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Biochimie 93 (2011) 91e100
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Biochimie
journal homepage: www.elsevier.com/locate/biochi
Mini-review
Unraveling algal lipid metabolism: Recent advances in gene identification
Inna Khozin-Goldberg*, Zvi Cohen
Microalgal Biotechnology Laboratory, French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research,
Ben-Gurion University of the Negev, Midreshet Ben-Gurion 84990, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 May 2010
Accepted 30 July 2010
Available online 13 August 2010
Microalgae are now the focus of intensive research due to their potential as a renewable feedstock for
biodiesel. This research requires a thorough understanding of the biochemistry and genetics of these
organisms’ lipid-biosynthesis pathways. Genes encoding lipid-biosynthesis enzymes can now be identified in the genomes of various eukaryotic microalgae. However, an examination of the predicted
proteins at the biochemical and molecular levels is mandatory to verify their function. The essential
molecular and genetic tools are now available for a comprehensive characterization of genes coding for
enzymes of the lipid-biosynthesis pathways in some algal species. This review mainly summarizes the
novel information emerging from recently obtained algal gene identification.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Microalgae
Lipids
LC-PUFA
Desaturases
Acyltransferases
1. Introduction
This review covers the recent discoveries in the lipid metabolism of eukaryotic photosynthetic microalgae, an extremely diverse
group of unicellular organisms dwelling in various environments.
Our goal is to cover some aspects which have not been described in
previous comprehensive reviews on algal lipid metabolism [1e3,
and ref therein]; thus mainly novel information on recently identified genes will be summarized. Numerous microalgal species are
used for the production of high-value lipid compounds, such as
long-chain polyunsaturated fatty acids (LC-PUFA) and carotenoid
pigments, mainly as a feed source in aquaculture and for nutraceutical applications. Oleaginous algae accumulate high quantities
of neutral storage lipids, mainly triacylglycerols (TAG), in response
to environmental stresses, such as nitrogen limitation, salinity or
high temperature, and their TAG are composed largely of saturated
and monounsaturated fatty acids [2,4 and ref. therein]. A rare
exception is the freshwater chlorophyte Parietochloris incisa which
accumulates an unprecedented proportion (60% of total fatty acids)
of the u6 LC-PUFA arachidonic acid (ARA) in its TAG [4,5]. We
hypothesized that one of the roles of LC-PUFA-rich TAG in certain
algal species, rather than serving only as an energy store, is to serve
as a reservoir that can be used for the rapid construction of PUFArich chloroplastic membranes, particularly under low temperatures
or during growth recovery after nitrogen starvation [4,6]. Indeed,
* Corresponding author. Tel.: þ972 8 6563478; fax: þ972 8 6596742.
E-mail address: [email protected] (I. Khozin-Goldberg).
0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2010.07.020
the role of lipid bodies in eukaryotic cells is now thought to be
much more complex than simply serving as a carbon store, because
stores of fatty acid moieties are also required for membrane
synthesis and remodeling [7,8]. Due to the high potential of
microalgae as a renewable feedstock for biodiesel production [for
further information refer to [9e12]], isolation and functional
characterization of genes encoding enzymes that mediate the key
steps in fatty acid and TAG biosynthesis, as well as in lipid catabolism, are of particular importance. The anticipated progress in
elucidating these steps is supported by the wealth of genetic
information on algae that has been revealed in the last decade.
Genome sequence information is currently available for seven
eukaryotic algae, including the red microalga Cyanidioshyzon
merolae, two marine diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum, several green microalgae, including Chlamydomonas reinhardtii, and marine picoeukaryotes of the class
Prasinophyceae: Ostreococcus tauri, Ostreococcus lucimarinus,
Micromonas pussila and Micromonas sp. A few more projects on
algal genomes are currently in progress (e.g. of the haptophyte
Emiliania huxleyi) which, together with numerous expressed
sequence tag (EST) studies of various algal species, are expected to
provide substantial novel information [11,13e15]. C. reinhardtii is
a model microalga for which microarray gene expression data are
available and tools for the transformation of plastids and nuclear
genomes, as well as for silencing by RNA interference (RNAi), have
been developed [13,16]. New approaches for analyzing gene
expression are expected to allow more efficient evaluation of the
conditions that favor biosynthesis of energy-rich products, such as
starch and TAG (http://www.jgi.doe.gov/sequencing/why/chlamy.
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I. Khozin-Goldberg, Z. Cohen / Biochimie 93 (2011) 91e100
html). Moreover, genetic transformation tools have also been
recently developed for diatom species [17,18], and the presence of
functional silencing machinery and feasibility of targeted
gene knockdown have been demonstrated in P. tricornutum
by introducing constructs that express anti-sense or invertedrepeat-containing RNAs [19]. Diatom expression vectors for the
validation of subcellular localization, immunodetection of proteins
of interest and overexpression studies have also been developed
[20]. Thus, the essential molecular and genetic tools are now
available for a comprehensive characterization of genes coding for
enzymes of the lipid-biosynthesis pathways.
2. Fatty acid biosynthesis and production of
precursors in the plastid
Genome information has enabled in silico analysis of fatty acid
and glycerolipid metabolism and a prediction of the genes encoding proteins involved in membrane and storage lipid biogenesis in
some microalgae [21e24]. For example, the major pathways of fatty
acid synthesis were reconstructed for C. reinhardtii [21e23]: based
on their similarity to proteins with experimentally verified functions in higher plants, all components of the plastidial multisubunit
acetyl coenzyme A (acetyl-CoA) carboxylase (ACCase), catalyzing
the first committed step in fatty acid biosynthesis, carboxylation of
acetyl-CoA to malonyl-CoA, and fatty acid synthase (FAS)
complexes were identified (Fig. 1). Higher plant orthologs of key
enzymes of the plastidial FAS complex are present in the genomic
and EST data bases of other algae [24,25]. Examination of EST
sequences of the thermo-acidophilic red microalga Galdieria sulphuraria [25] and of the genome of a primitive red microalga
C. merolae [24] revealed that their fatty acid biosynthesis is likely
similar to that of the green lineage, which includes green algae and
higher plants. Two types of ACCase were identified in these algae:
a prokaryotic-type multisubunit enzyme in the plastid and
a multifunctional homomeric enzyme in the cytosol [24]. The
plastid localization of the nuclear-encoded subunit of a multisubunit ACCase was confirmed by green fluorescent protein (GFP)fusion experiments. Moreover, genes encoding enzymes involved
in acetyl-CoA generation in the plastid (Fig. 1), such as pyruvate
kinase and pyruvate dehydrogenase complex (PDC), can also be
readily identified, for example, in the Chlamydomonas genome, by
sequence similarity to those of higher plants. PDC is a multienzyme
complex that catalyzes the oxidative decarboxylation of pyruvate,
leading to acetyl-CoA. The plastidial pyruvate kinase, which
converts phosphoenolpyruvate into pyruvate, was shown to be
important as a pyruvate supply for FAS via PDC. It should be noted
that the cellular localization of the putative plastid proteins is
generally predicted by web-based protein-targeting algorithms,
such as TargetP (http://www.cbs.dtu.dk/services/TargetP), and
sometimes these predictions produce uncertain probabilities of the
protein being targeted either to mitochondria or chloroplasts.
