Identification and Functional Analysis of Delta-9 Desaturase, a Key Enzyme in PUFA Synthesis, Isolated from the Oleaginous Diatom Fistulifera
Isolated from the Oleaginous Diatom Fistulifera. PLoS ONE 8(9): e73507. doi:10.1371/journal.pone.0073507
Identification and Functional Analysis of Delta-9 Desaturase, a Key Enzyme in PUFA Synthesis, Isolated from the Oleaginous Diatom Fistulifera
Masaki Muto 0
Chihiro Kubota 0
Masayoshi Tanaka 0
Akira Satoh 0
Mitsufumi Matsumoto 0
Tomoko Yoshino 0
Tsuyoshi Tanaka 0
Vladimir N. Uversky, University of South Florida College of Medicine, United States of America
0 1 Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology , Koganei, Tokyo , Japan , 2 JST, CREST, Sanbancho 5, Chiyoda-ku, Tokyo , Japan , 3 BT Development Group, Research and Development Section, Technology Center, Yamaha Motor Co. Ltd. , Fukuroi, Shizuoka, Japan, 4 Biotechnology Laboratory , Electric Power Development Co. Ltd. , Yanagisaki-machi, Wakamatsu-ku, Kitakyusyu , Japan
Oleaginous microalgae are one of the promising resource of nonedible biodiesel fuel (BDF) feed stock alternatives. Now a challenge task is the decrease of the long-chain polyunsaturated fatty acids (PUFAs) content affecting on the BDF oxidative stability by using gene manipulation techniques. However, only the limited knowledge has been available concerning the fatty acid and PUFA synthesis pathways in microalgae. Especially, the function of D9 desaturase, which is a key enzyme in PUFA synthesis pathway, has not been determined in diatom. In this study, 4 D9 desaturase genes (fD9desA, fD9desB, fD9desC and fD9desD) from the oleaginous diatom Fistulifera were newly isolated and functionally characterized. The putative D9 acyl-CoA desaturases in the endoplasmic reticulum (ER) showed 3 histidine clusters that are well-conserved motifs in the typical D9 desaturase. Furthermore, the function of these D9 desaturases was confirmed in the Saccharomyces cerevisiae ole1 gene deletion mutant (Dole1). All the putative D9 acyl-CoA desaturases showed D9 desaturation activity for C16:0 fatty acids; fD9desA and fD9desB also showed desaturation activity for C18:0 fatty acids. This study represents the first functional analysis of D9 desaturases from oleaginous microalgae and from diatoms as the first enzyme to introduce a double bond in saturated fatty acids during PUFA synthesis. The findings will provide beneficial insights into applying metabolic engineering processes to suppressing PUFA synthesis in this oleaginous microalgal strain.
Competing Interests: Akira Satoh is employed by Yamaha Motor Co., Ltd. and Mitsufumi Matsumoto is employed by Electric Power Development Co., Ltd.
There are no patents, products in development, or marketed products to declare. The authors declare that all other data and materials described in the
manuscript adhere to all the PLoS ONE policies on sharing data and materials as detailed online in the guide for authors.
Biodiesel fuel (BDF) has attracted considerable attention over
the past decade as a renewable and biodegradable fuel alternative
to fossil fuels. Commercially available BDFs are produced from a
variety of terrestrial plants, including soybean, rapeseed,
sunflower, castor seed, jatropha and palm oil. Terrestrial plants potentially
have a negative impact on food supply. Furthermore, they have
lower oil yield per area than oleaginous microalgae [1,2,3]. Based
on the reasons, recently, oleaginous microalgae have been
intensively studied as non-food biomass and high-triacylglyceride
(TAG) producer for efficient BDF production [1,4].
BDF is a series of fatty acid methyl esters (FAMEs) generated by
transesterification of TAG from feedstocks . The physical and
chemical properties of FAMEs are determined by its acyl
composition, with respect to both carbon chain length and the
number of double bonds. As the degree of unsaturation of fatty
acids in FAMEs particularly affects the oxidative stability of BDF
, the unsaturated fatty acid content in BDF is a primary
limitation to its commercial use . BDFs from soybean, sunflower
and grape seed contain high levels of polyunsaturated fatty acids
(PUFAs), resulting in poor oxidative stability [8,9]. On the other
hand, BDFs from rapeseed, olive, corn, almond and high oleic
sunflower oils show superior BDF properties because of their high
content of monounsaturated . Microalgal TAG mainly consists
of short and saturated fatty acids, however, non-negligible
quantities of long-chain PUFAs, such as methyl linolenate
(C18:3), eicosapentaenoic acid (EPA; C20:5) or docosahexaenoic
acid (DHA; C22:6) are also involved [10,11].
