Fatty acid transport in yeast

Uptake of fatty acids in yeast

Fatty acid import in yeast follows the same principle of vectorial acylation described for E. coli. In Saccharomyces cerevisiae, the import system is composed of Fat1 and Faa1 or Faa4. Mutations in these genes show hampered growth under fatty acid auxotrophic conditions (Black & Dirusso, 2003). Although S. cerevisiae cannot use fatty acids as growth substrate, it needs to incorporate exogenous fatty acids in case of inhibition of fatty acid synthesis by cerulenin or during anaerobic growth as desaturases require molecular oxygen to produce unsaturated fatty acids.
Fat1 is an ortholog of the mammal fatty acid transport proteins identified in murine species (Nils J. Færgeman, DiRusso, Elberger, Knudsen, & Black, 1997), and it has been described to be involved both in the import of fatty acids and in the activation of very-long-chain fatty acids (Zou, Dirusso, Ctrnacta, & Black, 2002). Fat1 is an integral membrane protein with two transmembrane domains (Obermeyer, Fraisl, DiRusso, & Black, 2007; Figure 4). The conserved ATP/AMP binding region characteristic of acyl-CoA synthetases is separated by a portion of the protein that is inserted into the membrane. The intracellular C-terminus contains a region conserved among other fatty acid transport proteins with very-long-chain acyl-CoA synthetase activity (VLACS). Finally, the soluble Faa1 has been observed to interact with the C-terminus of this protein to activate the imported fatty acids. The transport mechanism of Fat1 is unknown but several residues from Fat1 have been mutated to study their effects (Zou et al., 2002). These experiments have shown that although most residues affect both transport and acyl-CoA synthetase activity, the mutation of certain residues separates these two activities, suggesting that they follow different mechanisms. On one hand, F528A and L669R mutations abolish transport function, but retain some acyl-CoA synthetase activity. On the other hand, S258A and D508A mutations abolish acyl-CoA synthetase activity while retaining some transport activity. Fat1 has been proposed to be situated not only in the plasma membrane but also in lipid bodies, endoplasmic reticulum and peroxisomes (Van Roermund et al., 2012).
S. cerevisiae contains two main acyl-CoA synthetases for long-chain fatty acids, Faa1 and Faa4. These proteins interact with Fat1 to form a complex that combines transport and activation of fatty acids. Faa1 is responsible for most of the acyl-CoA synthetase activity observed in S. cerevisiae and it has been observed to interact with the carboxy terminal of Fat1 as described in several studies, including yeast two-hybrid experiments (Zou et al., 2003). Although the activity of Faa4 is lower than Faa4, it is the only acyl-CoA synthetase gene that can rescue fatty acid import activity in a ΔFaa1 mutant, suggesting that its mode of action is identical (Johnson, Knoll, Levin, & Gordon, 1994). Next to vectorial acylation, endocytosis plays a significant role in the uptake of exogenous fatty acids (Jacquier & Schneiter, 2010). The deletion of Ypk1, a protein-kinase involved in endocytosis was found to affect fatty acid import by reducing it by half compared to the wild-type. These results led to investigate the involvement of other proteins associated to endocytosis End3, Vrp1 and Srv2, whose deletion also hampered fatty acid import at the same extent. The involvement of all these genes stresses the importance of endocytosis for fatty acid import, probably due to the internalization of fatty acid-rich membrane domains.
Besides S. cerevisiae, the uptake of exogenous fatty has been studied in other yeasts. Cryptococcus neoformans is an important fungal pathogen that infects alveolar macrophages and it is responsible for increasing deaths in immunosuppressed individuals. This yeast has been observed to import exogenous fatty acids for the formation of lipid droplets; and the presence of oleic acid stimulates its replication, both in extracellular form and during macrophage infection (Nolan, Fu, Coppens, & Casadevall, 2017). However, no molecular mechanism or components has been described for this process. The acquisition of exogenous fatty acids in Candida albicans, another important fungal pathogen, has been studied at the molecular level. CaFaa4, the ortholog gene for Faa4 and Faa1 from S. cerevisiae, was characterized and observed to be essential for fatty acid import (Tejima, Ishiai, Murayama, Iwatani, & Kajiwara, 2018). Note that in contrast to S. cerevisiae , in C. albicans only one Faa gene seems to be involved. The same holds true for Y. lipolytica. Moreover, the mechanism behind fatty acid transport is not conserved across yeasts. Although Y. lipolytica possesses an ortholog of Fat1 from S. cerevisiae, this protein is not associated to fatty acid import and it has been suggested to be involved in fatty acid export from lipid bodies (R. Dulermo, Gamboa-Meléndez, Dulermo, Thevenieau, & Nicaud, 2014). Furthermore, the acyl-CoA synthetase fromY. lipolytica (YlFaa1), while being the only gene involved in fatty acid activation, it is not essential for growth on fatty acids (R. Dulermo, Gamboa-meléndez, & Ledesma-amaro, 2015).