Moreover, it was suggested that activities of dual-targeted proteins
in Chlamydomonas are required in both organelles [23]. Hence,
functional validation and characterization of annotated genes in
algal genomes requires further experimental evidence.
Bioinformatics analyses have indicated that the key regulatory
steps identified in eukaryotic microalgae for fatty acid biosynthesis
(Fig. 1) and for the import of fatty acids to the endoplasmic reticulum (ER) for further glycerolipid biosynthesis, are largely similar
to those in higher plant plastids [21e23]. Thus, several key strategic
steps for the metabolic engineering of microalgae aimed at
increasing TAG production, as required for sustainable biodiesel
production, have been proposed [9e11]. Among these are overexpression of ACCase and FAS enzymes, increased availability of
acetyl-CoA precursor for FAS by overexpression of the enzymes
involved in its production or downregulation of phosphoenolpyruvate conversion to oxaloacetate; interference of competing pathways leading to TAG degradation, e.g. inhibition of b-oxidation and
Fig. 1. A simplified scheme for the control of FAS formation in the plastid of green algae. ACCase, acetyl-CoA carboxylase; ACL, ATP-citrate lyase; FAS, fatty acid synthase; ME, malic
enzyme; MCAT, malonyl-CoA:ACP transacylase; PDC, pyruvate dehydrogenase complex; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PK, pyruvate kinase.
I. Khozin-Goldberg, Z. Cohen / Biochimie 93 (2011) 91e100
lipolysis (Fig. 1). Modification of fatty acid chain length and saturation levels to produce monounsaturated and saturated TAG, the
ideal form for biodiesel, can be further achieved via expression of
thioesterases and silencing of desaturases. These strategies are
based on the recently obtained thorough understanding of lipid
metabolism in higher plants [26] and the success in manipulating
lipid-biosynthesis pathways with molecular tools. Moreover, these
strategies can be applied to algae which produce TAG under
conditions of nutrient deficiency or environmental stress, and are
amendable to genetic transformation [10]. Another feasible
approach is the regulation of gene expression by transcription
factors identified in algae to modulate lipid metabolism [27,28].
3. Neutral lipids
There is a significant body of research on the effects of environmental stresses on neutral lipid biosynthesis in algae [1,2].
Conversely, the enzymes and pathways that trigger and control the
accumulation of storage TAG in microalgae have been little studied
at the molecular level. The first genetic engineering effort to
manipulate algal lipid production was made in the diatom Cyclotella cryptica by overexpressing the endogenous gene encoding
ACCase [29]. It was previously shown that an increase in ACCase
activity plays a significant role in the induction of TAG accumulation in nutrient-deprived C. cryptica [30]. However, as reported by
Sheehan et al. [31], overexpression of the additional copies of
ACCase did not have a significant effect on lipid synthesis in either
C. cryptica or a different diatom strain, Navicula saprophila.
C. reinhardtii has been recently used in several studies relating to
the isolation of genes and identification of proteins involved in
neutral lipid biosynthesis. Nitrogen depletion induces significant
ultrastructural changes in the cells of C. reinhardtii, such as reduction of stacked thylakoid membranes, accumulation of starch
granules, and the appearance of lipid droplets (otherwise referred
to as oil bodies) [32]. A lipid droplet-enriched fraction was purified
from C. reinhardtii, and 16 proteins potentially involved in lipid
biosynthesis were identified by mass spectrometry. These proteins
included predicted acyl-CoA synthetases and acyltransferases,
involved in the biosynthesis of TAG or the transfer of acyl groups
between the membrane and neutral lipids. A major protein,
designated major lipid droplet protein (MLDP), was isolated and its
mRNA abundance appeared to correlate with the accumulation of
TAG during the time course of nitrogen deprivation. The protein
was found to be specific to the green algal lineage of photosynthetic
organisms. MLDP was found to be a very hydrophobic protein,
although it was not similar to oleosindthe main structural
hydrophobic protein of plant oil bodies. The functional importance
of MLDP was further examined by RNAi silencing; for the first time
this technique had been employed to characterize a lipid gene in
algae. To this aim, an expression cassette containing the nitrate
reductase (NIT1) promoter, inducible by nitrogen deprivation, was
used to drive the expression of an MLDP genomic sense cDNA
antisense RNAi hairpin. An increase in average lipid droplet size
was observed in the MLDP-RNAi lines, but the levels of TAG or rate
of TAG mobilization upon nitrogen replenishment did not change,
indicating a structural role for MLDP in oil-body formation.
Stressful conditions, such as nutrient limitation or high light
intensity, trigger both starch and TAG accumulation in some algal
species. There is now substantial evidence that the shift of carbon
fluxes from starch to TAG biosynthesis in those species represents
an alternative and efficient approach to increasing oil production in
microalgae [33,34]. This is supported by the fact that the block in
starch biosynthesis leads to enhanced formation of lipid bodies and
TAG [33,34]. Starch biosynthesis is impaired in the sta6 mutant of
C. reinhardtii harboring a mutation that leads to inactivation of
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ADP-glucose pyrophosphorylase [35]. In the nitrogen-starved sta6
mutant, the cellular content of lipid bodies increased 30-fold [32],
providing direct evidence that genetic interference of the starch
biosynthesis pathway can enhance lipid accumulation. A further
study by Li et al. [34] reported that the starchless mutant of
C. reinhardtii enhances storage lipid accumulation under stressful
conditions, resulting in a 10-fold increase in cellular TAG production (i.e., from 2 to 20.5% of DW).
