marine drugs

Review
Bacterial Long-Chain Polyunsaturated Fatty Acids:
Their Biosynthetic Genes, Functions, and
Practical Use
Kiyohito Yoshida 1 , Mikako Hashimoto 2 , Ryuji Hori 3 , Takumi Adachi 4,5 , Hidetoshi Okuyama 6 ,
Yoshitake Orikasa 7 , Tadashi Nagamine 8 , Satoru Shimizu 9 , Akio Ueno 9 and Naoki Morita 4,5, *
1 Laboratory of Ecological Genetics, Section of Environmental Biology, Faculty of Environmental Earth
Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-0810, Japan; majin@ees.hokudai.ac.jp
2 Course in Ecological Genetics, Division of Biosphere Science, Graduate School of Environmental Science,
Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-0810, Japan; mkk_hsmt@eis.hokudai.ac.jp
3 Technical Solution Center First Group, J-OIL MILLS, Inc., Chuo-ku, Tokyo 104-0044, Japan;
ryuji.hori@j-oil.com
4 Laboratory of Environmental Microbiology, Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-8589, Japan; takumin_ikuno@ybb.ne.jp
5 Bioproduction Research Institute, Department of Life Science and Biotechnology, National Institute of
Advanced Industrial Science and Technology (AIST), Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan
6 Laboratory of Environmental Molecular Biology, Section of Environmental Biology, Faculty of
Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-0810, Japan;
hoku@ees.hokudai.ac.jp
7 Department Food Science, Obihiro University Agriculture Veterinary Medicine, Inada-cho, Obihiro,
Hokkaido 080-8555, Japan; yosori@obihiro.ac.jp
8 ROM Co. Ltd., Togashi Bld., Chuo-ku, Sapporo, Hokkaido 060-0062, Japan; nagamine@rom-ef.co.jp
9 Horonobe Research Institute for the Subsurface Environment, Northern Advancement Centre for Science
and Technology, 5-3, Sakae-machi, Horonobe, Teshio-gun, Hokkaido 098-3221, Japan;
satoru.shimizu@h-rise.jp (S.S.); akio.ueno@h-rise.jp (A.U.)
* Correspondence: morita.n@aist.go.jp; Tel.: +81-11-857-8963; Fax: +81-11-857-8992

Academic Editor: Andrew Desbois

Received: 14 January 2016; Accepted: 29 April 2016; Published: 12 May 2016

Abstract: The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids
(LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well
recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes.
Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria
compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular
biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably
diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA
synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme.
In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane
proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long
chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be
closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly
anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of
anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

Keywords: long-chain polyunsaturated fatty acids; pfa genes; polyunsaturated fatty acid synthase
(PUFA synthase); eicosapentaenoic acid (EPA); docosahexaenoic acid (DHA); arachidonic acid; very
long chain polyunsaturated hydrocarbons

Mar. Drugs 2016, 14, 94; doi:10.3390/md14050094 www.mdpi.com/journal/marinedrugs

Mar. Drugs 2016, 14, 94 2 of 23

1. Introduction
The functions and structural nature of biological membranes are provided by the physical and
chemical properties of their building blocks, lipids and proteins. The primary properties of lipids are
largely specified by their constituting fatty acids; in turn, the properties of fatty acids are critically
dependent on their chain length and degree of saturation. In bacterial cell membranes, saturated
16- and 18-carbon fatty acids are most common. Unsaturated fatty acids, usually containing one or two
double bonds, occur as frequently as saturated fatty acids. However, some limited groups of bacteria
have been demonstrated to produce distinct unsaturated fatty acids that have chain lengths longer
than 20 carbons and contain at least four double bonds; examples include arachidonic acid (ARA,
20:4 n-6), eicosapentaenoic acid (EPA, 20:5 n-3), and docosahexaenoic acid (DHA, 22:6 n-3), which are
collectively termed long-chain polyunsaturated fatty acids (LC-PUFAs) [1]. In nature there are two
major modes of LC-PUFA biosynthesis. One is the mechanism by which LC-PUFAs are aerobically
synthesized through the aerobic desaturation and elongation of saturated fatty acids that is typically
employed in eukaryotes [2]. The other, the so-called polyketide mode that has been specifically found
in bacteria, can anaerobically synthesize LC-PUFAs de novo [3]. The latter mode of reaction is catalyzed
by an enzyme complex, PUFA synthase; the genes coding for the members of this complex are termed
pfa genes [4].
The pfa genes that have been identified to be responsible for production of LC-PUFAs to date
display a broad diversity of gene structure. To gain a comprehensive view of such a complex gene
family, knowledge of the enzymatic domains that function in LC-PUFA biosynthesis is helpful.
Here, we outline the reactions and enzymes in fatty acid biosynthesis because both biosynthesis
pathways share many similar reactions [5]. Fatty acids, the constituents of lipids, and polyketides,
which are classified as secondary metabolites, are primarily biosynthesized through the common
type of carbon-chain building reaction wherein a carbon-carbon bond is formed by decarboxylative
condensation utilizing a Claisen-type chemical reaction between acetyl-CoA as a starter unit and
malonyl-CoA as an elongation unit. This reaction is carried out by three conserved functional
components: an acyltransferase (AT), which loads the appropriate acyl group onto a reaction scaffold,
a β-ketoacyl synthase (KS), which adds the loaded building block onto the growing acyl chain, and
an acyl carrier protein (ACP), whose phosphopantetheine prosthetic group serves as the scaffold for
the intermediate acyl chain during the entire elongation process. After condensation but prior to
the next round of chain extension, in the fatty acid synthesis pathway the resulting β-keto group is
processed via reduction and dehydration, which are performed by ketoreductase (KR), dehydratase
(DH), and enoyl reductase (ER) enzymes, producing a β-hydroxyl, an α, β double bond, and a
fully-reduced methylene, respectively. On the other hand, the polyketide biosynthesis pathways
modify the growing polyketone-chain intermediates in various ways by re-arranging the order and
combinations of these reductive enzymatic components to produce diverse final products including
antibiotics, toxins, pigments, and infochemicals [6]. In this review, we first describe the structure
and domain organization of the pfa genes, and then discuss the process of LC-PUFA biosynthesis
in bacteria.
Since the melting temperatures of LC-PUFAs are much lower than those of saturated and
monounsaturated fatty acids, appropriate membrane fluidity at low temperatures can be attained by
membrane phospholipids containing LC-PUFAs. Therefore, LC-PUFAs, particularly EPA and DHA,
have been believed to be efficient modulators for adjusting membrane fluidity. In fact, LC-PUFAs are
detected exclusively in bacteria that inhabit cold marine environments such as the Polar Regions, deep
seawater, and within sea fishes in general. These bacteria produce much higher levels of LC-PUFA
when grown at lower temperatures. In addition, it has been observed that DHA-producing bacteria
are more abundant in deeper seawater (a lower temperature environment) than are EPA-producing
bacteria [7]. This trend has been considered to be explained by the fact that the melting temperature
of DHA is lower than that of EPA. Therefore, the view that LC-PUFAs in the cell membrane are
important factors for cold adapted bacteria has been commonly shared by researchers. However, such
Mar. Drugs 2016, 14, 94 3 of 23

