Fatty acid transport in
bacteria
Fatty acid uptake systems
Import of fatty acids in E. coli:the FadL-FACS
system
The first discovered microbial transport systems for fatty acids was the
FadL-FACS system from E. coli for the import of exogenous
long-chain fatty acids. It was described in 1969 that the ability ofE. coli to degrade exogenous fatty acids was associated to the
presence of a fatty acid-CoA synthetase (FACS). Characterization of this
enzyme suggested the presence of another transport protein which was
discovered later in 1978 (Nunn & Simons, 1978). The gene encoding this
protein, named fadL , belongs to the fatty acid degrading
(fad) regulon containing eight catabolic genes for fatty acids
(Feng & Cronan, 2009), including fadD which codes for the FACS.
The regulon is controlled by the fadR gene, whose product
activates the fad genes upon binding to acyl-CoA. Both fadL andfadD are expressed in a basal level to allow detection of
exogenous fatty acids by fadR (Nils Joakim Færgeman & Knudsen,
1997).
FadL is a long-chain fatty acid-specific transporter with a β-barrel
structure present in the outer membrane of E. coli . The presence
of lipopolysaccharides in the outer membrane renders the cell
impermeable to hydrophobic molecules such as fatty acids. FadL is
necessary for the uptake of exogenous fatty acids so that they can be
used both as a source of energy and as a constituent of phospholipids
and triacylglycerols, relieving the bacteria from spending energy and
resources into synthetizing new fatty acids (Van Den Berg, 2005). The
structure and mechanism of FadL has been studied by Van der Berg et al
(Van Den Berg, Black, Clemons, & Rapoport, 2004). FadL is a long
14-strand β-barrel that contains special features: in the extracellular
region two loops containing α-helices form a hydrophobic groove; in the
intracellular region a hatch domain with three α-helices blocks the
channel; the N-terminus extends through the barrel towards the
extracellular regions; finally, the strand S3 shows a peculiar bend or
kink that disrupts the β-sheet formation and forms a lateral opening
(Figure 1A). Concerning the mechanism of transport, the authors
initially considered at a first stage that the fatty acids would bind
first to the hydrophobic groove, then they would diffuse to an internal
high-affinity binding pocket where they would lead to conformation
changes both in the N terminus and the hatch domain opening a path for
their liberation in the periplasmic space (Van Den Berg, 2005).
Nonetheless, this model was refuted after showing the rigidity of the
hatch domain (Hearn, Patel, Lepore, Indic, & Van Den Berg, 2009). The
importance of the lateral opening in the β-barrel caused by the S3 kink
led to support the hypothesis of lateral diffusion, in which the fatty
acids would not be liberated to the periplasmic space but rather through
the opening space in the β-barrel to the outer membrane (Lepore et al.,
2011; Figure 1B). A summary of studied residues from FadL can be found
in Table 1, all of them conserved in homologue fatty acids transporters
from other species.
Once the fatty acids have been transported by FadL through the outer
membrane, they move to the inner membrane. Even though there are reports
that proteins may be involved in the process (A. Azizan & Black, 1994),
it is likely that this step happens spontaneously. A piece of evidence
that supports passive diffusion from the outer membrane to the inner
membrane, as well as a flip-flop movement inside each membrane, is the
necessity of a proton motive force that can generate acidic conditions
in the periplasm leading to the protonation of fatty acid and the
subsequent increase in liposolubility (Azliyati Azizan, Sherin, DiRusso,
& Black, 1999). Once in the inner membrane, these fatty acids are
catalysed by FACS to form acyl-CoA molecules, which are destined to
further catabolic or anabolic processes. FACS is a soluble protein that
is recruited to the membrane to assist in the transport of fatty acids
(Overath, Pauli, & Schaire, 1969). The mechanism of this recruitment is
not known and it possibly happens due to conformational changes after
binding of ATP and it has been observed to be assisted by the presence
of D-lactate, among other conditions (Mangroo & Gerber, 1993). The
activity of FACS is necessary for the metabolism of exogenous fatty
acids and it is speculated that it promotes the transport of fatty acids
through vectorial acylation analogous to the vectorial phosphorylation
of sugars (Black & Dirusso, 2003).