3.1. Sterols
Ergosterol is the predominant sterol in the membranes of
C. reinhardtii, which is rarely found in higher plants but is common
in fungi. Bioinformatics analysis revealed, however, that C. reinhardtii likely synthesizes sterols via a pathway resembling the
higher plant pathway that uses cycloartenol as a precursor, rather
than lanosterol as in fungi [36]. This assumption was supported by
the fact that two key enzymes in the cycloartenol pathway are
found in the C. reinhardtii genome, namely cycloartenol cyclase and
cyclopropyl isomerase. This pathway also involves the action of the
C5 sterol desaturase (ERG3) responsible for introducing a double
bond at C5 in the B-ring of episteroldan essential step in ergosterol
biosynthesis. The ERG3 ortholog of C. reinhardtii was cloned and
functional validation was performed by heterologous expression in
the yeast Saccharomyces cerevisiae. To this aim, the ERG3 gene was
knocked out by homologous recombination. Functional complementation in ERG3-knockout strains of S. cerevisiae was able to
restore ergosterol biosynthesis and abolish the mutant’s characteristic sensitivity to cycloheximide.
3.2. Waxes
In higher plants, extracellular waxes play an important role as
a barrier controlling the flux of water and environmental hazards.
A unicellular phytoflagellate Euglena gracilis accumulates wax
esters to up to 62% of its total lipid content under anaerobic growth
conditions as an energy reserve. Wax esters produced by E. gracilis
are 20 to 36 C atoms in length, comprised of 12- to 18C-chain
saturated fatty acids and alcohols with myristyl myristate
(14:0e14:0) as the major species [37]. Two genes involved in the
biosynthesis of wax esters in E. gracilis were recently identified and
characterized: a fatty acyl-CoA reductase (EgFAR) involved in the
conversion of acyl-CoAs to fatty alcohols, and a wax synthase
(EgWS) catalyzing the esterification of acyl-CoAs and fatty alcohols
to yield wax esters. Functional expression in S. cerevisiae confirmed
their activity and medium-chain substrate preference, and the wax
biosynthesis pathway was reconstituted by coexpressing both
genes.
3.3. Acyltransferases
TAG biosynthesis and assembly is a complex process involving
diverse enzymatic activities. Acyltransferases of the Kennedy
pathway [38] reside in the ER and catalyze a sequential transfer of
acyl groups from the acyl-CoA pool to the different positions of the
glycerol-3-phosphate backbone. These enzymes play a key role in
determining the acyl composition of glycerolipids and the final
content of TAG [39e41]. Candidate genes which putatively encode
acyltransferases of the Kennedy pathway can be found in the
genomic databases for the sequenced algal species. These
include the acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT),
the acyl-CoA:lysophosphatidic acyltransferase (LPAAT) and
the acyl-CoA:diacylglycerol acyltransferase (DGAT). In addition,
acyl-CoA-independent reactions can contribute significantly to
the production of TAG in some plant species. The activities of
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I. Khozin-Goldberg, Z. Cohen / Biochimie 93 (2011) 91e100
phospholipid:diacylglycerol acyltransferase (PDAT) [42] and
DAG:DAG transacylase (DGTA) utilize diacylglycerol (DAG),
a central lipid intermediate, for direct incorporation into the TAG
molecule. The candidate genes for PDAT are identifiable in some
algal genomes; as for DGTA, no gene candidates have been identified in either higher plants or algae. The TAG assembly enzymes
play an important role in establishing the fatty acid patterns of the
major polar membrane lipids and TAG, and therefore the genes that
encode them are potential targets in the metabolic engineering of
lipid-biosynthesis pathways in both higher plants and algae.
As far as algae are concerned, one GPAT and two DGATs were
cloned and characterized based on the genome information and
annotation of the diatom T. pseudonana and the chlorophyte O. tauri
[43e45]. Detailed analysis of candidate genes of C. reinhardtii is
currently underway [Q. Hu, personal communication]. Proteomic
analysis of the lipid body-enriched fraction of C. reinhardtii as well
as of the eyespot apparatus, which is composed of carotenoid-rich
lipid globules inside the chloroplast, revealed the presence of
several proteins with similarity to acytransferases [32,46].
Microsomal GPAT catalyzes the initial acylation at the sn-1
position of glycerol-3-phosphate and produces lysophosphatidic
acid in the Kennedy pathway. In mammals, four homologous isoforms of GPAT, each the product of a separate gene, catalyze this
reaction, two of them residing in the ER [39 and references therein].
All four isoforms are thought to be able to initiate glycerolipid
biosynthesis and at least three of them are able to increase the
incorporation of fatty acids into TAG. There are several indications of
GPAT3’s high importance for TAG formation: GPAT3’s mRNA
expression increases 60-fold in adipocytes after differentiation,
indicating a key role in TAG formation [39,47]. In Arabidopsis thaliana, a family of eight related genes encodes GPAT proteins that are
similar in size and deduced amino acid sequence [48], and that
contain four conserved amino acid motifs characteristic of acyltransferases [49]. None of these isoforms were implicated in the
biosynthesis of storage TAG. However, a new ER-localized GPAT
isoform from A. thaliana was recently identified, and designated
GPAT9 [48]; the deduced protein was shown to be most similar to
the mammalian GPAT3 inferred to have role in TAG biosynthesis
[47]. Both human GPAT3 and A. thaliana GPAT9 contain an additional
fifth conserved motif. It is suggested that in plants GPAT may not
exhibit a strong substrate preference and acyl-CoA substrate availability determines the acyl composition of sn-1 position of TAG [41].
Due to the limited information on algal acyltransferases, in this
section we provide a detailed description of the pertinent research
efforts (Table 1). A candidate GPAT of T. pseudonana was identified
by searching the genome for DNA sequences with similarity to the
yeast GPATs (Gat1 and Gat2) [43]. The amino acid sequence of GPAT
of T. pseudonana showed 24% and 23% identity to yeast GPATs and
little homology to bacterial, mammalian or Arabidopsis GPATs.
Topology analysis predicted five transmembrane domains located
in both the N and C termini of the deduced protein and separated by
a stretch of more than 400 amino acids, including four catalytically
important domains common in acyltransferases. To provide
evidence for the predicted function, TpGPAT was expressed in
a yeast GPAT-deficient mutant (gat1), and a GPAT assay using [14C]
glycerol-3-phosphate as the acyl acceptor and palmytoyl-CoA as
the acyl donor was performed. This resulted in restoration of GPAT
function in the mutant, evidenced by a sevenfold increase in GPAT
activity relative to the control transformed with empty vector.