a classical concept for the function of LC-PUFAs has not been necessarily confirmed functionally in
LC-PUFA-producing bacteria or eukaryotic microorganisms.
Recent progress in genetic engineering has allowed these techniques to be applied to such bacteria
to elucidate the physiological roles of LC-PUFAs and has provided new findings regarding the function
of LC-PUFAs, particularly of EPA, in bacteria. In some EPA-producing psychrophilic and piezophilic
bacteria, EPA was found not to be involved in their adaption to cold-temperature and high-pressure
environments; rather, the presence of EPA constrains the membrane fluidity. Another unexpected new
function of EPA and DHA is that these LC-PUFAs have been shown to be involved in the resistance of
bacteria against extracellular oxidants such as H2 O2 . As LC-PUFAs have many bisallylic hydrogen
atoms, which are quite active toward reactive oxygen species (ROS) and free radicals [8,9], it was
conceivable that these compounds are apt to be easily oxidized by oxidants (or ROS). However,
LC-PUFAs located in cell membrane are not oxidized by (or are rather stable against) exogenously
added ROS [10]. The antioxidative function of LC-PUFAs is difficult to be conceptualized and its
molecular mechanism is to be solved. On the other hand, EPA is responsible for cell division only at
low temperatures in some EPA-producing marine bacteria suggesting that its presence is not entirely a
consequence of its membrane fluidity adjustment. These findings were provided by investigations
employing various EPA-producing marine bacteria and their EPA-deficient mutants in addition
to Escherichia coli (E. coli) recombinants transformed by the pfa genes responsible for EPA or DHA
biosynthesis. Recently, much attention has further been paid to the relationships between LC-PUFAs
and individual membrane functions such as membrane transport and the efflux activities of various
compounds. We describe here the functions of LC-PUFAs with an emphasis on their involvement in
the efflux of antibiotics in E. coli recombinants capable of producing EPA.
Hentriacontanonaene (C31 H46 ; C31:9), a very long-chain polyunsaturated hydrocarbon (LC-HC),
was originally discovered in a psychrophilic bacterium isolated from Antarctic sea ice cores [11].
It was subsequently demonstrated that all C31:9-producing bacteria are facultative anaerobes and
simultaneously produce EPA or DHA [12]. C31:9 is predicted to be synthesized by the condensation
of two 4,7,10,13-hexadecatetraenoic acid (16:4 n-3) molecules and subsequent reactions, which are
catalyzed by OleA, B, C, and D [13]. A medium chain polyunsaturated fatty acid, 16:4 n-3 is a product
of the PUFA synthase PfaA-E [12]. Since the amount of C31:9 significantly increases in cells grown at
decreased temperatures, this hydrocarbon has been suggested to play a role in cold adaptation [13].
On the basis of the fact that strictly anaerobic bacteria such as Geobacter bemidjiensis (G. bemidjiensis)
BemT contain a cluster of pfa and ole genes found in tandem, we finally discuss the physiological
significance of the Pfa and Ole protein products in strict anaerobes and the evolutionary implications
of the phylogenetic distribution of the pfa and/or ole genes and their homologs in bacteria.

2. Organization and Function of the pfa Genes

2.1. Cluster Structure and Domain Organization of the pfa Genes

Fatty acid or polyketide de novo biosynthesis is achieved by fatty acid (FAS) or polyketide (PKS)
synthases, respectively. According to the architecture and mode of employment of the enzymatic
components, FASs and PKSs are generally classified into three types. A type I synthase is a
multifunctional megasynthase consisting of multiple domains, each of which functions as an enzymatic
component linked together in a single polypeptide. In contrast, a type II synthase is a discrete and
monofunctional enzyme that functions as a dissociable complex. Additionally, a multifunctional
enzyme of the chalcone synthase group is classified as a type III PKS and can synthesize a final product
without the support of an ACP [14].
LC-PUFAs found in microorganisms are also biosynthesized de novo by the FAS/PKS like
enzyme system, namely PUFA synthase [3]. Since 2000, many pfa gene clusters have been identified,
primarily within marine microorganisms that produce LC-PUFAs, and it has been found that the gene
organization, order of genes, and composition of enzymatic domains are highly conserved [1]. Figure 1
Mar. Drugs 2016, 14, 94 4 of 23

Mar. Drugs 2016, 14, 94 4 of 23

shows representative gene structures and enzymatic domains of the pfa gene clusters responsible for
resembles
the that of
production ofFAS/PKS
LC-PUFAs.andThetheir multigene system
multidomain and theofgene
organization PUFAclustering is reminiscent
synthases of the
resembles that of
similar feature
FAS/PKS typical
and their of type IIsystem
multigene bacterial
andFASthesystems. This complex
gene clustering organization
is reminiscent of themight reflect
similar the
feature
evolutionary history of the pfa gene clusters and/or an adaptive or structural significance of PUFA
typical of type II bacterial FAS systems. This complex organization might reflect the evolutionary
synthases.
history of the pfa gene clusters and/or an adaptive or structural significance of PUFA synthases.

Figure 1. Examples of pfa and ole gene clusters. Gene clusters of pfa gene families and ole genes from
Figure 1. Examples of pfa and ole gene clusters. Gene clusters of pfa gene families and ole genes from
nine microbial genomes are shown. Horizontal lines indicate genome sequences. Lines split into
nine microbial genomes are shown. Horizontal lines indicate genome sequences. Lines split into several
several
parts partsthat
denote denote that separated
separated genome genome regions
regions are arein
located located in different
different locinot
loci or have or have not mapped.
yet been yet been
mapped. Gray colored boxes show pfa gene coding regions with their enzymatic
Gray colored boxes show pfa gene coding regions with their enzymatic domains indicated by colored domains indicated
by colored
boxes. Whiteboxes. Whiteboxes
pentagonal pentagonal
represent boxes
genes represent
unrelatedgenes
to pfaunrelated
genes. Pink pfa genes.
tocolored Pink colored
pentagonal boxes
pentagonal boxes are ole genes. The acute angles of the pentagonal boxes indicate
are ole genes. The acute angles of the pentagonal boxes indicate the direction of transcription. Gene the direction of
transcription. Gene names if assigned are listed on the boxes. The domain regions
names if assigned are listed on the boxes. The domain regions were located by NCBI BLASTP searches; were located by
NCBIinclude
these BLASTP searches;
β-ketoacyl these (KS),
synthase include β-ketoacylacyltransferase
malonyl-CoA synthase (KS), malonyl-CoA
(MAT), acyl carrieracyltransferase
protein (ACP),
(MAT), acyl carrier protein (ACP), ketoreductase (KR), polyketide
ketoreductase (KR), polyketide synthase dehydratase (PS-DH), acyltransferase synthase dehydratase (PS-DH),
(AT), dehydratase
acyltransferase
(DH), (AT), dehydratase
enoyl reductase (DH), enoyl reductase
(ER), phosphopantetheine transferase(ER), phosphopantetheine
(PPTase), transferase
1-acylglycerol-3-phosphate
(PPTase), 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT), and oleA–D.
O-acyltransferase (AGPAT), and oleA–D. DH’domains were also identified by NCBI BLASTP searches DH’domains were
also identified by NCBI BLASTP searches with amino acid sequences that
with amino acid sequences that span regions 1096–1305 and 1498–1755 of PfaC in Photobacterium span regions 1096–1305
and 1498–1755
profundum of PfaC
SS9 [15]. in Photobacterium
In the table, “+” denotes profundum SS9was
that C31:9 [15].detected,
In the table,
and“+”“´”denotes
denotesthat
thatC31:9 was
products
detected,
were and “−” denotes
not detected. ND means thatthat
products were not
the existence detected.
of C31:9 has ND means
not been that the existence
determined. pfa geneofclusters
C31:9 has
are
not been determined. pfa gene clusters are classified
classified into Types according to Shulse and Allen [16]. into Types according to Shulse and Allen [16].

The conserved arrangements of the pfa genes are observed not only in LC-PUFA producing
The conserved arrangements of the pfa genes are observed not only in LC-PUFA producing
bacteria but also in ecologically and phylogenetically widespread bacterial species. Shulse and Allen
bacteria but also in ecologically and phylogenetically widespread bacterial species. Shulse and
[16] identified pfa gene homologs from the various bacterial genomes of 86 species belonging to 10
Allen [16] identified pfa gene homologs from the various bacterial genomes of 86 species belonging to
phyla and classified them into 20 types (A–T) based on the combination of domain arrangements
10 phyla and classified them into 20 types (A–T) based on the combination of domain arrangements
and the final products, if known. Although the fatty acyl metabolites, designated as “secondary
and the final products, if known. Although the fatty acyl metabolites, designated as “secondary lipids”,
lipids”, synthesized by these pfa-like gene products are largely unknown, the gene types show a
synthesized by these pfa-like gene products are largely unknown, the gene types show a significant
significant correlation with diverse ecological and physiological properties of the microorganisms
correlation with diverse ecological and physiological properties of the microorganisms possessing these
possessing these genes. Therefore, the authors point out that a deeper understanding of secondary
genes. Therefore, the authors point out that a deeper understanding of secondary lipid biosynthesis
lipid biosynthesis pathways expands our insight into the ecological adaptation and evolution of
pathways expands our insight into the ecological adaptation and evolution of microorganisms.
microorganisms.
Since pfa gene structures vary considerably, we herein adopted the pfa gene cluster of Shewanella
oneidensis (S. oneidensis) MR-1 as a typical structure (Figure 1). The genes responsible for EPA
production in S. oneidensis, a mesophilic bacterium, consist of five open reading frames (ORFs)
denoting the pfaA–E genes, which form a gene cluster in the genome [17]. pfaA is a multi-functional
Mar. Drugs 2016, 14, 94 5 of 23