Engineering the fatty acid import system
of E. coli for biotechnological
applications
The import of fatty acid into cells is important for several industrial
processes, such as fermentation processes using fatty acids and waste
oils as carbon source. The cellular catabolism of fatty acids leads to
the formation of acetyl-CoA, which delivers the acetyl group to the
citric acid cycle providing energy and intermediates to the cell.
Furthermore, acetyl-CoA is a precursor for important metabolites, such
as branched-amino acids (Amorim Franco & Blanchard, 2017).
Engineering efforts to produce 3-hydroxypropionate (bioplastic
precursor) from fatty acids showed the importance of controlling the
expression of the FadL-FACS system (B. Liu et al., 2019). While using a
strong promoter for FACS resulted in an increase in productivity, it led
to an important decrease when used for FadL. However, the use of medium
or weak constitutive promoters for FadL allowed to increase the
productivity. This exemplifies the common problem associated to
overexpression of membrane proteins, which can increase membrane stress
and decrease cell growth and overall productivity (Kang &
Tullman-Ercek, 2018). The constitutive expression of FACS and FadL, next
to other metabolic modifications, led to a 3-hydroxypropionate yield of
1.56 g/g when grown in a 5L bioreactor using palmitic acid as substrate.
In another study where E. coli was engineered for the production
of lycopene (N. Liu et al., 2020), overexpression of FACS led to a small
increase in the lycopene titre (from 29 to 33 mg/g DCW). Nevertheless,
growth of the engineered strain in a mix of glucose, waste oil and yeast
extract allowed for a total yield of 94 mg/g DCW.
Another important set of applications for the import of fatty acids are
whole cell biotransformations. In these, bacteria grown previously to
reach a certain biomass density act as catalysers to modify fatty acids
or derived compounds through a limited number of enzymatic steps in
order to produce a compound of higher value. In such process, cells act
as capsules containing enzymes and are not grown during the
biotransformation process. Deletion of FACS and overexpression of FadL
showed an increase on fatty acid hydroxylation when expressing the
cytochrome P450 CYP153A from Marinobacter aquaeolei, a
heterologous cytosolic enzyme in E. coli, suggesting that other
proteins can replace the role of FACS in recruiting free fatty acids
from the inner membrane and modifying them (Bae, Park, Jung, Lee, &
Kim, 2014). Another study involving both fatty acids and hydroxy-fatty
acids determined the impact of FadL expression on the biotransformation
of these compounds to hydroxy-fatty acids and keto-fatty acids,
respectively (Jeon et al., 2018). Enhanced expression of FadL led to a
five-fold increase in the single step transformation of oleic acid and
10-hydroxyoctadecanoic acid to 10-hydroxyoctadecanoic acid and
10-keto-octadecanoic, respectively, as well as a two-fold increase in
the multistep transformation of ricinoleic acid to the ester molecule
((Z)-11-(heptanoyloxy)undec-9-enoic acid). This study also showed the
negative effects that excessive overexpression of FadL can have on
overall productivity. Finally, in another study FadL was overexpressed
in a strain expressing human Cav1 proteins (Shin et al., 2019). Cav1
proteins stimulate the formation of endosomes that excise from the inner
membrane. The formation of endosomes increased the uptake rates of
ricinoleic acid two-fold and caused a decrease of fatty acid toxicity in
the inner membrane. However, overexpression of FadL in this strain did
not lead to a further increase in fatty acid uptake.