A similar approach was used to characterize an activity encoded by
a putative GPAT of C. reinhardtii (CrGPAT): its overexpression in
a gat1 mutant increased TAG and phosphoinositol content [Q. Hu,
personal communication]. Moreover, the gene’s expression level
was upregulated under stress conditions, high light or nitrogen
starvation, inducing TAG production, indicating CrGPAT’s contribution to TAG biosynthesis in C. reinhardtii.
In vitro, TpGPAT could accept a range of tested acyl-CoA
substrates, with the preference for 16:0-CoA. TpGPAT did not utilize
14:0-CoA or 16:1-CoA; however, it should be noted that substantial
activity was also measured for 20:4-CoA, and lower levels determined for 18:0-, 18:1- and 22:6-CoAs. Fatty acid composition of the
transformed yeast also indicated TpGPAT’s preference for 16:0:
a significant increase in the share of 16:0 was determined in
phospholipids and TAG with a concomitant decrease in the
proportions of monounsaturated 16:1 and 18:1. The fatty acid
composition of T. pseudonana is characterized by a predominance
of 16:0, 16:1 and the u3 LC-PUFA EPA (20:5u3); C18 fatty acids are
present at low levels. Almost 60% of the molecular species of TAG in
the starved T. pseudonana contain 16:0 in combination with 16:1
and 14:0 [50]. This alga also contains some DHA and is capable of
partitioning u3 LC-PUFA (EPA and DHA) into TAG in the stationary
growth phase [51]. Indeed, these LC-PUFA were shown to be major
components of the higher molecular weight molecular species of
TAG detected in T. pseudonana cells that had been nitrogen- or
Table 1
Updated available information on algal acyltransferases involved in phospholipid and TAG biosynthesis.
Gene
Algal species
Functional characterization
(host species)
Validation parameter
Ref.
GPAT
T. pseudonana
C. reinhardtii
S. cerevisiae (gat 1 mutant)
S. cerevisiae (gat 1 mutant)
Increase in GPAT activity in yeast
Increased TAG contents in yeast
Increased transcript level in alga
Increased transcript level in correlation
with increased TAG content in alga
Extraplastidic localization (Arabidopsis protoplasts)
[43]
[Hu]a
P. incisa
DGAT2
O. tauri
T. pseudonana
S. cerevisiae H1236
(neutral lipid-deficient
quadruple mutant)
S. cerevisiae H1236
C. reinhardtii
[Khozin-Goldberg]a
Restoration of TAG biosynthesis, oil body
formation in yeast
[44]
Restoration of TAG biosynthesis, oil body
formation in yeast
Increased transcript level in the starchless mutant
in correlation with increased TAG content
[45]
[Hu]a
PDAT
C. reinhardtii
P. pastoris
Transacylase activity in yeast
Increased transcript level in alga
[Hu]a
LPCAT
T. pseudonana
S. cerevisiae (lpcat mutant)
Restoration of activity and phenotype in yeast
[60]
a
Personal communication.
I. Khozin-Goldberg, Z. Cohen / Biochimie 93 (2011) 91e100
silica-starved [50]. The ability of TpGPAT to incorporate LC-PUFA
into phospholipids and TAG was tested in yeast-feeding experiments. The expression of TpGPAT did not affect the incorporation of
supplied EPA and DHA into either lipid group in comparison to
the empty-vector control. It was thus proposed that TpGPAT catalyzes the incorporation of 16:0 at the sn-1 position of glycerol3-phosphate and may be of value for the production of saturated
TAG in genetically modified algae that have inherently higher PUFA
content than needed for biodiesel production.
Extraplastidic LPAAT catalyzes sn-2 acylation of LPA and the
formation of phosphatidic acid, a key intermediate in the biosynthesis of both membrane phospholipids and storage TAG. The key
role of the seed-specific LPAAT in channeling unusual fatty acids to
the sn-2 position of TAG in certain species of oilseed plants is well
established [41 and references therein]. Moreover, increases in seed
oil content have been reported following expression of foreign
microsomal LPAATs; in A. thaliana, for example, expression of two
microsomal LPAAT isoforms of Brassica napus resulted in enhanced
oil content in the seeds [52]. Overexpression of yeast LPAAT
(SLC1-1) in A. thaliana and B. napus was shown to increase seed oil
content as well as alter the fatty acid composition, with higher
accumulation of very long-chain fatty acids [53]. It should be noted
that bioinformatics approaches have revealed the presence of
candidate LPAATs in several algal genomes; however, their functions and acyl specificities have yet to be elucidated.
DGAT catalyzes the final, committed step in TAG assemblydthe
final acylation of diacylglycerol. DGAT is regarded as a bottleneck in
TAG biosynthesis since it has the lowest activity among the Kennedy pathway enzymes [41]. Two major isoforms, DGAT1 and
DGAT2, mediate this step in various organisms, including
mammals, higher plants and fungi. The genes encoding the putative
DGAT are present in the Chlamydomonas genome, but their functional assignment needs further characterization. Three putative
DGAT candidates were identified in the genome of O. tauri, all of
which appeared to belong to the DGAT2 family [44]. O. tauri is able
to synthesize LC-PUFA by sequentially alternating desaturation and
elongation steps which have been partially characterized by
molecular tools [54,55]. The major u3 LC-PUFA DHA (22:6)
accounts for more than 25 mol% of the TAG in this microalga and is
significantly less abundant within phospholipids and glycolipids
(less than 5 mol%) [44], indicating its specific channeling to TAG.
Among three putative DGAT2-like gene sequences, OtDGAT2A and
OtDGAT2B were most similar to those DGTA2 of higher plants that
engage in the channeling of unusual fatty acids from phospholipids
to TAG and thus prevent their accumulation in membrane lipids
[56]. OtDGAT2C showed high homology to mammalian monoacylglycerol acyltransferases. All three candidate genes were
expressed in a yeast mutant lacking DGAT1 and PDAT. Importantly,
significant codon optimization was necessary to obtain a high level
of algal protein expression in the heterologous host; nevertheless,
two of the candidate genes, OtDGAT2A and OtDGAT2C, were
virtually not active in the yeast system, even after complete
codon optimization. Only expression of OtDGAT2B with a codonoptimized N terminus resulted in successful complementation of
the TAG-deficient mutant phenotype, as determined by the
appearance of a TAG spot on TLC plates. Moreover, the expression of
OtDGAT2B in the quadruple S. cerevisiae mutant H1246, which is
unable to produce neutral lipids, TAG or steryl esters [57], restored
oil-body formation. TAG production was also observed in enzymatic DGAT assays in homogenates of transformed yeast cells; this
activity did not discriminate between dioleoyl- and linoleoyl-CoA.