Since pfa gene structures vary considerably, we herein adopted the pfa gene cluster of Shewanella
oneidensis (S. oneidensis) MR-1 as a typical structure (Figure 1). The genes responsible for EPA production
in S. oneidensis, a mesophilic bacterium, consist of five open reading frames (ORFs) denoting the pfaA–E
genes, which form a gene cluster in the genome [17]. pfaA is a multi-functional gene that includes
five domains: KS, malonyl-CoA acyltransferase (MAT), four repeats of ACP, KR, and PKS-like DH.
pfaB is mono-functional, possessing one AT domain. pfaC includes two KS and four FabA-like DH
domains (see below). The pfaD gene has only a single domain, ER. The last pfaE gene, which encodes
phosphopantetheine transferase (PPTase), is located upstream of the pfaA gene and is oriented in the
opposite direction. Based on this gene organization, this pfa gene cluster is classified into Type A [16].
In almost all eukaryotes, most unsaturated fatty acids including LC-PUFAs are commonly
produced by a combination of fatty acid elongation and oxygen-dependent desaturation. However,
some marine algae have the potential to synthesize LC-PUFAs using pfa genes whose organization
is similar to those of bacteria (Figure 1). Schizochytrium sp. and Aurantiochytrium sp. have a pfa
organization consisting of PFA1 (equivalent to pfaA), PFA2 (equivalent to pfaB + the anterior region of
pfaC + pfaD), and PFA3 (the posterior region of pfaC + pfaD). Notably, a haptophyte Emiliania huxleyi,
which produces mainly DHA, exhibits a quite unique pfa gene organization consisting of only one
ORF with a structure the same as that of the combined PFA1, PFA2, and PFA3 of Schizochytrium sp. and
Aurantiochytrium sp., in this order. These gene clusters are classified into Type E [16].
Recently, two new pfa genes have been experimentally verified to be responsible for LC-PUFA
biosynthesis. Aureispira marina (A. marina), which is classified into the phylum Bacteroides and was
isolated from a marine sponge and an alga that inhabit the coastal areas of Thailand, produces 30%–40%
of its total fatty acids (TFAs) as ARA [18]. The pfa gene homologs that were isolated from the bacterial
genome direct ARA biosynthesis when they are introduced into E. coli [19]. Although the genomic
structure of the genes highly resembles the Type A pfa gene cluster with respect to domain arrangement,
the region containing the KR domain is separate from the pfaA gene, and the AT domain region is
fused to the pfaC gene (Figure 1). Based on this domain organization and the production of ARA, the
pfa gene cluster in A. marina can be grouped into Type D. This category also includes the gene cluster
found in a marine Bacteroidetes species, Psychroflexus torquis [16], which is a psychrophilic species
isolated from Antarctica ice samples [20]. Despite remarkable difference in their habitats, the finding
that A. marina and Psychroflexus torquis share conserved pfa genes and ARA production might be of
considerable relevance toward understanding the physiological function of LC-PUFAs.
Although the pfa genes have been almost exclusively found from marine bacteria, another example
of a pfa gene cluster has been identified in terrestrial myxobacteria of the genus Aetherobacter [21].
Four species of Aetherobacter produce several types of LC-PUFA including ARA, EPA, DHA, and C18
PUFAs. The pfa gene clusters found in these bacteria consist of three ORFs (pfa1–3) and show several
structural differences when compared with a typical Type A cluster (Figure 1). The pfaD gene homolog
containing an ER domain is located upstream of the myxobacterial gene cluster, and the pfaB gene
segment is missing from the cluster of Aetherobacter spp.; instead, the AT domain present in the pfaB
gene is relocated in the middle of the pfa3 gene. In addition, the pfaE gene homolog that encodes
a PPTase is found at a distinct locus separated from the pfa gene cluster, as was seen in Moritella
marina (M. marina) MP-1 [22]. The most characteristic feature of the myxobacterial pfa genes is that the
pfa3 gene corresponding to the pfaC gene possesses a domain coding for 1-acylglycerol-3-phosphate
O-acyltransferase (AGPAT) at its carboxyl terminal. Judging from the gene organizational features, the
pfa genes of Aetherobacter spp. can be classified into Type L. It is unclear whether the myxobacterial
PUFA synthases can produce all of the types of LC-PUFAs found in these bacteria or whether they
exclusively produce only certain LC-PUFAs, which are then converted into the remaining LC-PUFAs
by other enzyme activities. Sorangium cellulosum (S. cellulosum), a closely related species to Aetherobacter,
produces only linoleic acid (LA) as a PUFA, but not any other LC-PUFAs [23]. Although both bacteria
share the highly conserved pfa genes, the pfa3 gene of S. cellulosum lacks the AT domain sequence [21].
This discrepancy might explain the inability to synthesize EPA and DHA in S. cellulosum.
Mar. Drugs 2016, 14, 94 6 of 23

2.2. Biosynthetic Process of LC-PUFAs in Bacteria

2.2.1. Initiation
The LC-PUFA biosynthesis reaction is initiated by activating the ACPs of PfaA from their
apo- to their active holo-form. This activity is catalyzed by PPTase (PfaE), which transfers a
41 -phosphopantetheine prosthetic group from CoA to a serine residue of ACP [24]. PfaE is considered
to utilize the ACP repeats of PfaA specifically to activate ACP. However, the heterologous production
of EPA was observed in E. coli transformed in combination with the pfaA–D gene from Shewanella
pneumatophori (S. pneumatophori) as well as with the E. coli entD gene encoding an Sfp-type PPTase
responsible for the biosynthesis of siderophore [25], indicating that the activation of PfaE can be
replaced partially by that of other types of PPTases.

2.2.2. Extension of Acyl Chains (Elongation, Double Bond Formation, and Final
Product Determination)
The activated ACP domains of PfaA, hereafter designated as PfaA(ACP), accept acetyl and
malonyl groups from acetyl- and malonyl-CoA and each reaction is respectively catalyzed by AT and
MAT. These two molecules on the PfaA(ACP) are condensed with the release of one molecule of CO2
by a Claisen condensation reaction catalyzed by KS. Subsequently, the β-oxo group is reduced by
KR and the β-OH group is formed. The latter undergoes a dehydration reaction catalyzed by DH to
form a ∆2 -trans double bond; this double bond then is reduced by ER to form a saturated C–C bond
or isomerized to ∆2 -cis and ∆3 -cis isomers by DH. The thus-formed cis double bonds are conserved
throughout LC-PUFA synthesis. The Type A PUFA synthase system has a PKS-DH on PfaA and a dual
FabA DH in the PfaC; however, their role-sharing has not been clarified. The cycle of condensation
by KS and modification by KR, DH, and ER extend the fatty acyl chains in two carbon increments.
These sequential reactions in LC-PUFA biosynthesis are speculated based on the functional domain
structures of the pfa genes. However, confirmation of the individual enzymatic reactions of the gene
products have not yet been fully determined.
Type A, B, and D pfa gene clusters are involved in the synthesis of EPA, DHA, and ARA,
respectively (Figure 1). Although the mechanism determining the final LC-PUFA product by the
PUFA synthase is not yet fully understood, the KS domain, which exists only in the PfaB of the Type B
pfa cluster, is known to be responsible for the synthesis of DHA [26]. Furthermore, the characteristic
domain order in the Type D cluster, in comparison with that in Type A, is that the position of the AT
domain moves between the carboxyl-terminal KS domain and the DH’ domain (Figure 1); thus, this
feature might be involved in determining the type of final LC-PUFA produced.