Import of fatty acids in other bacteria
Besides the fadL-FACS system from E. coli , other systems for the
import and assimilation of exogenous fatty acids have been studied in
other bacteria. All bacterial fatty acid import systems were found to be
dependent on energy supply whether in the form of ATP or in the form of
a proton gradient (Calmes & Deal, 1976). One of the first systems
studied was that of other gram-negative bacteria such asPseudomonas oleovorans and Caulobacter crecentus , which
showed similar characteristics to the fadL-FACS system (Toscano &
Hartline, 1973; Zalatan & Black, 2011). Due to the different
plasma membrane structure, it has been observed that import of fatty
acids in gram positive bacteria occurs differently than in gram-negative
bacteria (Figure 2A and 2B). Although fatty acid metabolism and
transport has not extensively been studied in gram positive model
bacteria, such as B. subtilis, some studies have been performed
on other gram-positive bacteria. In the lipophilic gram-positive
bacteria Nocardia asteroides (Calmes & Deal, 1976), the
fatty acid uptake system was, in contrast to the previously discussed
systems, found to be constitutively expressed and able to import fatty
acids as free fatty acids, which would be activated by a soluble FACS at
a later and independent stage. With a KM of 870 μM, the
transport process from N. asteroides was found to have much less
affinity than that of E. coli (KM between 15 and
34 μM). The import of fatty acids in another gram-positive bacterium,Streptomyces coelicolor (Banchio & Gramajo, 1997), also
showed a constitutive expression, as well as a dependency on pH levels
in the environment, decreasing the uptake of long-chain fatty acids with
increasing pH. The import system of this bacterium was found to be
specific for ionized fatty acids (at pH 7 or higher), while protonated
ones were claimed to use a passive process rather than an active one (at
a pH lower than 7).
In the fadL-FACS system, FACS is associated to the membrane, linking the
formation of acyl-CoA to transport. Nevertheless, the production of
acyl-CoA from exogenous fatty acids is not conserved in all bacteria,
and other ways of activating and incorporating exogenous fatty acids
have been studied, such as acyl-ACP synthase in Vibrio harveyi,or fatty acid kinases in gram positive bacteria (Cronan, 2014). The
different ways to in which exogenous fatty acids are activated and
metabolized has been reviewed elsewhere (Yao & Rock, 2017).
The study of fatty acid import in bacteria can serve different
interests, such as improving the fatty acid import capacities of
organisms of industrial importance or identifying new drug targets for
pathogenic bacteria able to import fatty acids from the host. Among the
different bacteria that are of interest for industry, the gram-positive
genus Rhodococcus has attracted special attention for their
ability to withstand and degrade a wide variety of pollutant, including
hydrocarbons and lignin-derived compounds (Kim, Choi, Yoo, Zylstra, &
Kim, 2018). Some species of this genus, such as Rhodococcus
opacus and Rhodococcus jostii, can be considered oleaginous,
being able to accumulate high amounts of triacylglycerol when growing in
carbon-excess conditions (Alvarez et al., 2019). A fatty acid importer
from R. jostii was found in a cluster with genes involved in
lipid metabolism after a homology search using known ACB-transporters
for hydrophobic compounds. The function of this gene (ro05645 or ltp1)
was confirmed after its overexpression, which led to an increased growth
in media containing fatty acids as substrate, as well as the uptake of
fluorescently labelled fatty acids (Villalba & Alvarez, 2014).
Furthermore, the overexpression of ltp1 was used to increase both
the growth (2.2-fold) and the lipid accumulation (3.5-fold) of R.
jostii when growing in olive mill waste (Herrero, Villalba,
Lanfranconi, & Alvarez, 2018).