By feeding the recombinant yeast with various non-radioactive
fatty acids, it was also shown that OtDGAT2B accepts a wide array of
acyl substrates, ranging from saturated fatty acids to PUFA and
LC-PUFA, indiscriminately incorporating both u3 and u6 substrates
95
into TAG. Thus, at this point, it is not likely that OtDGAT2B is
responsible for the targeting of DHA to TAG. However, the results of
this work suggest the emerging role of an acyl-CoA-independent
pathway in this process. Research in our laboratory has shown that
both acyl-CoA-dependent and independent pathways are potentially involved in TAG formation in P. incisa [58]. Microsomes and oil
bodies of P. incisa were able to synthesize TAG when 1,2- [14C]
oleoyl-DAG or 1-stearoyl -2- [14C]arachidonoyl-DAG were supplied
as the sole acyl donors, indicating transacylase activity. DGAT with
lower specific activity was also observed in both cellular fractions.
Another example of the DGAT cloned from algae is described in
a patent application by Zou et al. [45]. The gene coding for T.
pseudonana DGAT2 was isolated and used to transform the
quadruple S. cerevisiae mutant H1246; the same strain, transformed
with DGATl of A. thaliana, served as a positive control. It was
reported that the recombinant TpDGAT2 could efficiently incorporate DHA into TAG. The importance of DGAT2 of C. reinhardtii in
the hyper-accumulation of TAG in a starchless mutant of this alga
was demonstrated by the increased transcription of the gene in the
mutant, relative to the wild type, under conditions that induce TAG
biosynthesis [Q. Hu, personal communication].
Another recent report underscoring the intensive search for
algal genes mediating TAG biosynthesis relates to a putative PDAT
from C. reinhardtii (CrPDAT). The annotated gene, with homology to
PDAT, phospholipid:sterol acyltransferase and lecithin:cholesterol
acyltransferase was cloned, and its truncated sequence was
expressed in the methylotrophic yeast Pichia pastoris [Q. Hu,
personal communication]. Multiple catalytic functions are suggested for the recombinant CrPDAT, including transacylation of
phospholipids and DAG, or of two molecules of DAG, and even
lipase activity. Further information on such multifunctional
enzymes is definitely of interest to the scientific community.
The identification of acyltransferases with a very strong
substrate preference for EPA or DHA is important in the genetic
engineering of higher plants and algae to produce desired LC-PUFA
[59]. Lysophosphatidylcholine acyltransferase (LPCAT) plays a critical role in remodeling fatty acid and phosphatidylcholine (PC)
pools and is important for the effective channeling of LC-PUFA to
TAG. This enzyme mediates bidirectional exchange between the PC
and acyl-CoA pools, and the remodeled fatty acyl chains in the form
of acyl-CoA or esterified at the sn-2 position of PC can be used for
TAG biosynthesis [for further information refer to 59]. LPCAT was
identified in the alga T. pseudonana by a homology search of the
genome with LPCAT from S. cerevisiae [60], and the deduced protein
of T. pseudonana showed 27% identity to the yeast LPCAT. To confirm
its function, a LPCAT-deficient yeast strain was transformed with
the coding region of TpLPCAT. As described in the patent application, LPCAT function was restored in the mutant, and its sensitivity
to 1-O-alkyl-sn-glycero-3-phosphocholine, which is characteristic
of this mutant, was reduced. The LPCAT activity assay with yeast
microsomal fractions using [14C]lyso-PC and a range of unlabeled
acyl-CoAs revealed that TpLPCAT is capable of incorporating a wide
range of saturated, monounsaturated acyl-CoA, as well as 22:6
(DHA)-CoA; it showed highest preference, however, for 18:1-CoA.
3.4. Acyl-CoA synthase
Long-chain acyl synthase (LACS) activate free fatty acids to
produce acyl-CoAs and play an important role in regulating the size,
composition and availability of the cytoplasmic acyl-CoA pool for
various enzymatic activities, including TAG biosynthesis pathway.
Eight putative LACS genes were identified by analysis of the T.
pseudonana genome [61]. One of these putative genes (TplascA)
was characterized in detail and appeared to encode LC-PUFAspecific acyl-CoA synthase [61]. The ORF was heterologously
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I. Khozin-Goldberg, Z. Cohen / Biochimie 93 (2011) 91e100
expressed in the yeast deletion strain (faa4D) featuring low background levels of acyl-CoA activation and, particularly, for the
LC-PUFA, 20:5u3 and 22:6u3. It was shown that the recombinant
protein was efficient in the activation of both u6 and u3 PUFA:
18:3u6, 20:4u6, as well as 18:4u3 and 20:5u3. It was thus suggested that the algal LC-PUFA synthase activity might be of use to
improve the flux of acyl intermediates through the LC-PUFA
biosynthetic pathway to ensure the accumulation of DHA in the
genetically modified plant storage lipids.
4. Membranal glycerolipids
The biosynthesis of membrane lipids in eukaryotic microalgae
often involves cooperation between the plastid and the extraplastidial compartment, with the participation of enzymes of the ER
and chloroplast envelope. The current knowledge on membrane
lipid biosynthesis in a model alga C. reinhardtii is summarized and
described in detail elsewhere [21e23]. Extraplastidial membranes
of C. reinhardtii do not contain PC, but they do contain the nonphosphorous betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), which has similar physico-chemical properties, as
a major membrane component [1,21e23]. A bioinformatics
approach identified a putative protein in the genome of C. reinhardtii, BTA1Cr, which is sufficient for the biosynthesis of DGTS. It
was predicted by its sequence similarity to two proteins, BtaARs and
BtaBRs, of the bacterium Rhodobacter sphaeroides, required for the
biosynthesis of DGTS [21]. BTA1Cr appeared to encode a bifunctional protein that demonstrates functional homology to the
bacterial orthologs. Heterologous expression of BTA1Cr alone led to
DGTS accumulation in Escherichia coli. Interestingly, BTA1Cr was
present as the second major protein in the purified lipid droplet
fraction of C. reinhardtii [32]. It was therefore suggested that DGTS
may have role in lipid-droplet formation similar to that of PC for the
oil bodies of other organisms. This assumption seems very likely
since DGTS, which possesses properties analogous to PC, replaces
the latter in the extraplastidial membranes of C. reinhardtii.