Toward addressing the above issue, breeding efforts have been
done in terrestrial plants. The PUFA contents of TAG have been
successfully reduced by the suppression of desaturase gene
expression using RNA interference (RNAi) system in soybean,
cotton seed and brassica seed [12,13,14]. By contrast, in
microalgae, genetic modifications of FAME profiles have been
hampered by the limited knowledge available concerning the fatty
acid synthesis pathway (including PUFA synthesis) and/or by
difficulties in the genetic engineering approach [15,16]. Among
eukaryotic microalgal groups, diatoms are well-established in
terms of genomic and transgenic capabilities. Furthermore, the
enzymes involved in fatty acid synthesis have been primarily
identified in a model diatom, Phaeodactylum tricornutum [17,18,19].
In P. tricornutum, v3, D5, D6 and D12 desaturases were responsible
for PUFA synthesis [18,19]. The end-product of the pathway is
EPA. However, among various desaturases, the function of D9
desaturase from diatom has not been determined, although the
enzyme plays a key role in PUFA synthesis as the first enzyme to
introduce a double bond into saturated fatty acids .
A marine oleaginous diatom, Fistulifera sp. used in this study, has
been recognized as a potential candidate for BDF production 
because of its exceedingly high levels of intracellular TAGs (60%
w/w) and its rapid growth. High-cell-density cultivation and
outdoor mass cultivation of Fistulifera sp. have been demonstrated
in flat-type photobioreactors , and column-type and
racewaytype bioreactors . In this strain, the major fatty acids are
palmitate (C16:0; 3040% of total fatty acids), palmitoleate
(C16:1; 4050%) and eicosapentaenoic acid (EPA, C20:5; 4
20%) as a PUFA. Recently, genetic transformation for this strain
was performed . Metabolic engineering with the gene
manipulation technique is a promising approach to decrease the
PUFA content in TAG. One of the targets for genetic
transformation was D9 desaturase because they may play a key
role in fatty acid (and subsequent TAG) synthesis [12,13,14].
In this study, we report the screening of D9 desaturase genes in the
oleaginous diatom Fistulifera and their functional characterization
by expression in the yeast Dole1 mutant. Through the comparison
of the isolated D9 desaturases with those from other diatoms,
unique features of D9 desaturase genes in Fistulifera sp. were
determined. To our knowledge, this is the first study to confirm
Materials and Methods
Strains and Growth Conditions
The marine pennate diatom Fistulifera sp. was grown in
halfstrength Guillards f solution (f/2)  dissolved in artificial
seawater (Tomita Pharmaceutical Co. Ltd., Naruto, Japan).
Cultures were grown at 25uC under continuous and cool-white
fluorescent lights at 140 mmol?m22?s21 with aeration. Genes were
cloned in Escherichia coli TOP10 (Invitrogen, Carlsbad, CA, USA)
or E. coli DH5a (BioDynamics Laboratory Inc., Tokyo, Japan)
cultured in Luria broth (Merck, Darmstadt, Germany) containing
50 mg/mL kanamycin or ampicillin at 37uC.
Putative D9 desaturase genes were expressed in Saccharomyces
cerevisiae INVSc-1 (MATa/MATa, his3D1/his3D1, leu2/leu2,
trip1289/trip1-289, and ura3-52/ura3-52) (Invitrogen) or the yeast Dole1
mutant (MATa, his3D1, leu2D0, ura3D0, and ole1D::kanMX4) .
The yeast Dole1 mutant (MATa, his3D1, leu2D0, ura3D0, and
ole1D::kanMX4) was generated via the sporulation of the S. cerevisiae
YGL055W/BY4743 heterozygous strain (MATa/MATa, his3D1/
his3D1, leu2D0/leu2D0, lys2D0/+, met15D0/+, ura3D0/ura3D0, and
ole1D::kanMX4) (ATCC number: 4024422).
Isolation of D9 desaturase Genes from Fistulifera sp.