Intracellular trafficking of fatty acids in yeast

Fatty acids must be activated to acyl-CoA before they can enter a specific metabolic pathway. As described in the previous section, exogenous fatty acids are imported and converted to acyl-CoA nearly simultaneously. Acyl-CoA can also be derived from de novosynthesised fatty acids and from fatty acids contained in the neutral lipids stored in lipid bodies. The joint action of lipases and acyl-CoA synthetases, such as the Fat1 from S. cerevisiae and its ortholog in Y. lipolytica, leads to the mobilisation of fatty acids from lipid bodies (T. Dulermo, Thevenieau, & Nicaud, 2014). Acyl-CoA molecules must reach the destination inside the cell where they will be degraded, stored or used to build other molecules (DiRusso & Black, 1999). The intracellular trafficking routes for fatty acids and acyl-CoA molecules are shown in Figure 5. In S. cerevisiae , this intracellular transport is facilitated by an acyl-CoA binding protein coded by the gene acb1 . Although it has been observed that this protein facilitates transport of acyl-CoA to lipid bodies for the formation of triacylglycerol, it is not necessary for the survival of the cell (Schjerling et al., 1996). Therefore, either there are other transport mechanisms, or they are not needed for the diffusion of acyl-CoA molecules across the cytosol.
In yeast, fatty acids are catabolized by β-oxidation in peroxisomes. The transport of fatty acids in the peroxisome of S. cerevisiae has been studied, revealing the involvement of several proteins. Free fatty acids, mainly medium-chain fatty acids, can be imported into the peroxisome by passive diffusion or an unidentified system (Hettema et al., 1996). Long-chain fatty acids, in the form of acyl-CoA, are transported into the peroxisome through the heterodimeric transporter formed by Pxa1 and Pxa2 (Shani, Sapag, Watkins, & Valle, 1996). It has been proposed that the Pxa1/Pxa2 transporter would cleave the fatty acyl-CoA prior to transport and it would introduce only the free fatty acid portion into the peroxisome (Van Roermund et al., 2012). This mechanism prevents accumulation of CoA in the peroxisome and it has already been described in plants (Fulda, Schnurr, Abbadi, Heinz, & Browse, 2004). Fatty acids transported by Pxa1/Pxa2 need to be activated by re-conversion to acyl-CoA to enter the β-oxidation cycle. Two acyl-CoA synthetases have been associated to the peroxisomal activation of fatty acids: Fat1 and Faa2. Substitution of the Pxa1/Pxa2 transporter with the human orthologue showed that Fat1 must interact with the yeast Pxa1/Pxa2 transporter to be active (Van Roermund et al., 2012). While Fat1 is also found in other regions of the cell, such as the plasma membrane, Faa2 is found exclusively in peroxisomes. Faa2 accepts a wide range of fatty acids as substrate, but it has a preference for medium-chain fatty acids (Knoll, Johnson, & Gordon, 1994). The general model of peroxisomal fatty acid transport can be observed in Figure 5. The peroxisomal transport of fatty acids in Y. lipolytica has also been studied (R. Dulermo et al., 2015). The overall system seems similar to that of S. cerevisiae , with distinct routes for long and medium-chain fatty acids. Both Pxa1/Pxa2 and Fat1 are involved in the peroxisomal transport. However, Y. lipolytica lacks the Faa2 protein for the activation of medium-chain fatty acids, and it has been proposed that a coumarate ligase-like protein might fulfil this role instead (R. Dulermo et al., 2015).

Export of fatty acids in yeast

Just as for prokaryotic organisms, secretion of free fatty acids is no common natural process in yeasts or fungi. Yet, upon the engineered intracellular accumulation of free fatty acids, yeasts are able to secrete them to the extracellular medium (Arhar & Natter, 2019; Scharnewski, Pongdontri, Mora, Hoppert, & Fulda, 2008). Some proteins involved in the export of fatty acids in S. cerevisiae have been described. An omics analysis of fatty acid secreting mutants of S. cerevisiae identified potential fatty acid export protein Mrp8, though no experimental results are available for this transporter (Fang et al., 2016). An experimentally proven fatty acid export protein is Tpo1, from the MFS superfamily, that was initially identified as a polyamine transporter involved in the resistance of yeast towards spermidine (Albertsen, Bellahn, Krämer, & Waffenschmidt, 2003; Tomitori, Kashiwagi, Sakata, Kakinuma, & Igarashi, 1999). Further studies identified different substrates for this transporter, including medium-chain fatty acids (Legras et al., 2010). Other proteins involved in fatty acid export are the pathogen-related yeast (Pry) proteins.S. cerevisiae contains three Pry proteins, two of which are involved in the export of sterol molecules (Darwiche, El Atab, Cottier, & Schneiter, 2018). One of these proteins, Pry1, is also able to bind fatty acids and its deletion hampers the secretion of fatty acids in a fatty acid accumulating mutant strain (Darwiche, Mène-Saffrané, Gfeller, Asojo, & Schneiter, 2017).
Fatty acid export in yeast has been engineered to improve productivity of microbial cell factories. The heterologous expression of human transporter FATP1 in Y. lipolytica increased extracellular fatty acid titre from 60mg/L to 190 mg/L. Furthermore, this transporter also showed activity towards fatty alcohols, rising the percentage of extracellular fatty alcohols from 9% to 29% of total fatty alcohols produced (Hu, Zhu, Nielsen, & Siewers, 2018). In S. cerevisiae,Tpo1 has been engineered through directed evolution to increase medium-chain fatty acid export rate (Zhu et al., 2020). The mutants were obtained by selecting for increased resistance against medium-chain fatty acids through two rounds of enrichment in selective media containing decanoate of a library of Tpo1 mutants obtained by error-prone PCR. The mutations F322L, T45S, and I432N increased resistance against both decanoate and octanoate and F322L was identified as the mutation with the highest impact. Integration of two copies of this engineered Tpo1 in S. cerevisiae increased the extracellular fatty acids with a chain length of 6 to 10 carbon atoms about 2-fold and those with 12 and 14 carbon atoms about 4-fold.