2.2.3. Chain-Termination of LC-PUFA Biosynthesis and Their Integration into Lipids

The LC-PUFA synthesis process is accomplished when the final products are incorporated into
lipids. Newly synthesized LC-PUFAs are transferred to 1-acyl-3-gycerophosphate by AGPAT, forming
phosphatidic acid, and the final acceptor molecules of these constitute phospholipids in bacteria or
phospholipids and triacylglycerols in eukaryotes.
Shewanella livingstonensis (S. livingstonensis) Ac10, which produces EPA, possesses five types of
genes homologous to E. coli plsC (AGPAT). Notably, the bacterial mutant for one of these (plsC1)
synthesizes significantly reduced levels of EPA [27], indicating that EPA is transferred following
completion by PlsC1. Thus, two schemes for the chain-termination and transfer route of LC-PUFAs are
generally considered in bacteria (Figure 2). One is the pathway wherein the LC-PUFAs synthesized on
PfaA(ACP) are directly utilized as a substrate of transacylation to generate phosphatidic acid. In this
case the transfer of acyl groups catalyzed by AGPAT is regarded as a chain-terminating step in the
synthesis of LC-PUFA. AGPAT in LC-PUFA producing bacteria might use acyl-ACP and/or acyl-CoA
for its substrate because of its high similarity to the E. coli PlsC, which utilizes either compound as
Mar. Drugs 2016, 14, 94 7 of 23

a substrate. This raises the possibility that AGPAT recognizes LC-PUFA-PfaA(ACP) as a substrate
Mar. Drugs 2016, 14, 94
as well. 7 of 23

Figure 2. Possible processes for LC-PUFA biosynthesis via PUFA synthase and their incorporation
Figure 2. Possible processes for LC-PUFA biosynthesis via PUFA synthase and their incorporation
into phospholipids by membrane-bound 1-acyl-3-phosphatidic acid acyltransferase (PlsC). Fatty acid
into phospholipids by membrane-bound 1-acyl-3-phosphatidic acid acyltransferase (PlsC). Fatty acid
LC-PUFA biosynthesis is initiated by activation of the acyl carrier protein (ACP) domains of the Pfa
LC-PUFA biosynthesis is initiated by activation of the acyl carrier protein (ACP) domains of the Pfa
enzyme PUFA synthase via 4′-phosphopantetheine transferase (PPTase) (1. Initiation). The extension
enzyme PUFA synthase via 41 -phosphopantetheine transferase (PPTase) (1. Initiation). The extension
of an acyl chain is carried out by the combined β-ketoacyl synthase (KS), ketoreductase (KR), and
of an acyl chain is carried out by the combined β-ketoacyl synthase (KS), ketoreductase (KR), and
bifunctional dehydratase (DH) activities (2. Extension). The growing fatty acids (blue wavy line) with
bifunctional dehydratase (DH) activities (2. Extension). The growing fatty acids (blue wavy line)
a Δ2-trans2 double bond are reduced to form saturated fatty acids catalyzed by enoyl reductase (ER),
with a ∆ -trans double bond are reduced to form saturated fatty acids catalyzed by enoyl reductase
and those with a Δ2-cis or2 Δ3-cis double bond are isomerized positionally and geometrically to form
(ER), and those with a ∆ -cis or ∆3 -cis double bond are isomerized positionally and geometrically to
unsaturated fatty acids catalyzed by bifunctional PKS or FabA DH. In the termination step, the
form unsaturated fatty acids catalyzed by bifunctional PKS or FabA DH. In the termination step, the
matured LC-PUFA molecule, represented by EPA in this figure (red wavy line), which is
matured LC-PUFA molecule, represented by EPA in this figure (red wavy line), which is accommodated
accommodated
in the Pfa protein in isthe Pfa directly
either protein used
is either directly of
as substrate used as substrate
the PlsC of thephospholipids
to synthesize PlsC to synthesize
(route
phospholipids
A; black arrow) (route A; black
or released toarrow) or released
free acid to free acid
by thioesterase (TE) by thioesterase
encoded (TE)
by orf6. In encoded
the latterby orf6.free
step, In
the latter step, free LC-PUFAs are converted to CoA/ACP derivatives by acyl-CoA/ACP
LC-PUFAs are converted to CoA/ACP derivatives by acyl-CoA/ACP synthetase, which are then used synthetase,
which are then used
for phospholipid for phospholipid
synthesis synthesis
by PlsC (route by arrow)
B; white PlsC (route B; white arrow) (3. Termination).
(3. Termination).

The second possibility is that thioesterase (TE) works as a chain-terminator in the biosynthesis
The second possibility is that thioesterase (TE) works as a chain-terminator in the biosynthesis
cycle of LC-PUFA. Type I FAS/PKS multidomain enzymes generally have TE domains to cleave their
cycle of LC-PUFA. Type I FAS/PKS multidomain enzymes generally have TE domains to cleave their
final products from ACP domains. In PUFA synthase systems, Rodriguez-Guilbe et al. [28] found
final products from ACP domains. In PUFA synthase systems, Rodriguez-Guilbe et al. [28] found
that bacteria possessing pfa gene clusters belonging to Type A or B share highly conserved TE genes;
that bacteria possessing pfa gene clusters belonging to Type A or B share highly conserved TE genes;
in particular, Photobacterium profundum (P. profundum) SS9 (Type A) and M. marina MP-1 (Type B)
in particular, Photobacterium profundum (P. profundum) SS9 (Type A) and M. marina MP-1 (Type B)
genomes possess TE genes located immediately upstream of their pfaA genes. They also showed that
genomes possess TE genes located immediately upstream of their pfaA genes. They also showed that
the purified TE proteins exhibit a TE activity specific for long-chain fatty acyl-CoAs such as
the purified TE proteins exhibit a TE activity specific for long-chain fatty acyl-CoAs such as palmitoyl-
palmitoyl- or eicosapentaenoyl-CoA.
or eicosapentaenoyl-CoA.
The action of TE as a chain-terminator releases the LC-PUFA molecules synthesized as
The action of TE as a chain-terminator releases the LC-PUFA molecules synthesized as acyl-ACP
acyl-ACP derivatives on the PfaA(ACP). The released free LC-PUFAs must be then activated to form
derivatives on the PfaA(ACP). The released free LC-PUFAs must be then activated to form
LC-PUFA-CoA by acyl-CoA synthetase or LC-PUFA-ACP by acyl-ACP synthetase, which utilizes an
LC-PUFA-CoA by acyl-CoA synthetase or LC-PUFA-ACP by acyl-ACP synthetase, which utilizes an
ACP as an acceptor for free LC-PUFA. Acyl-CoA synthetase genes are found in both Type A and B
bacterial genomes and acyl-ACP synthetase genes are present in Type A, suggesting that in these
bacteria the LC-PUFAs released by TE are loaded onto either ACPs or CoAs by these synthetases
and are then transferred into phospholipids by AGPAT. If this scenario is correct, TE must be an
important enzyme for the PUFA synthase system. Therefore, we propose here to use orf6 to
Mar. Drugs 2016, 14, 94 8 of 23

ACP as an acceptor for free LC-PUFA. Acyl-CoA synthetase genes are found in both Type A and B
bacterial genomes and acyl-ACP synthetase genes are present in Type A, suggesting that in these
bacteria the LC-PUFAs released by TE are loaded onto either ACPs or CoAs by these synthetases and
are then transferred into phospholipids by AGPAT. If this scenario is correct, TE must be an important
enzyme for the PUFA synthase system. Therefore, we propose here to use orf6 to designate the TE
gene, because the corresponding TE gene of P. profundum SS9 has been called as orf6 [28].
In addition, EPA can be synthesized in E. coli recombinants carrying only the pfaA–E genes from
S. pneumatophori but without orf6 [29], indicating that the orf6 gene is not essential for LC-PUFA
biosynthesis. E. coli exhibits minimal acyl-ACP TE activity; therefore, these recombinants would not
have been able to release LC-PUFAs from PfaA(ACP) via endogenous bacterial enzymes, implying
that the heterologously expressed PUFA synthase exhibits LC-PUFA releasing activity, acting as a
TE. This postulated third mechanism is suggested by several studies. First, an E. coli recombinant
expressing the DNA sequence region covering the DH domain of the P. profundum SS9 pfaC gene was
shown to be capable of producing up to a 5-fold increase in TFAs over the negative control, although
the dehydration reaction had not previously been identified as a limiting step in bacterial fatty acid
biosynthesis [30] and overexpression of the native dehydratase from E. coli, FabA, did not increase
the production of fatty acids [31]. Therefore, it was proposed that this DH domain also catalyzes
the reaction of thioester hydrolysis (TE activity). In addition, the corresponding DH domain of the
PfaC family is composed of four contiguous hotdog fold domains, described in detail in Section 2.3,
characteristic of dehydratases and thioesterases and this family of structural domains has elsewhere
been implicated in both DH and TE activity [32].
Furthermore, since the intrinsic TE activity and the distinct orf6 function can be compatible with
each other, some LC-PUFA producing bacteria are likely to contain both of these chain-termination
modes simultaneously.