Mycobacterium tuberculosis is the causing agent of tuberculosis,
one of the deadliest diseases in the world. This bacterium is adapted to
survive for long periods of time inside the human host, partially thanks
to its ability to metabolize host-derived fatty acids. This ability is
especially important, as these cells tend to reside within lipid-rich
sites, such as inside foamy macrophages (Lovewell, Sassetti, &
VanderVen, 2016). M. tuberculosis presents a cell envelope
containing mycolic acid and glycolipids which acts as a barrier for
hydrophobic molecules. A multiprotein complex was observed to be
involved in the uptake of fatty acids during macrophage infection as
well as in monocultures (Figure 2C). The first member of this complex to
be identified was LucA, a protein also involved in the uptake of
cholesterol (Nazarova et al., 2017). It was observed that a mutant
lacking LucA was unable to incorporate fatty acids, and a transcriptomic
analysis of such mutant revealed the involvement of genes from the Mce1
locus in the uptake of fatty acids. From this locus, gene rv0167coding for the putative permease YrbE1A was found to be directly
involved in fatty acid import. A further screening of mutants lacking
the ability to assimilate fatty acids when infecting macrophages
revealed the participation of other proteins (Nazarova et al., 2019),
such as MceD1, MceG and OmamB. While the function of OmamB is not known,
a similar protein OmamA was found to interact with LucA and stabilize
the fatty acid import complex. MceG is a putative ATPase that may be
involved in providing the energy for the transport of the fatty acids.
Finally, MceD1 and other proteins from the same locus such as MceA1 and
MceC1, may have structural roles in the formation of the fatty acid
import complex. In addition to the complex Mce1, another gene
(rv1272 ) from M. tuberculosis showing homology to theltp1 gene from R. jostii as well as to the lipid A export
gene msbA from E. coli was confirmed to import fatty acids
when expressed in E. coli (Martin & Daniel, 2018). A Blast
search of rv1272 against the SwissProt database showed that this
gene is homologous (39.39% identity) to the uncharacterized transporter
YfiC from Bacillus subtilis, suggesting that the role of this
transporter might be associated to the transport of fatty acids or other
hydrophobic compounds.
Fatty acid export systems
The fatty acid export system in E.
coli and its
engineering
In its natural environment, E. coli must face high concentrations
of fatty acids and other hydrophobic compounds and therefore it must
contain mechanisms to prevent toxic effects, such as export proteins. At
the same time, secretion of endogenous fatty acids is not detected for
wild type strains under normal conditions. Nevertheless, fatty acid
secretion in E. coli was observed for the first time when
expressing a thioesterease from the plant Umbelleria californica(Voelker & Davies, 1994). This enzyme was expressed to generate
free fatty acids inside the cell. In normal conditions, free fatty acids
do not accumulate intracellularly in bacteria, as they are
directly transferred from acyl-ACP to glycerol-3-phosphate (Magnuson et
al., 1993). On the other hand, plants possess thioesterases to liberate
fatty acids from acyl-ACP and these free fatty acids are used for
anabolic processes (Gerhardt, 1992). The oilseed from U.
californica accumulates medium-chain fatty acids, mainly lauric acid
(C12:0) (Davies, Anderson, Fan, & Hawkins, 1991), and
the expression of its thioesterase in E. coli leads to the same
fatty acid profile (Voelker & Davies, 1994). However, the accumulation
of fatty acids was only observed when the β-oxidation pathway was
disabled. Secretion of lauric acid was shown by the observation of
extracellular laurate crystals when growing on solid media. Later
engineering of E. coli showed that saturated and monounsaturated
fatty acids with 12 till 18 carbon atoms can be secreted in an efficient
way reaching 40 mg/L of extracellular fatty acids, but no specific
efflux protein for fatty acids was identified (H. Liu et al., 2012).
The accumulation of medium-chain fatty acids is toxic to bacteria due to
their ability to destabilize the membrane and interfere with essential
activities, such as creation of proton gradients (Lennen et al., 2011).