To obtain the putative D9 desaturase genes of Fistulifera sp., a
homology search using BlastX was performed with reference to
Figure 2. Amino acid sequence alignments of four D9 desaturases from Fistulifera sp. The ClustalW program was used for the
alignment. The conserved amino acids are on gray backgrounds. The 3 histidine clusters are framed.
the 19,859 genes from the draft genome sequence of Fistulifera sp.
. The full-length cDNAs of putative D9 desaturase genes were
obtained by 59- and 39-RACE using a Smarter RACE cDNA
amplification kit (Clontech, Palo Alto, CA, USA). Partial
sequences of these genes predicted by the AUGUSTUS program
were used for designing gene-specific primers to amplify the 59 and
39 ends of the target genes (Table S1). The PCR products were
cloned into the pCR-Blunt II-TOPO vector (Invitrogen). The
fulllength cDNA sequences were assembled based on the 59- and
Conserved histidine sequences
Number of predicted TMHs
Amino acid sequence alignments of D9 desaturases from
different organisms were generated using the ClustalW program
(http://www.genome.jp/tools/clustalw/). The phylogenetic tree
was constructed via the neighbor-joining method and evaluated
with 1,000 rounds of bootstrapping using MEGA4. D9 desaturase
amino acid sequences were retrieved from the databases of the
whole genome of P. tricornutum  and Thalassiosira pseudonana 
by a BlastX search using the D9 desaturase genes of Fistulifera sp. as
the query sequence. Cloned sequences and other putative diatom
sequences were also investigated to determine whether the protein
has N-terminal signal peptides; SignalP 4.0 (http://www.cbs.dtu.
dk/services/SignalP/) , TargetP 1.1 (http://www.cbs.dtu.dk/
services/TargetP/) , and HECTAR (http://www.sb-roscoff.
fr/hectar/)  were used for this analysis. TMHMM 2.0 (http://
www.cbs.dtu.dk/services/TMHMM)  and TMHTOP 2.0
(http://www.enzim.hu/hmmtop/index.php)  were used for
the prediction of transmembrane domains.
Functional Characterization of D9 Desaturases in the
Yeast Dole1 Mutant
For functional characterization, 4 D9 desaturase genes (fD9desA,
fD9desB, fD9desC, and fD9desD) with the Kozak sequence  in
front of the start codon were cloned into the yeast expression
vector pYES2.1/V5-His-TOPO (Invitrogen) (Table S1). The yeast
Dole1 mutant was transformed with plasmid DNA with a
polyethylene glycol/lithium acetate protocol . The yeast cells
harboring the control pYES2.1/V5-His/lacZ were used as a
negative control. All transformants were selected by uracil
prototrophy on a selective dropout media (SD) plate lacking
uracil. For functional expression, SD medium containing 2% (w/v)
galactose, 1% Tergitol Type NP-40 (Invitrogen), and 500 mM
C16:1 or C18:1 fatty acids was inoculated with the
pYES2.1FsDES9 transformants and grown at 20uC for 96 h in a
water bath shaker. Cell pellets were sequentially washed with 1%
Tergitol Type NP-40 and 0.5% Tergitol Type NP-40,
freezedried, and subject to fatty acid analysis.
Complementation Assay in the Yeast Dole1 Mutant
Each transformant harboring the plasmid for the expression of
D9 desaturases was suspended in distilled water and adjusted to an
OD600 of 1 and 0.1. The resulting 2.5 mL yeast solution was
spotted on an SD agar plate lacking uracil but containing 2%
galactose and (a) no fatty acids; (b) 500 mM C16:1 fatty acid and
1% Tergitol Type NP-40; or (c) 500 mM of the C18:1 fatty acid
and 1% Tergitol Type NP-40. Cell growth was evaluated after
72 h at 30uC to examine the unsaturated fatty acid requirement
for the growth of yeast Dole1 mutant transformants.
Fatty Acid Analysis Using GC/MS
The freeze-dried yeast cells were directly transmethylated with
1.25 M hydrochloric acid in methanol (1 h at 100uC) to prepare
the FAMEs. The FAMEs were extracted in n-hexane and analyzed
Table 2. Substrate specificity analysis of D9 desaturases (fD9desA, fD9desB, fD9desC, and fD9desD) from Fistulifera sp. on the basis
of expression in the yeast Dole1 mutant (n = 3).
