Fatty acid export (FAX) proteins contribute to oil production in the green microalga Chlamydomonas reinhardtii

Abstract

In algae and land plants, transport of fatty acids (FAs) from their site of synthesis in the plastid stroma to the endoplasmic reticulum (ER) for assembly into acyl lipids is crucial for cellular lipid homeostasis, including the biosynthesis of triacylglycerol (TAG) for energy storage. In the unicellular green alga Chlamydomonas reinhardtii, understanding and engineering of these processes is of particular interest for microalga-based biofuel and biomaterial production. Whereas in the model plant Arabidopsis thaliana, FAX (fatty acid export) proteins have been associated with a function in plastid FA-export and hence TAG synthesis in the ER, the knowledge on the function and subcellular localization of this protein family in Chlamydomonas is still scarce. Among the four FAX proteins encoded in the Chlamydomonas genome, we found Cr-FAX1 and Cr-FAX5 to be involved in TAG production by functioning in chloroplast and ER membranes, respectively. By in situ immunolocalization, we show that Cr-FAX1 inserts into the chloroplast envelope, while Cr-FAX5 is located in ER membranes. Severe reduction of Cr-FAX1 or Cr-FAX5 proteins by an artificial microRNA approach results in a strong decrease of the TAG content in the mutant strains. Further, overexpression of chloroplast Cr-FAX1, but not of ER-intrinsic Cr-FAX5, doubled the content of TAG in Chlamydomonas cells. We therefore propose that Cr-FAX1 in chloroplast envelopes and Cr-FAX5 in ER membranes represent a basic set of FAX proteins to ensure shuttling of FAs from chloroplasts to the ER and are crucial for oil production in Chlamydomonas.

Plastid fatty acid export (FAX) proteins inArabidopsis thaliana- the role of FAX1 and FAX3 in growth and development

“…Relatives to At-FAX4 could be detected in dicots, monocots, Selaginella moellendorfii (spikemoss) and Physcomitrium, but not in Chlamydomonas (Supplementary Fig. S5; Peter et al [2022]).…”

“…In the green microalga we further could show that Cr-FAX1 in the chloroplast envelope and Cr-FAX5 in the ER membrane indeed function in the same metabolic pathway -i.e. in production of the neutral storage lipid TAG (Peter et al, 2022). Further, all FAX1 proteins in plants are characterized by a prominent stretch of two glycines, one asparagine, followed by two to three prolines and two to three lysine residues (GGNPPKK-motif) after helix four (Fig.…”

Co-Expression of Lipid Transporters Simultaneously Enhances Oil and Starch Accumulation in the Green Microalga Chlamydomonas reinhardtii under Nitrogen Starvation

“…However, FA biosynthesis and incorporation of FA into TAG occur mainly within the chloroplast and the endoplasmic reticulum (ER), respectively [ 13 , 14 ]. During the consecutive FA transport from chloroplast to ER, FA exporters (FAX1 and FAX2) and ATP binding cassette (ABC) transporters of subfamily A (ABCA2) were found to play critical roles in the lipid metabolism of Chlamydomonas [ 15 , 16 , 17 ]. FAX1 is plastid-located, while FAX2 (also named Cr-FAX5) is targeted to ER.…”

“…ABCA2 is an ER-located lipid transporter and is involved in lipid accumulation in Chlamydomonas [ 15 ]. The single over-expressor (FAX1- or ABCA2-OE) showed an increase in oil content [ 15 , 17 ]. However, in the strains with increased levels of FAX2 only, the TAG content was not improved [ 17 ].…”

“…The single over-expressor (FAX1- or ABCA2-OE) showed an increase in oil content [ 15 , 17 ]. However, in the strains with increased levels of FAX2 only, the TAG content was not improved [ 17 ]. Whether the TAG accumulation in these single over-expressors is limited by the functional diversity or spatiotemporal expression regulation of these lipid transporters remains unknown.…”