This toxic activity was used to identify proteins involved in fatty acid
export that would protect the cells from the accumulation of
medium-chain fatty acids when expressing the thioesterase from U.
californica (Lennen, Politz, Kruziki, & Pfleger, 2013). The
genes whose knock out led to important effects on cell viability under
these conditions were rob, acrAB, tolC and, at a lower extent,emrAB . The activity of the first three genes is tightly linked,
as rob is a regulatory gene that induced acrAB expression
among other genes, and TolC is an unspecific porin from the outer
membrane that forms a complex with acrAB. The AcrAB-TolC complex
is a transporter from the Resistance-Nodule-Division (RND) superfamily
that spans from the intracellular space until the outer side of the
bacterial membrane, forming a channel whose mechanism is regulated by
allosteric changes and a proton gradient (Wang et al., 2017; Zgurskaya
& Nikaido, 1999). The complex is formed by three TolC components, six
AcrA components and three AcrB components (Figure 3). The mechanism
behind the binding of ligands to AcrB is not precisely known, but due to
the large number of different substrates that the AcrAB-TolC complex can
transport it is thought that there might be several binding sites in the
same binding pocket (Nakashima, Sakurai, Yamasaki, Nishino, &
Yamaguchi, 2011). In any case, the binding of a substrate changes the
conformation of the AcrB monomer from the relaxed or access state to a
binding state, which induces conformational changes in the rest of the
complex. Through several interactions with AcrB, AcrA continues the
conformational changes to TolC, so that it can change from the closed
state to the open state, opening the channel. Finally, the AcrB subunit
bound to the ligand changes to another conformation (open state),
liberating the substrate into the open channel for its liberation to the
extracellular medium (Wang et al., 2017).
Due to the broad specificity of the AcrAB-TolC system, the AcrB
component has been engineered through directed evolution to improve the
export rate of different hydrophobic molecules, such as medium-chain
alcohols, alkanes and alkenes (Chen, Ling, & Chang, 2013; Fisher et
al., 2014; Mingardon et al., 2015). However, no protein engineering of
AcrB to improve fatty acid export has been documented. Genetic
engineering to improve medium-chain fatty acid export in E. coliwas performed by overexpressing several potential transporters (J. Wu et
al., 2019). Overexpression of either of three transport proteins, namely
AcrE, MdtC and MdtE, was found to increase medium-chain fatty acid
extracellular concentrations from 600 mg/L to values between 800 and
1100 mg/L. One must realize that these proteins are part of larger
protein complexes whose other components were not overexpressed in the
study.
The AcrEF complex displays homology to the well-studied AcrAB-TolC
complex. Yet, the AcrEF complex has been observed to be expressed at
lower levels than the AcrAB complex and it has shown an important
function in cell division and chromosome segregation (Lau & Zgurskaya,
2005). Nevertheless, the AcrAB and AcrEF complexes are from a structural
point of view highly related: the amino acid sequence of acrF shares
87% similarity with acrB, and acrE is homologous to acrA (80% AA
similarity). Therefore, it is likely that acrE fulfils the same role as
acrA in the acrEF-TolC complex as the channel that connects acrF and
TolC. Yet, when overexpressing acrA and acrB from the acrAB-TolC complex
separately, no increased fatty acid export was observed. Hence, the
mechanism behind the overexpression leading to increased export activity
of a channel protein that connects the inner membrane pump (which
contains the substrate binding) and the outer membrane porin remains
unsolved. Furthermore, in the same study three-fold higher extracellular
medium-chain fatty acid concentrations were achieved when overexpressing
the three transport proteins (acrE, mdtC and mdtE) simultaneously, while
observing only a 10% reduction in the OD (J. Wu et al., 2019).
Export of fatty acids in other
bacteria
As observed in the previous section, fatty acid export in E. coliseems to be mainly associated to protection against membrane-related
toxic effects caused by large concentrations of medium and long-chain
fatty acids (Desbois & Smith, 2010). Medium-chain fatty acids have
antimicrobial effects affecting a wide range of bacteria (Huang,
Alimova, Myers, & Ebersole, 2011). Therefore, it is expected that many
bacteria present export systems similar to AcrAB-TolC to export toxic
fatty acids, although the specific systems have not been studied to
date.