INVSc-1+ control vector
Supplementation of C18:1
Supplementation of C16:1
The yeast strain INVSc-1 transformed with the control vector pYES2.1/V5-His/lacZ (Control vector) served as the positive control. The yeast Dole1 mutant transformed
with pYES2.1/V5-His/lacZ served as the negative control.
aThe relative amount of each fatty acid was expressed as a percentage of total fatty acids (6 SD) after subtracting the amount of the supplemented fatty acid from the
by GC/MS (QP2010 Plus; Shimadzu, Kyoto, Japan) with
FAMEWAX (RESTEK, Bellefonte, PA, USA) in the electron
impact mode. FAMEs were identified using the F.A.M.E. Mix,
C4C24 Unsaturates (Sigma-Aldrich, Dorset, UK). Each sample
was analyzed in 3 independent experiments.
Sequence Analysis of Putative D9 Desaturase Genes from
The 6 D9 desaturase candidate genes (Gene ID: g5394, g10778,
g10781, g12958, g19483 and g19486) were identified with the
BlastX algorithm from all 19,859 genes of this strain as the query
sequence from a non-redundant protein sequences database
[Manuscript in preparation]. Because the predicted D9 desaturases
from draft genome sequence seemed to be partial ORFs due to the
lack of the conserved motifs of histidine box and cytochrome b5
domain, the full-length sequences of putative acyl-CoA D9 desaturase
cDNA were sequenced from the products obtained by rapid
amplification of cDNA ends (RACE) PCR. The cDNAs
containing predicted gene regions were verified to be 996 bp for g10778
and g19483 and 1,002 bp for g10781 and g19486; these were
designated as fD9desA, fD9desB fD9desC, and fD9desD, respectively
(Fig. 1). The fD9desA nucleotide sequences exhibit high identity
with the fD9desB (96%), and the fD9desC had 93% identity with the
fD9desD. The amino acid sequences of fD9desA and fD9desB were
identical, while fD9desC and fD9desD showed the differences of 2
amino acid residues. The 2 proteins encoded by g5394 and
g12958 had 49% and 47% identity, respectively, with D9
desaturase from the plant Asclepias syriaca (GenBank accession no.
AAC49719.1) . The 4 proteins encoded by g10778, g10781,
g19483 and g19486 showed 78%, 69%, 72% and 71% identity,
respectively, with the putative D9 desaturase from a diatom, P.
tricornutum (EEC47008.1). A phylogenetic tree of D9 desaturase
amino acid sequences from different organisms, prepared using
ClustalW, showed that the g5394 and g12958 genes appeared in
the cluster of the plastidial acyl-ACP desaturase group, while the
remaining genes were categorized in acyl-CoA D9 desaturase
groups localizing in the endoplasmic reticulum (ER) (Fig. 2). As the
PUFA synthesis occurs in the ER of eukaryotic cells , these 4
genes in the acyl-CoA D9 desaturase groups were further
investigated. In model strains of pennate and centric diatoms, P.
tricornutum (EEC47008), and Thalassiosira pseudonana (EED91785
and EED86245), ER acyl-CoA desaturases and plastidal acyl-ACP
desaturases were also identified by a BLAST search using the
predicted genes from Fistulifera sp. as the query sequence (Table 1).
A unique histidine motif is generally found in D9 desaturases
. Four D9 desaturases genes in Fistulifera sp. possessed 3 histidine
motifs (HxxxxH, HxxHH, and HxxHH) involved in the
coordination of the di-iron center as the active site of desaturation
(Table 1). On the other hand, the cytochrome b5 domain, which is
expected to transfer electrons away from the histidine motif, was
not observed in the identified D9 acyl-CoA desaturase. These
unique motifs of D9 desaturases were compared with those in 2
diatoms, P. tricornutum and T. pseudonana . These enzymes from
the 3 diatoms were well conserved, possessing the histidine
sequences without the cytochrome b5 domain. The findings
concerning the lack of cytochrome b5 in desaturases is in
agreement with those of a previous report about alternative
desaturation activity in the absence of the cytochrome b5 domain
in 2 other desaturases from T. pseudonana . Furthermore, the
results of truncation and disruption experiments for the
cytochrome b5 domain D9 desaturases have suggested that this domain
is not strictly required for fatty acid desaturation .