2.3. Tertiary Structures of the Domains

The determination of three-dimensional enzyme structure helps to understand enzyme kinetics
and reaction mechanisms. Although the conformation of PUFA synthases has not yet been solved,
Baerga-Ortiz and his colleagues have recently reported the tertiary structures of two domains, DH and
ACP, from the PUFA synthase [15,33]. Intensive sequence analysis in the C-terminal region of the pfaC
gene revealed two hidden pseudo-DH domains, which show relatively high sequence similarity to each
other but lack a conserved active site His residue. Each pseudo-DH domain is located immediately
upstream of two DH domains previously known to be related to a FabA DH domain. Furthermore, an
estimated three-dimensional modeling of these DH and pseudo-DH domains showed that not only
the DH domains but also two pseudo-DH domains were predicted to form so-called “hotdog” folds.
This conserved structural motif, which is recognized in many dehydratases such as bacterial FabA and
FabZ and thioesterases, possesses a long central α-helix “sausage” that nestles in a seven-stranded
antiparallel β-sheet “bun” [32]. Although it has not yet been demonstrated experimentally how
these four tandemly arranged hotdog folds are formed into the functional assembly, if these domains
comprise the two pseudodimeric DHs in the LC-PUFA biosynthesis process, each postulated DH
dimer might catalyze different reactions, i.e., trans-2,2-cis isomerization and trans-2,3-cis isomerization,
respectively, required to introduce double bonds.
Normally, each FAS/PKS ketosynthase module contains a single ACP domain whereas PUFA
synthases are characterized by the presence of multiple ACP domains, ranging from two to over
10. An increased number of ACP domains in PUFA synthases appear to contribute to an additive
effect of LC-PUFA yield [34]. Furthermore, in the PKS for curacin biosynthesis in cyanobacteria
evaluation of the contribution of each triplet ACP domains revealed that individual ACP domains
act in parallel and are functionally equivalent [35]. To understand the role of multiple ACP domains,
Trujillo and co-workers reconstructed three-dimensional models of the five ACP domains tandemly
arranged in the PUFA synthase of P. profundum [33]. In this study, small-angle X-ray scattering (SAXS)
Mar. Drugs 2016, 14, 94 9 of 23

analysis was used to predict the solution structure of the tandem ACP [36]. The most plausible model
calculated from the SAXS data shows a monomer form and an elongated beads-on-a-string structure.
In addition, the SAXS analysis revealed that the tandem ACP could adopt several conformations
in solution, suggesting that the structural flexibility of the tandem ACP would have additive and
parallel effects on LC-PUFA production. The crystallization of modular multidomain proteins with
flexible linkers is often problematic owing to their higher flexibility in solution, making it difficult
to analyze their structure by X-ray crystallography. Therefore, the SAXS technique, in which sample
crystallization is unnecessary, and sophisticated modeling programs that utilize SAXS data are likely
to be increasingly applied to the structural determination of flexible multidomain proteins including
PUFA synthases [37].

3. Functions of LC-PUFAs in Bacteria and Their Practical Use

3.1. Involvement of LC-PUFAs in Cold Adaptation

Most LC-PUFA-producing bacteria have been isolated from marine sources such as seawater,
fish, and sediments and have been commonly psychrophilic (psychrotrophic) and/or piezophilic in
nature. Therefore, the functions of LC-PUFA in such bacteria have been discussed in relation to their
adaptation to low-temperature and high-pressure environments. The relevant bacteria spontaneously
increase their content of LC-PUFA like EPA or DHA when grown under lower temperatures or
higher pressures [38]. Therefore, the function of LC-PUFAs in these bacteria has been regarded as the
adjustment of cell membrane fluidity under these conditions [39] although this classical concept has yet
to be experimentally confirmed in either bacterial or eukaryotic LC-PUFA-producing microorganisms.
Recently it was evidenced that EPA is not essential in some EPA-producing bacteria and that the loss of
EPA appears to be readily compensated by increased levels of monounsaturated fatty acids (MUFAs)
or branched chain fatty acids (BCFAs) (see below). For example, the psychrotolerant piezophilic
deep-sea bacterium P. profundum SS9 produces EPA (8% of TFAs) and the content of EPA increases
when grown under low temperatures and high pressures [40]. However, growth inhibition of the
P. profundum SS9 mutant strain EA10, which is defective in EPA synthesis, is completely compensated
in vivo by the increased content of MUFAs. In contrast, the P. profundum SS9 mutant strain EA3, which
is deficient in the production of MUFAs, becomes sensitive to low temperature and high pressure
and this sensitivity is relieved by exogenously added MUFA but not by EPA [40]. In addition, the
EPA-producing psychrotrophic bacterium Shewanella marinintestina (S. marinintestina) IK-1, isolated
from the intestine of a squid, has an EPA content representing approximately 15% of TFAs at 4 ˝ C but
which is almost negligible at 27 ˝ C. We observed that mutant IK-1∆8 cells, deficient in EPA production,
grow as well as the original IK-1 cells even at 4 ˝ C [41], indicating that EPA does not play an important
role in the growth of this species at lower temperature. Together, these studies using EPA-deficient
mutants indicate that EPA is not primarily responsible for adjusting the membrane fluidity at low
temperature or high pressure conditions.
EPA is absolutely required for the growth of bacteria at low temperatures and high pressures
in some Shewanella species. S. livingstonensis Ac10, an isolate from seawater of the Antarctic sea,
is a psychrotrophic bacterium capable of producing EPA [42]. The EPA content of the membrane
phospholipids of S. livingstonensis Ac10 grown at 4 ˝ C is approximately 5% of TFAs whereas the EPA
content is almost negligible at 18 ˝ C. Ac10 EPA-deficient mutants grow as well as the wild-type strain at
18 ˝ C. However, mutant growth is significantly inhibited at 4 ˝ C and this growth inhibition is recovered
only when EPA-containing phospholipids are added into growth medium, suggesting that EPA is
important for S. livingstonensis Ac10 to grow at lower temperature [42]. Shewanella violacea (S. violacea)
strain DSS12 is a deep-sea bacterium isolated from the Ryukyu Trench. It exhibits moderate piezophily
with optimal growth at 30 MPa and 8 ˝ C but it can also grow at 0.1 MPa. S. violacea DSS12 cells
contain a substantial amount of EPA (15% at 8 ˝ C) in their membrane [43]. Usui et al. [44] measured
the dynamic properties of these cells using a new system that enables the measurement of fluorescence
Mar. Drugs 2016, 14, 94 10 of 23

anisotropy under high pressure and demonstrated that in this bacterium EPA appears to structurally
maintain cell membrane rigidity by affecting membrane hydration over a wide range of pressure
conditions. EPA also plays a role in S. violacea DSS12 cell division under high pressure because the cell
shape of an EPA-deficient DSS12 mutant becomes filamentous at 30 MPa. In comparison, Shewanella
piezotolerans (S. piezotolerans) WP3, which was isolated from a sediment sample of the western Pacific
Ocean, is a psychrotolerant and piezotolerant bacterium that grows optimally at 15–20 ˝ C under
pressures of 0.1–20 MPa [45]. This bacterium also requires EPA for its growth at low temperatures and
high pressures. Its EPA content is approximately 14% and 6% of TFAs at 4 ˝ C and at 20 ˝ C, respectively,
and much higher levels of EPA are detected in cells grown under pressures of 20 MPa than under
0.1 MPa. In this bacterium, however, there is a much greater requirement for branched iso-13:0 and
iso-15:0 fatty acids (BCFAs), whose combined levels are approximately 50% and 22% of the total at 4
˝ C and at 20 ˝ C, respectively, than for EPA [46]. This differs from P. profundum SS9, which absolutely