Besides medium chain fatty acids, polyunsaturated fatty acids display
antimicrobial properties and in reaction to this, several pathogenic
bacteria have developed systems to prevent their toxicity. Some of these
systems are the degradation of exogenous fatty acids or their
incorporation into phospholipids (Jiang et al., 2019). However, the
environments where some pathogenic bacteria must thrive in, such as the
skin or the mouth, contain unsaturated fatty acids and therefore they
need more advanced protection mechanisms such as fatty acid export
systems (Choi et al., 2013; Parsons, Yao, Frank, Jackson, & Rock,
2012). In the gram-positive bacterium Staphylococcus aureus , an
opportunistic pathogen found on the skin, a fatty acid export system was
found when screening for strains resistant to linoleic acid (Alnaseri et
al., 2019). The strain S. aureus FAR7 showed a mutation in the
transcription factor farR, which led to the upregulation of thefarE gene, encoding an efflux pump from the RND
superfamily. Also the physiological response to PUFAs of the
gram-negative pathogenic bacteria Acinetobacter baumannii, was
studied and revealed the upregulation of the adeJ gene, a
component of the multidrug efflux pump adeIJK (Jiang et al., 2019). It
was observed that the deletion of this gene increased the susceptibility
to PUFAs, leading to a six-fold increased growth delay in the presence
of docosahexaenoic acid. However, the mutation did not lead to an
accumulation of PUFAs in the cell. Nevertheless, growth experiments
showed the ability of adeIJK to affect membrane lipid homeostasis and to
export lipids to the extracellular medium. These results suggested that
the adeIJK pump fulfils a similar role as the emhABC pump fromPseudomonas fluorescens , which controls lipid homeostasis through
the efflux of endogenous long-chain fatty acids, both saturated and
monounsaturated, in response to temperature changes (Adebusuyi & Foght,
2011). Another studied fatty acid export system from a pathogenic
bacterium is the farAB system from Neisseria gonorrhoeae , which
shows high similarity to the emrAB system from E. coli, and whose
deletion leads to susceptibility to the long-chain fatty acids oleic
acid, linoleic acid and palmitic acid. The minimal inhibitory
concentration decreased from 1600 μg/ml to 50 μg/ml in the case of
unsaturated fatty acids and from 100 μg/ml to 12.5 μg/ml in the case of
palmitic acid (Lee & Shafer, 1999).
Photosynthetic microorganisms are of special interest due to their
ability to fix carbon from the atmosphere to produce industrially
relevant compounds in a more efficient way than plants.Synechocystis sp. PCC 6803 is the model organism for
cyanobacteria and it has been observed to secrete long-chain fatty
acids, up to 13% of the cellular biomass, without any genetic
modifications (X. Liu, Sheng, & Curtiss, 2011). The deletion ofSynechocystis sp. PCC 6803 genes sll0180 andslr2131, homologous to the respective E. coli genesacrA and acrB, showed an effect in the fatty acid
secretion of an engineered strain of Synechocystis sp. PCC 6803
specialized in the extracellular production of fatty acids (Bellefleur,
Wanda, & Curtiss, 2019). While the complementation with acrA did
not lead to a recovery of the fatty acid secretion rates, the
complementation of slr2131 with acrB allowed for an
increase in both extracellular and intracellular fatty acid
concentrations.
Another fatty acid export mechanism has been identified in another
cyanobacteria, Synechococcus elongatus a. This system was
found through the genomic and transcriptomic analysis of a mutant able
to produce free fatty acids but resistant to their toxicity.
Inactivation of this export system, composed by the genes rndA1and rndB1 , led to susceptibility to exogenous saturated
medium-chain fatty acids and unsaturated long-chain fatty acids.
Orthologs of rndB1 are found in most genomes from cyanobacteria,
but not in those of Synechocystis sp. PCC 6803. The RndA1B1
system from S. elongatus allows for efficient secretion of oleic
acid, but palmitate is not transported and therefore it accumulates in
the cells (Kato et al., 2015).