Localization of general D9 desaturases in the ER membrane are
predicted from N-terminal amino acid sequences . Three
algorithms for subcellular targeting prediction, TargetP 1.1,
SignalP 4.0 and HECTAR, were used in this study. The D9
desaturase candidates were predicted to have neither N-terminal
signal sequences nor internal cleavable signal sequences. All 4 D9
desaturases were expected to possess 25 transmembrane
domains, according to a prediction by TMHMM 2.0 and
HMMTOP 2.0 (Table 1). This prediction supports the existence
of transmembrane helices in D9 desaturases from Fistulifera sp.,
although further analysis is needed to confirm the number of
Functional Characterization of Putative D9 Desaturases in
the Yeast Dole1 Mutant
In order to confirm the function of D9 desaturases, 4 putative
genes were cloned in the protein expression vector,
pYES2.1/V5His-TOPO, and transformed in the yeast Dole1 mutant. Dole1 is a
D9 desaturase knockout mutant that requires supplementation with
C16:1 or C18:1 fatty acids to grow. When the putative D9
desaturases work properly in the synthesis of monounsaturated
C16:1 and/or C18:1 fatty acids, the transformants can grow on an
agar plate without supplementation with exogenous unsaturated
fatty acids. The transformed yeast Dole1 mutant with the control
vector (in the absence of any desaturase gene), serving as a negative
control, did not grow in the absence of fatty acid supplementation
(Fig. 3(a), no UFA). The INVSc-1 cells possessing the native ole1
gene in the genome, serving as a positive control, grew well in the
absence of fatty acid supplementation (Fig. 3(b), no UFA) because
the cell can generate these essential desaturated fatty acids
endogenously. The fD9desA, fD9desB, fD9desC, and fD9desD genes
failed to complement the yeast ole1 mutation in the absence of
16:1/18:1 fatty acid supplementation (Fig. 3(c)-(f), no UFA).
Additionally, the growth of transformants with 16:1/18:1 fatty
acid supplementation was also confirmed. In a previous study, a
D9 desaturase (D9-3) from the fungus Mortierella alpina failed to
complement the yeast Dole1 mutant when transformants were
grown in the absence of monounsaturated fatty acids
supplementation . It is likely that the D9-3 desaturase from M. alpina and
D9 desaturases from Fistulifera sp. did not have sufficient activity for
the complementation in yeast, respectively.
Next, to further investigate the in vivo function and specificity of
these D9 desaturase genes, the fatty acid profiles of the yeast Dole1
transformant-carrying gene expression vector with the D9 desaturase
genes were evaluated by gas chromatography and mass
spectrometry (GC/MS) analysis (Table 2). Two D9 desaturases from
Fistulifera sp., fD9desC and fD9desD, showed activity for C16:0 as
a substrate (0.6% and 0.3% desaturation to C16:1, respectively)
(100 6 product/substrate+product) during growth with C18:1
supplementation (Table 2) but did not show activity for C18:0.
The remaining 2 D9 desaturases, fD9desA and fD9desB, showed
similar activities to each another with C16:0 as a substrate (0.8%
and 1.2% desaturation to C16:1, respectively). However, the ideal
substrate of fD9desA and fD9desB was C18:0 converted to C18:1
(6.9% and 5.5% desaturation to C18:1). The desaturation
efficiencies of fD9desA and fD9desB for C18:0 were significantly
higher than those for C16:0. The detected value of desaturation by
D9 desaturases from Fistulifera sp in yeast was similar to that of
house cricket (Acheta domesticus) (5% desaturation to 18:1) .
These heterogeneous gene expressions and functional analyses in
the model organism may cause the negative effect for the activity,
exhibiting these relatively low activities.
Six D9 desaturase genes were identified in Fistulifera sp. by
bioinformatic analysis. These genes were categorized as 4 ER
acyl-CoA desaturases (fD9desA, fD9desB, fD9desC, and fD9desD) and
2 plastidial acyl-ACP desaturases (g5394 and g12958). By way of
comparison, P. tricornutum has only 1 ER acyl-CoA desaturase and
1 plastidial acyl-ACP desaturase, and T. pseudonana has 2 ER
acylCoA desaturases and 1 plastidial acyl-ACP desaturase (Table 1).
