
fusion desaturases confirmed as front-end PUFA
desaturases, we have also identified a sphingolipid
D8-desaturase, TpDESB This is the first example of a
sphingolipid long chain baseD8-desaturase from a
mar-ine algal species It has previously been observed that
this class of sphingolipid desaturase displays a
paralo-gous relationship to the PUFA desaturases, though the
evolutionary significance of this is still unclear [29] In
that respect, the availability of the T pseudonana
gen-ome sequence may provide further insights into the
ancestry of these cytochrome b5 fusion desaturases, not
least of all as this diatom represents the first example
of an organism which carries out both front-end
LCPUFA desaturation (up to and including the
synthesis of DHA) and sphingolipid D8-desaturation However, there are several subtle difference between the TpDESB sphingolipid desaturases and the predom-inant form of the enzyme found in higher plants Firstly, TpDESB has a strong preference for dihydrox-ylated LCB substrates (i.e sphinganine), whereas almost all higher plant sphingolipidD8-desaturases dis-play greater activity towards trihydroxylated LCBs (i.e phytosphingosine) [30] A recent example of a higher plant sphingolipid desaturase with activity towards sphinganine was reported from Aquilegia vulgaris [24] The introduction of theD8-desaturation into dihydroxy-lated substrates may represent the first step in the syn-thesis of sphingadienine-containing sphingolipids (i.e containing d18:2D4t,8c⁄ t LCBs), by the subsequent
D4-desaturation of the D8-desaturated LCB This bio-synthetic route has been invoked to explain the absence
of sphingosine (i.e D4-desaturated dihydroxysphingo-sine) in many plant species, even though higher plant LCBD4-desaturases are clearly present, as witnessed by the high levels of sphinga-4,8-dienine present in plant sphingolipids Interestingly, we have detected a pre-sumptive ortholog of the dihydrosphingosine D4 -desaturase [22] in the T pseudonana genome sequence,
as well as the presence of sphingadienine LCBs (Michaelson and Napier, unpublished data) It there-fore seems likely that the biosynthesis of unsaturated sphingolipids in T pseudonana occurs in the manner initially proposed for higher plants, i.e viaD8 -desatura-tion of dihydroxy substrates, followed by D4 -desatura-tion This is in contrast to that reported for animal systems (which lack a sphingolipid D8-desaturase), where D4-desaturation occurs on an N-acylated dihy-droxylated LCB (i.e dihydroceramide) [31] Although TpDESA clustered closely to TpDESB, expression of TpDESA in either wild-type or sur2D yeast strains failed to reveal any activity as an LCB desaturase The second feature of TpDESB is that this is the first cloned example of stereo-selective sphingolipid D8 sphinganine desaturase; previous examples of the higher plant sphingolipid desaturases with this regio-specificity are stereo-unselective in terms of the double bond introduced (i.e a nonequal mixture of cis and trans configurations), though a stereo-specific D8t phy-tosphingosine desaturase has been reported from the yeast Kluyveromyces lactis [32] (see Sperling and Heinz,
2003 [30] for an excellent review of the topic of LCB desaturation) The enzymatic basis for this higher plant stereo-unselective is currently unclear, but has been hypothesized to result from a syn-elimination of two vicinal hydrogen atoms from two different substrate conformers, making this form of sphingolipid LCB desaturation distinct from the D4-desaturation which
Trang 9yields sphingosine [30] In that respect, it is perhaps
surprising to find a more ‘precise’ form of
stereo-speci-fic LCB desaturation in the unicellular diatom T
pseu-donana, which may be due to an as yet unknown role
It is also currently unclear as to the role of
sphingo-lipid LCB D8-desaturation, though it has been
hypo-thesized to be involved in chilling tolerance in some
plant species and we have observed some variation in