requires MUFAs at low temperatures. Furthermore, the growth retardation of an EPA-deficient

mutant of S. piezotolerans (WP3∆EPA ) at 4 ˝ C is partly compensated by increased levels of BCFAs [46].
Accordingly, the exact function of EPA is still unknown in this bacterium. However, EPA might affect
the activity of the branched-chain amino acid ABC transporter involved in BCFA biosynthesis in
S. piezotolerans WP3, because this transport system is upregulated only at low temperatures [46].
Therefore, although these examples indicate that EPA is required for the growth of various Shewanella
species in low temperature and/or high pressure conditions, its role is likely specific to individual
membrane functions but not to overall membrane fluidity (see below for details).
Certain Schizochytrium and related heterotrophic eukaryotic microorganisms are known to produce
and accumulate notable levels of LC-PUFAs (up to 50% of TFAs) through PUFA synthases encoded
by the bacterial pfa-like genes (Figure 1). These organisms have a capacity to produce LC-PUFAs at
25 ˝ C or much higher temperatures. To examine the role of LC-PUFAs, Lippmeier et al. [47] generated
DHA-deficient mutants of Schizochytrium sp. (ATCC 20888) by disrupting one of the three PFA genes.
The resulting DHA-deficient mutants became auxotrophic to LC-PUFA and required supplementation
of unsaturated fatty acids such as ARA, EPA, and DHA for its normal growth [47]. As Schizochytrium sp.
ATCC 20888 lacks a ∆12 fatty acid desaturase activity, LC-PUFAs synthesized through the pfa-like genes
are therefore absolutely necessary in this organism to maintain the membrane structure and functions
even at moderate temperature, which is in complete contrast with bacteria cells wherein LC-PUFAs
are required only under low temperature and high pressure conditions. However, the function of
LC-PUFAs in Schizochytrium sp. ATCC 20888 and other eukaryotic microorganisms possessing the
pfa-like genes has not been certified.

3.2. Antioxidative Functions of LC-PUFAs

Because ROS such as H2 O2 are freely membrane-permeable compounds, bacteria cope with ROS
attacks by using catalase or other types of ROS scavenging enzymes. Since the cell membrane of
most bacteria is composed of saturated and monounsaturated fatty acids [48], these fatty acids in the
membrane are not regarded as the primary targets of oxidative stress caused by exogenous ROS agents.
In contrast, PUFAs including LC-PUFAs are readily susceptible to oxygen or ROS; thus, bacterial
membranes with LC-PUFAs are considered much more susceptible to oxidants. Notably, however,
bacteria with LC-PUFAs are unexpectedly more resistant to ROS than those without LC-PUFAs [10,49].
The antioxidative function of LC-PUFAs in bacteria was reported for the first time by
Nishida et al. [10]. E. coli recombinants genetically engineered to produce EPA (designated EPA+)
become more resistant to exogenously supplied ROS such as H2 O2 and tert-butyl hydroperoxide
(t-BHP) compared to the parent strain that produces no EPA (designated EPA´). The antioxidative
function of EPA was confirmed using natively EPA-producing S. marinintestina IK-1 and its EPA-less
mutant (IK-1∆8) [50–52]. Recently, Tilay and Annapure [53] successfully isolated EPA-producing
and ARA-producing bacteria from seawater samples by evaluating resistance to H2 O2 . This report
supports the concept of an antioxidative function of not only n-3 LC-PUFAs but also n-6 LC-PUFAs
Mar. Drugs 2016, 14, 94 11 of 23

such as ARA as well. Since the resistance of E. coli DH5α recombinants with EPA to H2 O2 depends on
their cellular levels of EPA [10], relatively high levels of EPA in membrane phospholipids might be
required for the antioxidative activity in bacteria. Consistent with this hypothesis, catalase-deficient
E. coli UM2 mutant cells transformed with the pfaA–E genes can produce EPA at levels of 7%–8% of the
TFAs and become more resistant than the reference strain without EPA [49].
As a possible mechanism responsible for the antioxidative function of EPA and other LC-PUFAs,
a cell membrane-shielding effect of LC-PUFAs has been proposed [50]. In this mechanism, a more
hydrophobic interface (region) of the alkyl chain is suggested to be formed between the phospholipid
bilayers when cell membrane phospholipids are acylated in combination with a PUFA and a
medium-chain saturated or monounsaturated fatty acid [54]. The resulting hydrophobicity renders
it difficult to pass extracellular hydrophilic compounds through the membrane. Simultaneously, the
hydrophobicity allows the ready passage of hydrophobic compounds. Thus, using this relationship
we can evaluate the membrane-shielding effect by measuring its hydrophobicity. To examine this
effect, we obtained several E. coli DH5α recombinant cell lines that exhibit different levels of EPA by
introducing different combinations of the pfa genes and found that the cell line with the highest EPA
level exhibited higher cell hydrophobicity than that with the lowest EPA level [55]. The antioxidative
function of EPA (and of other LC-PUFAs) is predicted to be based on their common structural and
hydrophobic characteristic in the bacterial cell membranes.

3.3. Involvement of LC-PUFAs in Specific Membrane Functions

S. livingstonensis Ac10 requires EPA for proper growth at lower temperatures as described above.
Notably, its EPA-defective mutants display almost the same levels of cell membrane fluidity as that seen
in normal cells [42]. However, the mutant cells at lower temperature show an abnormally elongated
cell morphology during cell division and a significantly lower growth rate [42]. Tracer experiments
in this bacterium at 4 ˝ C using fluorescence-labeled phospholipids containing EPA revealed that the
fluorescent signals localized between two nucleoids at the cell center during cell division, whereas
no particular localization was observed for labeled phospholipids containing oleic acid [56]. This
localization of EPA-containing phospholipids (EPA-PLs) suggests that these are required for cell
membrane curvature at the occlusion site during cell division or, alternatively, that they interact with
membrane proteins at the site of cell division. The latter possibility might be supported by the result
that the outer membrane protein Omp74, which is induced at lower temperatures in S. livingstonensis
Ac10, requires liposomes with EPA-PLs for the acceleration of its proper folding and formation of its
β-sheet structure in vitro [57]. These findings suggest that at lower temperature environments EPA-PLs
act as a chemical chaperon for membrane proteins and contribute to the formation of a microdomain
to facilitate cell division.
Both E. coli DH5α that heterologously express EPA and S. marinintestina IK-1 that intrinsically
produce EPA show increased resistance against antibiotics [51,55]. This multidrug resistance
mechanism might be explained by two possible EPA effects on the cell membrane: One such possibility
is the cell membrane-shielding effect of EPA as described above. The other is an interaction of EPA
with membrane proteins involved in drug resistance. To distinguish between these, we investigated
the influence of EPA on mutants in proteins that affect drug permeation and efflux, and examined
the cell membrane-shielding effect of EPA on these mutants [41]. E. coli expresses the Omp system,
which is involved in membrane permeation and includes the outer membrane porin proteins, OmpC
and OmpF [58]. E. coli also contain the AcrAB-TolC multidrug efflux system [59], which consists of
AcrA, AcrB, and TolC, and utilizes MarA as a regulator for the acrA and acrB genes. We generated
two groups of bacterial lines; the first group (EPA+) included EPA-producing E. coli K12 recombinants
and its mutants deficient in ompC, ompF, acrA, acrB, tolC, or marA, which were transformed with the
pfaA–E genes, and the second group (EPA´) served as a control group that included the same strains
but which were alternatively transformed with the pfaA–D genes without the pfaE gene and thus were
unable to produce EPA. We evaluated the resistance of the EPA+ and EPA´ lines against t-BHP as an
Mar. Drugs 2016, 14, 94 12 of 23