These results indicate that Fistulifera sp. has more D9 desaturase
genes than other diatoms do. The higher number of D9 desaturase
genes in Fistulifera sp. suggests that the oleaginous strain may have
well-developed genome organization for fatty acid metabolism to
enable considerable accumulation of TAG endogenously. The
identified ER acyl-CoA desaturases show different substrate
specificities (Table 2). Two D9 desaturases (fD9desA and fD9desB)
converted both C16:0 and C18:0 to C16:1 and C18:1,
respectively, and the other 2 D9 desaturases (fD9desC and fD9desD)
converted C16:0 to C16:1. The sequences of the D9 desaturases
showed the presence of highly conserved histidine cluster motifs
 (Fig. 2), and a difference of 1 amino acid residue in the first
histidine motif box was observed among these 4 D9 desaturases.
The former group of D9 desaturases (fD9desA and fD9desB) has
the HRLWSH sequence, and the latter group of D9 desaturases
(fD9desC and fD9desD) has the HRLWAH sequence, with the
serine substituted with alanine. In other words, D9 desaturase pairs
from Fistulifera sp. have the same histidine motifs and desaturation
specificities (Table 2). In the oil-producing fungus M. alpina, a
different amino acid sequence in the first histidine box was found
in 3 D9 desaturases. Different D9 desaturases with different
sequences in the first histidine box showed varying specificities for
fatty acid desaturation . Therefore, the difference in amino
acid sequences is considered to reflect the substrate specificity of
D9 desaturases from Fistulifera sp. P. tricornutum has only 1 ER
acylCoA desaturase with the amino acid sequence of HRLWSH in the
first histidine motif, while T. pseudonana has 2 ER acyl-CoA
desaturases with the same amino acid sequence (HRLWSH;
Table 1). On the basis of these results, we hypothesized that all the
D9 desaturases in both diatoms possess the same substrate
specificity, although a detailed functional analysis should be
performed to confirm this hypothesis. If it is confirmed, this would
indicate that the varying specificities of D9 desaturases from
Fistulifera sp. may be related to the specific fatty acid metabolism in
this strain. In addition, the D9 desaturation activity was assayed for
the substrates of 16:0 and 18:0. In the case of mouse D9 desaturase,
for wide range of saturated fatty acids from 12:0 to 19:0, the
desaturation activity was detected . To fully confirm the
functional specificity of D9 desaturase from Fistulifera sp. for various
substrates, further analysis should be provided in the future.
In conclusion, 4 ER D9 acyl-CoA desaturase and 2 plastidial D9
acyl-ACP desaturase genes were identified from the oleaginous
diatom Fistulifera sp., and gene homologs were also observed in
other diatoms. Among the D9 desaturase genes, four D9 acyl-CoA
desaturases showed desaturation activity of the saturated fatty acids
of C16:0 and/or C18:0 in complementation assays using the yeast
Dole1 mutant. This study is the first functional confirmation of D9
desaturase from diatom and from oleaginous microalgae. While
the in vivo function of the D9 desaturase genes in the Fistulifera sp.
should be separately addressed, this study provides information
that will help in the regulation of the fatty acid profiles in the
List of primers used for this study.
Conceived and designed the experiments: M.Muto MT TY TT.
Performed the experiments: M.Muto CK AS M.Matsumoto. Analyzed
the data: M.Muto CK M.Matsumoto MT TY TT. Contributed reagents/
materials/analysis tools: AS M.Muto. Wrote the paper: M.Muto MT TT.
1. Chisti Y ( 2007 ) Biodiesel from microalgae . Biotechnol Adv 25 : 294 - 306 .
2. Mata TM , Martins AA , Caetano NS ( 2010 ) Microalgae for biodiesel production and other applications: A review . Renew Sust Energ Rev 14 : 217 - 232 .
3. Guarnieri MT , Nag A , Smolinski SL , Darzins A , Seibert M , et al. ( 2011 ) Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga . Plos One 6 : e25851 .
4. Sforza E , Simionato D , Giacometti GM , Bertucco A , Morosinotto T ( 2012 ) Adjusted light and dark cycles can optimize photosynthetic efficiency in algae growing in photobioreactors . Plos One 7 : e38975 .
5. Leung DYC , Guo Y ( 2006 ) Transesterification of neat and used frying oil: Optimization for biodiesel production . Fuel Process Technol 87 : 883 - 890 .