However, contamination in the oil-body preparations of the
microalgae can not be excluded.
Previously, two other enzymes important for phosphoethanolamine biosynthesis were cloned and characterized from C. reinhardtii: diacylglycerol:CDP-ethanolaminephosphotransferase (EPT)
and CTP:phosphoethanolamine cytidylyltransferase (ECT) [62,63].
A cDNA encoding EPT was cloned from C. reinhardtii and its function
was demonstrated by functional complementation in a yeast
mutant deficient in both cholinephosphotransferase and ethanolaminephosphotransferase. Importantly, the EPT of C. reinhardtii
was capable of synthesizing both PC and phosphoethanolamine;
yet PC is not present in C. reinhardtii cells. The deduced protein of
ECT of C. reinhardtii is longer than the same protein in yeast, rat or
humans due to 75 amino acids forming a hydrophobic N-terminal
extension. Analysis of ECT activity distribution in cellular fractions
indicated mitochondrial localization of the protein in Chlamydomonas cells in agreement with the presence of a predicted mitochondrial-targeting sequence. Thus, the subcellular localization of
ECT enzyme appeared to be different between mammals and
photosynthetic C. reinhardtii. The function of ECT was confirmed by
heterologous expression in E. coli which demonstrated the
production of CDP-ethanolamine from phosphoethanolamine and
CTP.
5. Desaturases and elongases
Due to the interest in metabolic engineering of oilseed plants for
the production of nutritionally valuable LC-PUFA [59], a number
of PUFA desaturases and elongases with various substrate and
regio-selectivities have been cloned from LC-PUFA-producing algal
species in the last decade [54,55,64e74]. Microalgae are primary
producers of LC-PUFA, and many algal species therefore possess the
machinery for sequentially alternating desaturation/elongation
steps. The numerous efforts made to clone and characterize desaturases and elongases involved in LC-PUFA biosynthesis are
described elsewhere, as well as their use in genetic modification of
oilseed plants to reconstitute LC-PUFA biosynthesis [59 and
references therein]. The main practical focus of these investigations is to provide a sustainable feedstock for human consumption
as an alternative to fish oils, whose supply is decreasing and quality
deteriorating. At the same time, such research has enabled an
elucidation of the constraints and bottlenecks in LC-PUFA production in heterologous organisms [for further information refer to 75].
Microalgal PUFA elongases are structurally similar to the ELO
family of enzymes that catalyze the condensation step of fatty acid
elongation in animals and fungi [67]. These elongases are different
from the higher plant condensing enzymes that participate in
microsomal elongation of saturated and monounsaturated fatty
acids. Three major types of PUFA elongases, regarding
substrate specificity, were characterized from LC-PUFA-producing
microalgae: D6 C18-PUFA-specific elongases were shown to be
involved in the elongation of C18 PUFA (18:3u6 and 18:4u3)
and the production of C20 LC-PUFA, ARA and EPA, while D5 C20PUFA-specific elongases are engaged in the elongation of 20:5u3
(EPA) in the pathway of DHA biosynthesis, in some marine species. In
an alternative route, C18-D9-specific elongation of 18:2u6 and
18:3u3 to the respective C20 intermediates precedes sequential D8
and D5 desaturations to form ARA and EPA, respectively. For functional characterization, these enzymes are expressed in yeast where
the recombinant proteins are suggested to accomplish a condensing
step in conjunction with host activities of ketoreduction, dehydration and enoyl reduction. For example, an elongase gene from the
marine haptophytes of the Pavlova family, being expressed in yeast,
catalyzed the conversion of EPA into DPA (22:5 u3) [66,70]. This
enzyme appeared to be specific towards both u6 and u3 C20-PUFA
substrates and did not show activity towards C18- or C22-PUFA
substrates. The consecutive activity of D4 desaturase acting on DPA is
necessary to accomplish DHA biosynthesis in microalgae, differently
from the D4 desaturase-independent Sprecher pathway [76] operating in mammals and fish. In the latter pathway, DPA is further
elongated to 24:5u3, followed by D6 desaturation to 24:6u3 and
chain shortening via b-oxidation to DHA in peroxisomes [67]. Several
genes encoding for D4 desaturases were cloned from DHAproducing microalgal species [65,66,69]. The PsD4Des gene of
P. salina was able to desaturate both 22:4D7,10,13,16 and 22:5D7,10,13,16,19
at the D4 position when expressed in yeast [66]. The D4 desaturase of
Euglena gracilis showed strict D4-regioselectivity and required the
presence of a D7-double bond in the substrate; the enzyme featured
a broad substrate specificity and activity was not limited to C22 PUFA
and also efficiently desaturated C16 PUFA such as 16:3 3D7,10,13 [65].
Some those candidate genes were used as a terminal activity to
reconstitute DHA biosynthesis in higher plants.
In some primitive marine eukaryotes of the Thraustochytriaceae
family, the alternative polyketide synthase pathway (PKS) catalyses
the production of LC-PUFA [77]. The PKS pathway does not require
aerobic desaturation while the double bonds are introduced during
the process of fatty acid synthesis. The PKS pathway is predominant
in Schizochytrium, whereas a desaturation/elongation pathway acts
in Thraustochytrium of the same family [78,79]. The homology
between the prokaryotic Shewanella and eukaryotic Schizochytrium
PKS genes suggested that the PUFA PKS pathway has undergone
lateral gene transfer [77].