T pseudonanaLCB profiles according to culture
condi-tions Interestingly, the ratio of cis⁄ trans forms of D8
-desaturated LCBs in higher plants varies according to
subcellular location, with a very much enhanced level
of D8t-LCBs found in plasma-membranes and
deter-gent-resistant membranes (lipid rafts) [33] Our
obser-vation of a D8t-specific dihydroxy-LCB desaturase in
T pseudonana may therefore provide an additional
tool with which to investigate the functionality and
importance of sphingolipid LCB heterogeneity [34]
In conclusion, this comprehensive study on the
T pseudonana front-end desaturases demonstrates the
value of whole genome sequence for gene discovery
programmes Whether the complete set of PUFA
desaturases from T pseudonana represent valuable
tools for metabolic engineering of the PUFA
biosyn-thetic pathway into oil crops remains to be established
The additional activities exhibited by the D5- and
D6-desaturases could prove problematic as it would be
preferable to limit the introduced enzymatic activities
to those essential for EPA and DHA production in
order to avoid the presence of additional fatty acids in
an end product Nevertheless, this set of desaturases
along with the recently characterized D5-elongase from
the same organism represents an attractive biotechno-logical resource As highlighted in recent publications [10], a critical issue for the development of a commer-cially viable product will be the final yield of EPA and DHA in the engineered vegetable oil and this will most likely require the introduction of additional activities such as acyltransferases and acyl-CoA synthetases [35] Further mining of the T pseudonana genome should lead to identification of genes encoding these addi-tional enzyme activities
Experimental procedures
Cultivation of T pseudonana, RNA extraction and RT-PCR analysis of gene expression
T pseudonana was cultivated as described previously [18] Total RNA was extracted from cells harvested at different stages of growth using an RNeasy plant mini kit (Qiagen, Valencia, CA, USA) First-strand cDNA was synthesized from three lg of DNAse treated RNA using a Prostar First-strand RT-PCR kit (Stratagene, La Jolla, CA, USA) PCR with primer pairs specific to each T pseudonana desaturase gene (Table 1) were performed using gDNA, or undiluted and five-fold serial dilutions of cDNAs as fol-lows: the reactions were heated to 95C for 5 min followed
by 35 cycles at 95C for 30 s, 30 s at temperatures ranging between 50 and 70C according to the primer pair used and 72C for 2 min, then a single step at 72 C for 10 min The 18S rRNA gene was used to ensure that the same quantity of cDNA was used for PCR on the different RNA samples Aliquots of PCR reactions were electrophoresed through a 1% agarose gel Identity of the diagnostic
frag-Table 1 Primers used in this study Sequence of the primers is given in the 5¢ to 3¢ orientation Restriction site used for cloning in the yeast plasmid pYES2 are in bold.
RT-PCR
Yeast expression
Trang 10ment for TpdesO, TpdesM, TpdesN and TpdesG was
veri-fied by sequencing after cloning in the pGEM-T
EasyVec-tor (Promega, Madison, WI, USA)
5¢- and 3¢-RACE experiment
The GeneRacerTM kit (Invitrogen, Carlsbad, CA, USA)
was used to reverse transcribe T pseudonana RNA and
cDNA was used to amplify the 5¢-end of TpdesE and the
3¢-end of the TpdesB gene Fragments generated by nested
PCR were cloned into the pGEM-T EasyVector (Promega)
and sequenced
Functional characterization of T pseudonana
putative front-end desaturases in Saccharomyces
cerevisiae
cDNA of the entire desaturase coding region was
synthes-ized from T pseudonana RNA using the SuperScriptTMIII
RNase H– Reverse Transcriptase (Invitrogen) or the
Enhanced Avian Reverse Transcriptase (Sigma) and gene
specific primers pairs (Table 1) Forward primers for
TpdesA, TpdesB, TpdesI and TpdesO gene were designed to
contain an alanine codon (GCT) just downstream of the
start codon not present in the original algal sequences
