Fatty acid transport in
microalgae
Export of fatty acids in
microalgae
Microalgae release fatty acids to the extracellular medium in their
natural habitat, proposedly as allelopathic compounds (Allen, Ten-hage,
& Leflaive, 2018; Sushchik, Kalacheva, Zhila, Gladyshev, & Volova,
2003). Although allelopathic compounds can be have a wide chemical
diversity, fatty acid and derivatives are common in water ecosystems and
their production is increased under conditions that do not allow for
optimal growth (such as nitrogen or phosphate limitation) but allow for
efficient photosynthesis (Allen et al., 2018). Microalgae fatty acid
production has been linked to allelopathic effects on competitor
organisms due to the capacity of certain fatty acids to alter membrane
permeability (J. T. Wu, Chiang, Huang, & Jane, 2006). For example, the
green algae Uronema confervicolum can secrete 1.45 μg/L of free
fatty acids, 77% of which correspond to linoileic and linolenic acid
(Allen et al., 2018). In the same study, these fatty acids were observed
to inhibit growth of the diatom Fistulifera saprophila , although
at higher concentrations that those produced by U. confervicolum .
The green alga Chlorella vulgaris has also been observed to
produce fatty acids at a concentration of 0.85
mg/L/106 cells under phosphate limiting conditions
(DellaGreca et al., 2010). The mixture of fatty acids changes
drastically when comparing two scientific reports and is reflected in
the production of palmitic acid, which can be absent from the fatty acid
mixture or be the most abundant depending on the growth conditions,
possibly due to the mode of CO2 supply or different
medium compositions (DellaGreca et al., 2010; Sushchik et al., 2003).
The fatty acid mixture produced by C. vulgaris has been observed
to be toxic to the alga Raphidocelis subcapitata , resulting in
the extinction of this alga when grown in a coculture with C.
vulgaris (DellaGreca et al., 2010). While microalgae are
observed to secrete fatty acids, no uptake has been described, probably
due to the autotropic nature of microalgae.
Fatty acid secretion in microalgae can be increased by means of genetic
engineering. Knockdown of long-chain acyl-CoA synthetase genes cracs1
and cracs2 in Chlamydomonas reinhardtii cc849 (a cell wall
deficient strain) increased extracellular fatty acid production from
2.93 mg/109 cells to 8.19 mg/109cells and 9.66 mg/109 cells, respectively (Jia et al.,
2016). Overexpression of transcription factor NobZIP1 inNannochloropsis oceanica increased the extracellular fatty acid
content by 40%(D. Li et al., 2019). The study of the effects of
NobZIP1 revealed that one of the negatively regulated targets,
UDP-glucose dehydrogenase, an enzyme involved in cell wall polymer
metabolism, is linked to lipid metabolism. Silencing this enzyme through
interference RNA increased the extracellular fatty acid content by 20%
(D. Li et al., 2019). Although the secretion of fatty acids in algae has
been widely studied, no export system has been identified to date.
Intracellular trafficking of fatty acids in
microalgae
Microalgae, analogous to plants, synthesize fatty acids in plastids.
However, synthesis of triacylglycerol and other lipids takes place in
the endoplasmic reticulum and therefore the newly synthesized fatty
acids must be export from the plastid to the cytosol. A plastid fatty
acid exporter acting on free fatty acids, AtFAX1, has been identified
and studied in Arabidopsis thaliana (N. Li et al., 2015).Orthologues of this protein can be found in microalgae, and their
function has been studied in some of them. The green-alga modelChlamydomonas reinhardtii contains two orthologues, CrFAX1 and
CrFAX2, whose overexpression respectively increased neutral lipid
content 15% and 17% under nitrogen limiting conditions (N. Li et al.,
2019). Overexpression of these genes produced more and larger lipid
droplets, increasing the content of intracellular triacylglycerol by
38%. The fatty acid composition did not show significant variation,
showing that these transporters are involved in the transport of both
saturated and unsaturated fatty acids. A fatty acid plastid exporter,
CmFAX1, has also been identified in an extremophilic red microalgae,Cyanidioschyzon merolae , that inhabits sulfuric acid hot springs
(Takemura, Imamura, & Tanaka, 2019). This exporter was confirmed to be
located in the plastid membrane through targeted immunofluorescence and
its fatty acid transport activity was verified through complementation
experiments in yeast cells lacking the FAT1 transporter. Deletion of
CmFAX1 in C. merolae increased free fatty acid content in the
total cell extract 2.5-fold and CmFAX1 overexpression increased lipid
droplet formation 2.4-fold. In contrast to the CrFAX transporters, the
overexpression of CmFAX1 introduced significant changes in the fatty
acid composition of the triacylglycerol molecules, incorporating C14:0,
C14:1 and C20:0 fatty acids and increasing the content of C18:2
(Takemura et al., 2019).
After the free fatty acids have abandoned the plastid, they are
incorporated into lipid droplets in the form of glycerolipids. For this,
they need to be activated to acyl-CoA by a long-chain acyl-CoA
synthetase. C. reinhardtii encodes three putative long-chain
acyl-CoA synthetase and two of them were found to be associated to lipid
droplets in a proteomic study (Nguyen et al., 2011). The deletion of one
of them, LCS2, led to a 2-fold increase in triacylglycerol rich in
polyunsaturated fatty acids (e.g. 52:10 or 54:9) showing that this
enzyme is mainly associated to the activation of saturated fatty acids
produced de novo in the plastids (X. Li et al., 2015).
Once the growth conditions change and the lipid reserves are needed for
survival and growth, fatty acids get mobilised from lipid droplets by
lipases and long-chain acyl-CoA synthetases. The resulting acyl-CoA
molecules must be incorporated into peroxisomes for their degradation
(Kong et al., 2017; Figure 6). This is speculated to happen in similar
way to plants and yeast, via a transporter with thioesterase activity
and the later participation of a peroxisomal long-chain acyl-CoA
synthetase. While there have been no studies to date on microalgae to
support this hypothesis, C. reinhardtii possesses a putative
fatty acid peroxisomal transporter, ABCD1 (Kong, Romero, Warakanont, &
Li-Beisson, 2018). An overview of the intracellular trafficking of fatty
acids and acyl-CoA molecules in C. reinhardtii can be observed in
Figure 6.