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.