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.