Presence of a G at position +4 has been shown to improve
translation initiation in eukaryotic cells [36] In the case of
TpDesK, activity was detected in S cerevisiae when a full
length cDNA that did not contain the alanine codon was
used The Expand High Fidelity PCR system (Roche,
Indi-anapolis, IN, USA) was employed to minimize potential
PCR errors The amplified product was gel purified and
restricted with KpnI and SacI for TpdesA and TpdesB,
EcoRI and BamHI for TpdesI, HindIII and XbaI for
TpdesO, and BamHI and XbaI for TpdesK Each desaturase
fragment was then cloned into the corresponding sites
behind the galactose-inducible GAL1 promoter of pYES2
(Invitrogen) to yield the plasmids pYDESA, pYDESB,
pY-DESI, pYDESO, and pYDESK The fidelity of the cloned
PCR product was checked by sequencing The vectors
con-taining the T pseudonana sequences were then transformed
into S cerevisiae strain Invsc1 (Invitrogen) (or W303A1 for
pYDESK) by a lithium acetate method Transformants
were selected on minimal medium plates lacking uracil
In order to monitor LCBs sphingolipids synthesis,
pYDESA, pYDESB, pYDESI, pYDESO, and pYDESK
were also transformed into the yeast mutant sur2D
(Euro-scarf, http://www.uni-frankfurt.de/fb15/mikro/euroscarf/
index.html) As a positive control for the sphingolipid
D8-desaturation of LCBs in both WT and sur2D mutant,
the yeast expression construct containing the borage D8
-desaturase was used, as described previously [23,24]
For PUFA feeding experiment, individual transformants
were grown at 25C in the presence of 2% (w ⁄ v) raffinose
and 1% (w⁄ v) Tergitol NP-40 (Sigma, St Louis, MO, USA) Expression of the transgene was induced at D600¼ 0.2–0.3 by supplementing galactose to 2% (w⁄ v) At that time, the appropriate fatty acids were added to a final con-centration of 50 lm Incubation was carried out at 25C for 3 days and then 15C for another 3 days For the co-feeding experiment, the same conditions were applied, except that both substrates were added to 25 lm final con-centration Each feeding experiment was repeated twice, and FA analysis was carried out on triplicate samples For functional characterization of Tpdes genes in the sur2D background, cultures were grown at 22C with sha-king in the presence of 2% (v⁄ v) raffinose and induction was carried out as previously described [37] All cultures were then grown for a further 48 h unless indicated All analysis was performed on triplicate samples and replicated three times
Fatty acid analysis Microalgae or yeast cells were harvested by centrifugation Total fatty acids were extracted and transmethylated as previously described [14] Fatty acid methyl esters (FAMEs)
of methyl pentadecanoate (15:0) or methyl heptadecanoate (17:0) were included as internal standards to enable quanti-fication PUFA FAMEs were identified by comparing chro-matographic traces with transmethylated commercial Menhaden oil (Supelco, Gillingham, Dorset, UK), and by identification of picolinyl ester and dimethyl disulphide adduct structures by GCMS as previously described [18]
Sphingoid base analysis Sphingolipid analysis of yeast cells was carried out essen-tially as described previously [21] LCBs were liberated from yeast cells by alkaline hydrolysis and extracted with chloroform⁄ dioxane ⁄ water (6 : 5 : 1, v ⁄ v ⁄ v) The LCB frac-tion was converted to dinitrophenol derivatives, extracted with chloroform⁄ methanol ⁄ water (8 : 4 : 3, v ⁄ v ⁄ v), purified
by TLC on silica plates and analysed by reversed-phase HPLC using an Agilent 1100 LC system, with MS analysis carried out on a Thermoquest LCQ system with an APCI source Standards were obtained from Matreya Inc USA,
or using previously authenticated in vivo synthesized LCBs [23,24]
Acknowledgements
Financial support for this work was provided by the Department for Environment, Food and Rural Affairs grant no NF 0507 and the EU Sixth Framework Pro-gramme Integrated Project LipGene (Contract FOOD-CT-2003–505944) RQ is a visiting scholar from