Further, we provide a few examples of unusual algal desaturases
that are not involved in LC-PUFA biosynthesis and an update of the
I. Khozin-Goldberg, Z. Cohen / Biochimie 93 (2011) 91e100
latest developments in the cloning of acyl-CoA-dependent desaturases from microalgae. An unusual front-end desaturase (containing a cytochrome b5-like domain at its N terminus was cloned
from the marine diatom T. pseudonana based on the set of genomic
DNA sequences putatively encoding front-end desaturases in this
alga [80]. The deduced amino acid sequence of TpDESN displayed
typical features of microsomal desaturases involved in the
biosynthesis of LC-PUFA, i.e. a fused N-terminal cytochrome b5
domain and three conserved histidine-rich motifs. However, this
desaturase was specifically active on saturated 16:0, desaturating it
to 16:1D11 when expressed in yeast, but was not active on supplemented PUFA. Interestingly, this D11-desaturase activity had been
previously detected only in insect cells. The identification of such
a novel enzyme in photosynthetic organisms expands the functional repertoire of the membrane-bound algal desaturases.
Another interesting case is a FAD6 from the diatom P. tricornutum [81]. This alga is widely used in aquaculture due to its content
of u3 eicosapentaenoic acid (EPA; 20:5D5,8,11,14,17). The pathways of
EPA biosynthesis have been previously described and genes
involved in EPA biosynthesis have been cloned and characterized
[64]. The chloroplastic lipids of this alga also contain an unusual
isomer of u6 hexadecatrienoic acid (16:3D6,9,12), rather than the u3
hexadecatrienoic acid (16:3D7,10,13) and a-linolenic acid
(18:3D9,12,15) which are common in chloroplast membrane lipids of
higher plants. The gene encoding PtFAD6 was cloned and expressed
in a strain of the cyanobacterium Synechococcus, which is characterized by a simple fatty acid composition and the absence of diand trienoic fatty acids. The heterologous expression of PtFAD6 in
the photosynthetic organism resulted in the production of 16:2D6,9,
in support of the suggestion that it encodes a plastidial enzyme
which requires ferredoxin/ferredoxin oxidoreductase as electron
donors. In contrast, the microsomal desaturase PtFAD2 was shown
to be active when expressed in yeast and specific for oleic 18:1D9
acid, indicating that PtFAD2 requires cytochrome b5/cytochrome
b5 oxidoreductase and is likely involved in the biosynthesis of EPA
in the ER. PtFAD6 appeared to have a longer N-terminal extension
than the protein encoding a plastidial FAD6 desaturase from Brassica napus. The N terminus of PtFAD6 contains two domains: one is
basic with an Arg in the third position and contains a hydrophobic
portion similar to a classical ER-targeting signal. This domain is
followed by a domain rich in hydroxylated amino acids (Ser and
Thr), which is typical for chloroplast transit peptides involved in
transport into the complex plastids of diatoms, surrounded by four
membranes. The plastid origin of PtFAD6 was confirmed when the
N-terminal 113 amino acids were fused with EGFP (enhanced green
fluorescent protein) and expressed in P. tricornutum cells.
Elucidation of algal genome sequences has accelerated the
identification of PUFA desaturases and their functional characterization in yeast and higher plants. Recently, it was shown that some
algal front-end D6 and D5 desaturases act on PUFA esterified to
CoA, similar to mammalian front-end desaturases. Currently, the
examples include D6 desaturases from the microalgae O. tauri [54],
O. lucimarinus [71] and M. pusilla [74], and D6 and D5 desaturases
from Mantoniella squamata [55]. The acyl-CoA-dependent enzymes
insert a double bond into CoA-activated fatty acid from the
extraplastidial acyl-CoA pool; this is distinctly different
from phospholipid-linked desaturases, which require a fatty acid
to be attached to PC or phosphoethanolamine. This feature
is of great importance considering current intensive use of acylCoA-dependent desaturases in research on genetic modification of
oilseed plants. The use of the acyl-CoA-dependent enzymes with
the correct substrate specificities may eliminate the requirement
for the rate-limiting acyl-exchange with PC and thus
avoid “substrate dichotomy” for lipid-linked desaturases and acylCoA-dependent elongases [for further information refer to 59,75].
97
Moreover, most of the recently cloned algal acyl-CoA-dependent
desaturases are highly specific for their u3 substrates when
expressed in yeast [55,74], apart from the D6 desaturase of the alga
O. tauri which desaturated u6 and u3 substrates with similar efficiency [54]. The strong and beneficial preference for the u3
substrate was utilized in research aiming to the metabolic engineering of u3 LC-PUFA (EPA and DHA) biosynthesis in plants
[55,74]. The D6 desaturase from the marine microalga M. pusilla
was shown to be very efficient in heterologous systems: this
enzyme displayed 3.5-fold higher conversion efficiency for the u3
substrate a-linolenic acid (18:3D9,12,15) over the u6 substrate linolenic acid (18:2D9,12) [74]. The preference for u3 substrate was also
confirmed in a competition experiment employing both substrates.
This enzyme was used for metabolic engineering of an EPAbiosynthesis pathway in planta: it was coexpressed with two more
algal enzymes, the D6 elongase of the microalga Pyramimonas
cordata and the D5 desaturase of the haptophyte Pavlova salina
(Fig. 2). This was performed by simultaneous expression of single
genes in Cauliflower mosaic virus (35S CMV)-driven constructs in
tobacco (Nicotiana benthamiana) leaf tissue in the presence of the
P19 viral suppressor and DGAT1 of A. thaliana to direct produced
fatty acids into TAG. The novel leaf-based assay [82] was developed
for the rapid transient expression of multistep pathways to identify
effective gene combinations for the production of desired VLCPUFA and indeed, it resulted in 26% EPA in the leaf TAG [74]. In
addition, preference for u3 was also observed in stable Arabidopsis
transformations with an u3/u6 ratio as high as 4.5 in T2 seeds. This
pathway was further extended to the production of DHA by the
addition of the D5 C20 PUFA elongase and the D4 desaturase [83]
(Fig. 2). Earlier study by the same group demonstrated the
production of u6 and u3 LC-PUFA, ARA, EPA and DHA from
endogenous tobacco linoleic and a linolenic acid by five transgeneencoded reactions [82]. The recombinant u6 and u3 pathways
were initiated with the action of Isochrysis galbana D9 elongase on
18:3
9,12,15
(ALA)
6Des Micromonas pusilla
18:4
6, 9,12,15
(SDA)
6 PUFA elongase Pyramimonas cordata
20:4
8,11,14,17
(ETA)
5Des Pavlova salina
20:5
5,8,11,14,17
(EPA)
5 PUFA elongase Pyramimonas cordata
22:5
7,10,13,16,19
(DPA)
4Des Pavlova salina
22:6
4,7,10,13,16,19
(DHA)
Fig. 2. Transient reconstitution of the EPA- and DHA-biosynthesis pathway, consisting
of algal enzymes, in tobacco leaf [74]. The pathway initiates with the action of M.
pussila D6 desaturase with a u3 preference on the endogenous ALA of tobacco leaves
[82,83]. ALA, a-linolenic acid; SDA, stearidonic acid; ETA, eicosatetraenoic acid; EPA,
eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid.