agent for ROS and against antibiotics (ampicillin, kanamycin, chloramphenicol, and nalidixic acid) by
measuring their minimum inhibitory concentration (MIC) values (Figure 3). In antibiotic resistance
experiments, the MIC value of EPA+ K-12 cells was higher than that of EPA´ K-12 cells, consistent
with previous reports [51,55]. The same effects were found even if ompC and ompF mutants were used,
indicating that the antibiotic resistance effect of EPA was not influenced by the Omp system. However,
Mar.
this Drugs was14,
effect2016, 94 detected in the AcrAB-TolC mutants, suggesting that the AcrAB-TolC drug 12
not of 23
efflux
system is required for the action of EPA on antibiotic resistance. In the antioxidative effect experiments,
mutants
all mutantsexhibited the same
exhibited levels
the same of resistance
levels for t-BHP
of resistance as wasasseen
for t-BHP wasinseenwild-type K-12 cells,
in wild-type K-12except
cells,
for marA mutant cells. These results suggest that although the AcrAB-TolC complex
except for marA mutant cells. These results suggest that although the AcrAB-TolC complex system system is not
involved in theinantioxidative
is not involved effecteffect
the antioxidative of EPA, MarA
of EPA, MarAplays a role
plays ininthis
a role thiseffect
effectthrough
through its
its protein
protein
regulatory function. Considering that the AcrAB-TolC system is widely
regulatory function. Considering that the AcrAB-TolC system is widely distributed in Gram-negative distributed in
Gram-negative bacteria including EPA-producing bacteria, similar antibiotic resistance
bacteria including EPA-producing bacteria, similar antibiotic resistance effects of LC-PUFA would be effects of
LC-PUFA
observed alsowould be observed
in bacteria also in bacteria
that natively producethat natively produce LC-PUFAs.
LC-PUFAs.

Figure 3. Effect of tert-butyl hydroperoxide (t-BHP) and various antibiotics on the growth of
Figure 3. Effect of tert-butyl hydroperoxide (t-BHP) and various antibiotics on the growth of Echerichia
Echerichia coli (E. coli) K-12 and its mutants transformed with the clustered pfa genes for EPA
coli (E. coli) K-12 and its mutants transformed with the clustered pfa genes for EPA biosynthesis. To
biosynthesis. To perform the growth inhibition tests, 96-well microtiter plates were used as described
perform the growth inhibition tests, 96-well microtiter plates were used as described previously [51].
previously [51]. The plates were incubated for 4 days at 20 °C. EPA+ and EPA− exhibited EPA
The plates were incubated for 4 days at 20 ˝ C. EPA+ and EPA´ exhibited EPA production and lack
production and lack of production, respectively. Amp, ampicillin; Kan, kanamycin; Cm,
of production, respectively. Amp, ampicillin; Kan, kanamycin; Cm, chloramphenicol; Nal, nalidixic
chloramphenicol; Nal, nalidixic acid. MIC, minimum inhibitory concentration. E. coli K-12 and its
acid. MIC, minimum inhibitory concentration. E. coli K-12 and its mutants used in this study were
mutants used
purchased in the
from thisColi
study were purchased
Genetic from
Stock Center, theUniversity.
Yale Coli Genetic Stock Center, Yale University.

3.4.
3.4. Commercial
Commercial Production
Production and Use of
and Use of LC-PUFAs
LC-PUFAs
LC-PUFAs
LC-PUFAs suchsuch as
as ARA,
ARA, EPA,
EPA, and
and DHA
DHA represent
represent essential
essential nutritional
nutritional factors
factors for
for humans.
humans. In
In
particular, the n-3 LC-PUFAs EPA and DHA are associated with the suppression of various
particular, the n-3 LC-PUFAs EPA and DHA are associated with the suppression of various diseases diseases
such ascardiovascular
such as cardiovascularhealth
health [60],
[60], Alzheimer’s
Alzheimer’s disease
disease [61],[61], allergic
allergic diseases
diseases [62,63][62,63] and [64,65],
and cancer cancer
[64,65], and therefore many countries recommend daily EPA and DHA intakes of 0.3–0.5 g [66]. The
main source of EPA and DHA in the human diet is sea-fish oil. However, there is a limit to the
amount of fish that can be sustainably harvested from available resources. Therefore, readily
available and sustainable alternatives in place of fish oil are required.
Bacteria and eukaryotic microorganisms have been regarded as a promising source of
Mar. Drugs 2016, 14, 94 13 of 23

and therefore many countries recommend daily EPA and DHA intakes of 0.3–0.5 g [66]. The main
source of EPA and DHA in the human diet is sea-fish oil. However, there is a limit to the amount
of fish that can be sustainably harvested from available resources. Therefore, readily available and
sustainable alternatives in place of fish oil are required.
Bacteria and eukaryotic microorganisms have been regarded as a promising source of LC-PUFAs.
The benefits to using microorganisms include a stable supply of LC-PUFAs and the fact that microbial
oils are not associated with marine pollution, fish odor, or fish allergies. Currently, some LC-PUFAs
are produced industrially using eukaryotic microorganisms in the US and in EU countries. However,
LC-PUFA-producing bacteria have not generally been utilized commercially because of their low
LC-PUFA productivity.
In comparison, photosynthetic microalgae such as Nannochloropsis oculata (N. oculata) and
Phaeodactylum tricornutum (P. tricornutum) accumulate EPA in triacylglycerols (TAGs) and EPAs
extracted from N. oculata are now commercially available [67]. The levels of EPA produced by the
genetically engineered N. oculata ST-6 represent 38%–39% of TFAs under optimal conditions and
2%–3% of its dry cell weight (DCW) is occupied by EPA [68]. For P. tricornutum UTEX 640, EPA
represents 31% of TFAs and 5% of DCW [69]. On the other hand, the soil fungus Mortierella alpina
1S-4 [70], which was first isolated as a remarkably ARA-accumulating organism [71], and the budding
yeast Saccharomyces cerevisiae FS01699 [72] are now being utilized to increase EPA productivity through
genetic modifications. In these microorganisms, LC-PUFAs are most likely produced by the common
combination of oxygen-dependent desaturation and fatty acid elongation but not by PUFA synthase.
In addition, various kinds of heterotrophic microalgae such as Aurantiochytrium limacinum
SR21 [73], thraustochytrid strain 12B [74], Ulkenia sp., and Crypthecodinium cohnii (C. cohnii) [67] are
known as good DHA sources. DHA from Schizochytrium sp., Ulkenia sp., and C. cohnii is commercially
purified; these exhibit high levels of DHA contents reported as 45%–52% of TFAs and 20%–24% of
DCW for Schizochytrium sp. [75], 10%–23% of TFAs and 5% of DCW for Ulkenia sp. [76], and 53%–57%
of TFAs and 5%–6% of DCW for C. cohnii ATCC 30556 [77]. It should be noted that some of these
microalgae also contain homologs of PUFA synthase [47,78,79] (Figure 1).
As a potential substitute for EPA and DHA supplied in TAG forms, the phospholipid (PL) forms of
EPA and DHA derived from Antarctic krill (Euphausia superba) oil have attracted considerable amounts
of attention because these are considered to be more effective as a nutritional source for human health
than are the TAG forms of EPA and DHA that are rich in fish oil [80–82]. However, the use of krill as a
sustainably available resource of LC-PUFAs is limited. Notably, the TAG accumulating DHA and ARA
forms can be easily converted in vivo to the PL form in cells of the DHA-producing thraustochytrid
strain 12B [83] and in an ARA-producing fungus [84], respectively, by the glucose starvation culture
technique in which the cells are first cultivated in high concentrations of glucose (or other carbon
sources) to accumulate LC-PUFA-rich TAG followed by cultivation in medium supplemented with no
glucose. This technique is considered promising to produce various kinds of LC-PUFA such as the
PL form.
Overall, the production of LC-PUFAs using microorganisms is considered to be both a feasible
and a promising technology. DHA-enriched milk from cattle [85], the muscle tissue of pigs [86],
and the egg yolk of layers (hens) [87] are produced using microbial DHA as feedstuffs. The only
task to be overcome for the effective production of microbial LC-PUFAs is the reduction of its cost.
Furthermore, considering that the lipid and fatty acid compositions of heterotrophic microorganisms
such as Schizochytrium and Aurantiochytrium, which are capable of producing LC-PUFAs via PUFA
synthase, are much simpler than those of fish and krill, these microorganisms might be more suitable
for the production of highly purified LC-PUFAs as a fine chemical.
Mar. Drugs 2016, 14, 94 14 of 23