6. Lim DKY , Garg S , Timmins M , Zhang ESB , Thomas-Hall SR , et al. ( 2012 ) Isolation and evaluation of oil-producing microalgae from subtropical coastal and brackish waters . Plos One 7 : e40751 .
7. Stournas S , Lois E , Serdari A ( 1995 ) Effects of fatty-acid derivatives on the Ignition quality and cold flow of diesel fuel . J Am Oil Chem Soc 72 : 433 - 437 .
8. Ramos MJ , Fernandez CM , Casas A , Rodriguez L , Perez A ( 2009 ) Influence of fatty acid composition of raw materials on biodiesel properties . Bioresour Technol 100 : 261 - 268 .
9. Tyson KS , McCormick RL ( 2006 ) Biodiesel handling and use guidelines .
10. Griffiths MJ , van Hille RP , Harrison STL ( 2012 ) Lipid productivity, settling potential and fatty acid profile of 11 microalgal species grown under nitrogen replete and limited conditions . J Appl Phycol 24 : 989 - 1001 .
11. Nascimento IA , Marques SSI , Cabanelas ITD , Pereira SA , Druzian JI , et al. ( 2013 ) Screening microalgae strains for biodiesel production: lipid productivity and estimation of fuel quality based on fatty acids profiles as selective criteria . Bioenerg Res 6 : 1 - 13 .
12. Knutzon DS , Thompson GA , Radke SE , Johnson WB , Knauf VC , et al. ( 1992 ) Modification of brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene . Proc Natl Acad Sci USA 89 : 2624 - 2628 .
13. Singh AK , Fu DQ , El-Habbak M , Navarre D , Ghabrial S , et al. ( 2011 ) Silencing genes encoding omega-3 fatty acid desaturase alters seed size and accumulation of bean pod mottle virus in soybean . Mol Plant Microbe In 24 : 506 - 515 .
14. Liu Q , Singh SP , Green AG ( 2002 ) High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing . Plant Physiol 129 : 1732 - 1743 .
15. Radakovits R , Jinkerson RE , Fuerstenberg SI , Tae H , Settlage RE , et al. ( 2012 ) Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana . Nat Commun 3 : 686 .
16. Lu J , Sheahan C , Fu PC ( 2011 ) Metabolic engineering of algae for fourth generation biofuels production . Energ Environ Sci 4 : 2451 - 2466 .
17. Bowler C , Allen AE , Badger JH , Grimwood J , Jabbari K , et al. ( 2008 ) The Phaeodactylum genome reveals the evolutionary history of diatom genomes . Nature 456 : 239 - 244 .
18. Domergue F , Lerchl J , Zahringer U , Heinz E ( 2002 ) Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis . Eur J Biochem 269 : 4105 - 4113 .
19. Domergue F , Spiekermann P , Lerchl J , Beckmann C , Kilian O , et al. ( 2003 ) New insight into Phaeodactylum tricornutum fatty acid metabolism. Cloning and functional characterization of plastidial and microsomal Delta 12-fatty acid desaturases . Plant Physiol 131 : 1648 - 1660 .
20. Matsumoto M , Sugiyama H , Maeda Y , Sato R , Tanaka T , et al. ( 2010 ) Marine diatom, Navicula sp. strain JPCC DA0580 and marine green alga, Chlorella sp. strain NKG400014 as potential sources for biodiesel production . Appl Biochem Biotech 161 : 483 - 490 .
21. Satoh A , Ichii K , Matsumoto M , Kubota C , Nemoto M , et al. ( 2013 ) A process design and productivity evaluation for oil production by indoor mass cultivation of a marine diatom, Fistulifera sp . JPCC DA0580 . Bioresour Technol 137 : 132 - 138 .
22. Sato R , Maeda Y , Matsumoto M , Yoshino T , Tanaka T ( 2013 ) Seasonal variation of biomass and oil production of marine diatom Fistulifera sp . in outdoor vertical bubble column and raceway-type bioreactors . Algal Res (Submitted).
23. Muto M , Fukuda Y , Nemoto M , Yoshino T , Matsunaga T , et al. ( 2013 ) Establishment of a genetic transformation system for the marine pennate diatom Fistulifera sp. strain JPCC DA0580-A high triglyceride producer . Mar Biotechnol 15 : 48 - 55 .