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I. Khozin-Goldberg, Z. Cohen / Biochimie 93 (2011) 91e100
the endogenous fatty acids of tobacco leaves. The algal enzymes
used in that study were the I. galbana D9 elongase, which elongates
18:2D9,12 to 20:2D11,14 and 18:3D9,12,15 to 20:3D11,14,17 in the so-called
alternative pathway, and four sequential enzymes engaged in
the sequential processing of 20:2D11,14 and 20:3D11,14,17, D8 desaturase, D5 desaturase, D5 elongase and D4 desaturase of P. salina.
Analysis of leaf TAG demonstrated that it contained 37% newly
synthesized LC-PUFA, including 7% ARA, 6% EPA and 3% DHA.
The focus of current research into modifying the fatty acid
composition of seed oils consists of the search for novel highly
efficient enzymes from low eukaryotes, including algae, the search
for efficient means to avoid bottlenecks in LC-PUFA production and
for channeling them towards TAG assembly [59]. The similar
approaches are applicable to the genetic modification of microalgae
for a desired LC-PUFA composition.
6. Microsomal elongation of saturated and
monounsaturated fatty acids
Elongation of saturated and monounsaturated fatty acids
(mainly 16:0 and 18:1) to very-long-chain C20 and C22 fatty acids
(VLC-FAs) in higher plants occurs in the ER and is mediated by the
fatty acid elongase complex. Fatty acid elongation involves the
activities of four consecutive enzymatic reactions (condensation,
reduction, dehydration, and a second reduction) and results in
a two-carbon extension per cycle. In higher plants, microsomal
elongation activity is important for the production of long-chain
saturated and monounsaturated fatty acids (LC-FA), which are
common in the cuticle, surface waxes and TAG of some oilseed
plants. The first enzyme of the fatty acid elongase complex, bketoacyl-coenzyme synthase (KCS), catalyzes the rate-limiting,
condensation reaction of the acyl primer with malonyl-CoA [84].
The other three components of the elongase complex (the 3ketoacyl-CoA reductase, the 3-hydroxyacyl-CoA dehydratase, the
enoyl-CoA reductase) were identified and characterized in yeast
and A. thaliana [26 and reference therein]. The activities of Arabidopsis genes were confirmed by means of a reverse-genetics
approach utilizing yeast deletion mutants deficient in each of the
activities.
In A. thaliana the KCS gene family, consisting of 21 members,
was discovered by genome annotation [85]. Several of these
enzymes were shown to be functional, as they were expressed in
yeast [86] and revealed distinct patterns of substrate selectivity and
product chain length, as well as differential sensitivity to oxyacetamide and chloroacetanilide herbicides [87]. To date, relatively
little is known about the genes and enzymes mediating microsomal
elongation of saturated and monounsaturated fatty acids in
microalgae. Some species of green algae demonstrate very high
sensitivity to herbicides targeting KCS. Severe inhibition of radiolabeled 18:1 incorporation into the non-soluble “non-lipid” fraction
occurred in some green algal species as a result of administration of
chloroacetamide [84]. This fraction appeared to contain sporopollenin, an acid-resistant complex material of the cell wall in green
algae. It was further proposed that oleic acid serves as a precursor
of sporopollenin and that KCS activity is important for its formation
[88,89]. In addition, effects of chloroacetamides on fatty acid
composition of membrane lipids were also demonstrated.
Furthermore, it was suggested that C18 fatty acids are produced
by KCS-mediated elongation of 16:0-CoA in the ER of the marine
haptophyte Pavlova lutherii. The elongation of 16:0-CoA was
implicated as a key intermediate step in the biosynthesis of the
LC-PUFA EPA and DHA [90]. Immunodetection analysis with antibody raised against a fusion protein containing the central portion
of the KCS of Brassica napus showed that a KCS-like enzyme
is present in the microsomes of the alga. The activity of the
condensing enzyme assayed in the microsomal fraction of the alga
preferred 16:0-CoA over shorter, longer or monounsaturated acyl
substrates. The gene Plelo1 (Pavlova lutheri elongase 1) was cloned
and its deduced protein showed strong similarities to higher plant
KCS. It was further suggested that the highly active elongation
reaction provides additional substrate (18:0-CoA) for further
desaturation and consequently, for VLC-PUFA biosynthesis. Several
lines of evidence supported this assumption: the abundance of
16:0-CoA in the acyl-CoA pool, and the increased transcript abundance of Plelo1 in the stationary phase which was associated with
a high level of microsomal elongase activity. However, no data on
heterologous expression have yet been provided.
The salt-inducible KCS was cloned from the halotolerant green
microalga Dunaliella salina exposed to 3.5 M NaCl [91]. Similar to
the KCS activity of P. lutheri, that of D. salina favored saturated
medium-chain acyl-CoA, with a preference for 16:0-CoA over the
monounsaturated 16:1- and C18:1-CoAs. The function of the
D. salina KCS gene product was demonstrated by the presence of
condensing activity in the recombinant E. coli. It was suggested that
the C16 elongation activity in conjunction with further desaturation steps is important for the adaptive membrane changes
required for proper membrane function at high internal glycerol
concentrations [91]. The putative KCS that was presumably
involved in microsomal elongation of medium-chain fatty
acids was also isolated from the green microalga P. incisa (KhozinGoldberg, unpublished). Homologs of higher plant KCS can also be
found in the genome of C. reinhardtii and of several diatoms. It
should be noted that KCS genes of green algae code for deduced
proteins with an unusual N-extension upstream of the two transmembrane regions, typical of higher plants’ microsomal KCS.
7. Conclusions
In conclusion, our knowledge on lipid metabolism at the
molecular level in eukaryotic microalgae is largely incomplete.
Genes encoding lipid-biosynthesis enzymes are present in various
algal genomes. However, their biochemical analysis is essential to
verify their function. We believe that in the near future, research
into lipid metabolism of microalgae will advance rapidly, thanks to
the development of indispensable molecular tools for algal gene
characterization.
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