24. Guillard RRL , Ryther JH ( 1962 ) Studies of marine planktonic diatoms: I. Cyclotella nana hustedt, and Detonula confervacea (cleve) gran . Can J Microbiol 8 : 229 - 239 .
25. Winzeler EA , Shoemaker DD , Astromoff A , Liang H , Anderson K , et al. ( 1999 ) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis . Science 285 : 901 - 906 .
26. Tanaka T , Fukuda Y , Yoshino T , Maeda Y , Muto M , et al. ( 2011 ) Highthroughput pyrosequencing of the chloroplast genome of a highly neutral-lipidproducing marine pennate diatom , Fistulifera sp. strain JPCC DA0580 . Photosynth Res 109 : 223 - 229 .
27. Armbrust EV , Berges JA , Bowler C , Green BR , Martinez D , et al. ( 2004 ) The genome of the diatom Thalassiosira pseudonana: Ecology , evolution, and metabolism. Science 306 : 79 - 86 .
28. Petersen TN , Brunak S , von Heijne G , Nielsen H ( 2011 ) SignalP 4.0: discriminating signal peptides from transmembrane regions . Nat Methods 8 : 785 - 786 .
29. Emanuelsson O , Nielsen H , Brunak S , von Heijne G ( 2000 ) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence . J Mol Biol 300 : 1005 - 1016 .
30. Gschloessl B , Guermeur Y , Cock JM ( 2008 ) HECTAR: A method to predict subcellular targeting in heterokonts . BMC Bioinformatics 9 : 393 .
31. Krogh A , Larsson B , von Heijne G , Sonnhammer ELL ( 2001 ) Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes . J Mol Biol 305 : 567 - 580 .
32. Tusnady GE , Simon I ( 2001 ) The HMMTOP transmembrane topology prediction server . Bioinformatics 17 : 849 - 850 .
33. Kozak M ( 1991 ) An analysis of vertebrate messenger-RNA sequences - intimations of translational control . J Cell Biol 115 : 887 - 903 .
34. Ito H , Fukuda Y , Murata K , Kimura A ( 1983 ) Transformation of intact yeastcells treated with alkali cations . J Bacteriol 153 : 163 - 168 .
35. Cahoon E , Coughlan S , Shanklin J ( 1997 ) Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed . Plant Mol Biol 33 : 1105 - 1110 .
36. Shanklin J , Whittle E , Fox BG ( 1994 ) Eight histidine-residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-coa desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase . BiochemistryUs 33 : 12787 - 12794 .
37. Itoh R , Toda K , Takahashi H , Takano H , Kuroiwa T ( 1998 ) Delta-9 fatty acid desaturase gene containing a carboxyl terminal cytochrome b5 domain from the red alga Cyanidioschyzon merolae . Curr Genet 33 : 165 - 170 .
38. Tonon T , Sayanova O , Michaelson LV , Qing R , Harvey D , et al. ( 2005 ) Fatty acid desaturases from the microalga Thalassiosira pseudonana . FEBS J 272 : 3401 - 3412 .
39. Mitchell AG , Martin CE ( 1995 ) A novel cytochrome b5-like domain is linked to the carboxyl-terminus of the Saccharomyces cerevisiae delta-9 fatty-acid desaturase . J Biol Chem 270 : 29766 - 29772 .
40. MacKenzie DA , Carter AT , Wongwathanarat P , Eagles J , Salt J , et al. ( 2002 ) A third fatty acid Delta 9-desaturase from Mortierella alpina with a different substrate specificity to ole1p and ole2p . Microbiol-Sgm 148 : 1725 - 1735 .
41. Vanhercke T , Shrestha P , Green AG , Singh SP ( 2011 ) Mechanistic and structural insights into the regioselectivity of an acyl-coa fatty acid desaturase via directed molecular evolution . J Biol Chem 286 : 12860 - 12869 .
42. Hashimoto K , Yoshizawa AC , Okuda S , Kuma K , Goto S , et al. ( 2008 ) The repertoire of desaturases and elongases reveals fatty acid variations in 56 eukaryotic genomes . J Lipid Res 49 : 183 - 191 .
43. Miyazaki M , Bruggink SM , Ntambi JM ( 2006 ) Identification of mouse palmitoyl- coenzyme A Delta 9-desaturase . J Lipid Res 47 : 700 - 704 .