4. Evolution of C31:9 and LC-PUFA-Producing Bacteria

4.1. Possible Role of LC-HCs in Anaerobic Environments

EPA and DHA are often called “marine lipids” owing to their preferential distribution in marine
environments [52]. However, recent studies show that the pfa genes encoding the PUFA synthase
proteins and their homologs are also widely distributed in terrestrial bacteria including aerobic bacteria
such as Rhodococcus spp. and strictly anaerobic bacteria including Geobacter and Desulfobacterium
spp. [16].
Furthermore, some bacteria classified as obligate anaerobes are capable of synthesizing ROS
scavenging enzymes such as superoxide dismutases, catalases [88–90], superoxide reductases, and
peroxidases [91–93]. The NADH:rubredoxin oxidoreductase (NORO) complex is found in Clostridium
species and an oxygen reductase is present in Desulfovibrio vulgaris to reduce cytoplasmic oxygen, NO,
and ROS [94]. Geobacter sulfurreducens can also grow using oxygen as a terminal electron acceptor
and can tolerate long-term exposure to oxygen [95]. These findings suggest that the ability to tolerate
oxygen exposure is an important feature for the survival of these anaerobic bacteria when they are
transiently exposed to aerobic circumstances [95,96].
The metal-reducing obligate anaerobic bacterium G. bemidjiensis BemT , which was isolated from
a terrestrial aquifer, carries the pfaD, A, B, C, oleA, B, C, D and pfaE genes in tandem (Figure 1).
Although the metabolites produced by these pfa and ole gene products in the anaerobic bacterium are
largely unknown, G. bemidjiensis BemT has been shown to produce a very long chain polyunsaturated
hydrocarbon hentriacontanonaene (C31:9) [97]. It is speculated that PUFA synthases are involved in the
production of intermediate polyunsaturated fatty acids and that these are subsequently transformed
into C31:9 by the Ole proteins [12,13]. Although no direct evidence has been available, C31:9 and
related LC-HCs of obligate anaerobic bacteria might be responsible for providing an antioxidative
function such as performed by the LC-PUFAs in facultatively anaerobic bacteria such as Shewanella
species when oxygen or exogenous ROS are encountered [50–52].
Finally, manganese and iron reduction in sediments are known to play important roles in the
biogeochemical cycle of many elements including carbon, sulfur, phosphorus, and several trace
elements [98]. The process by which microorganisms couple the reduction of manganese or iron oxides
to the oxidation of organic compounds (or H2 ) is often referred to as dissimilatory Fe(III) or Mn(IV)
reduction [99]. Microbial manganese and iron reduction coupled to organic matter oxidation requires
the direct contact of bacteria with the oxide surface [100]. Iron-reducing bacteria are assumed to exist in
the aerobic-anaerobic interface where bacterial Fe(II) oxidation and Fe(III) reduction activities are likely
to have coexisted within the same horizon [101]. Fe(II) is also subject to spontaneous chemical oxidation
by oxygen and Mn(IV) oxides at circumneutral pH [102–105]. A conceptual model of this interface is
depicted in Figure 4a. G. bemidjiensis BemT for example is assumed to exist in the aerobic-anaerobic
interface in sediments, where the bacterium could utilize Fe(III) as the terminal electron acceptor, and
where it, in turn, is likely to be exposed to oxidative stress. One possible explanation regarding why
G. bemidjiensis BemT possesses the ole and pfa genes might be that this bacterium utilizes LC-HCs to
protect itself against the occasional incursion of oxidative stress in the aerobic-anaerobic interface.
Specifically, the pfa-like and ole genes might be evolutionarily obtained and conserved to survive under
such an environment. Although this notion is still speculative and studies on the relationship between
oxidative stress and LC-PUFAs and LC-HCs in anaerobic bacteria remain rudimentary, it is likely that
further examination of the antioxidative functions of LC-PUFAs and LC-HCs in anaerobic bacteria
might contribute to the elucidation of the evolutionary process of pfa-like genes.
oxidative stress in the aerobic-anaerobic interface. Specifically, the pfa-like and ole genes might be
evolutionarily obtained and conserved to survive under such an environment. Although this notion
is still speculative and studies on the relationship between oxidative stress and LC-PUFAs and
LC-HCs in anaerobic bacteria remain rudimentary, it is likely that further examination of the
antioxidative functions
Mar. Drugs 2016, 14, 94 of LC-PUFAs and LC-HCs in anaerobic bacteria might contribute to the
15 of 23
elucidation of the evolutionary process of pfa-like genes.

Mar. Drugs 2016, 14, 94 15 of 23

Figure 4. (a) Schematic diagram of possible functions of LC-PUFAs and LC-HCs. The conceptual
Figure 4. (a) Schematic diagram of possible functions of LC-PUFAs and LC-HCs. The conceptual
model of the aerobic-anaerobic interface is taken from Roden et al. [101]. Metal-reducing bacteria
model of the aerobic-anaerobic interface is taken from Roden et al. [101]. Metal-reducing bacteria
(FeRB) harboring the pfa-like and ole genes (for example, Geobacter bemidjiensis BemT) Tare assumed to
(FeRB) harboring the pfa-like and ole genes (for example, Geobacter bemidjiensis Bem ) are assumed
produce LC-PUFAs and/or LC-HCs. Those genes might have been evolutionarily obtained (via
to produce LC-PUFAs and/or LC-HCs. Those genes might have been evolutionarily obtained (via
HGT?) or conserved as descendant genes from ancestral bacteria that harbored the pfa-like and ole
HGT?) or conserved as descendant genes from ancestral bacteria that harbored the pfa-like and ole
genes. FeRBs utilize Fe(III) (amorphous Fe(III) oxide) as the terminal electron acceptor and reduce it
genes. FeRBs utilize Fe(III) (amorphous Fe(III) oxide) as the terminal electron acceptor and reduce it to
to Fe(II) under an anaerobic environment. Fe(II), in turn, is oxidized by iron-oxidizing bacteria
Fe(II) under an anaerobic environment. Fe(II), in turn, is oxidized by iron-oxidizing bacteria (FeOB) or
(FeOB) or
oxygen (O2oxygen (O2). The environment
). The environment becomes
becomes more more
aerobic asaerobic as it is separated
it is separated from the
from the center center
of the of
circle
the circle environment;
(anaerobic (anaerobic environment;
brown circle).brown
FeRBscircle). FeRBstoare
are exposed exposed
oxidative to at
stress oxidative stress at the
the aerobic-anaerobic
aerobic-anaerobic interface, which might be potentially alleviated by LC-PUFA
interface, which might be potentially alleviated by LC-PUFA and/or LC-HCs. (b) A possible and/or LC-HCs. (b)
route
A possible route for conservation of the pfa-like gene in anaerobic bacteria. This conceptual
for conservation of the pfa-like gene in anaerobic bacteria. This conceptual model, though speculative, model,
thoughthe
shows speculative,
possibilityshows
of the the possibility
pfa-like of the
gene being pfa-like in
harbored gene being harbored
anaerobic bacterium incapable
anaerobic bacterium
of producing
capable ofand/or
LC-PUFA producing LC-PUFA
LC-HCs. and/or LC-HCs.
For comparison, Forroutes
possible comparison, possible
of pfa-like routes of pfa-like
gene conservation gene
in marine
conservation in marine and aerobic bacteria are
and aerobic bacteria are also depicted in this figure. also depicted in this figure.

4.2. Evolution of pfa-Like Genes

Shulse and Allen argue that the patchy distribution of secondary lipid pathways among
Mar. Drugs 2016, 14, 94 16 of 23

4.2. Evolution of pfa-Like Genes

Shulse and Allen argue that the patchy distribution of secondary lipid pathways among bacteria
and eukaryotic phyla might be attributed to horizontal gene transfer (HGT) [16]. We discuss here one
possibility of HGT of the pfa and ole genes in bacteria from the viewpoint of the defense mechanisms
against oxidative stress in surface and subsurface environments (Figure 4b).
HGT of the pfa-like and ole-like gene prototypes is assumed to have occurred frequently in the
ancient Earth environment among a wide variety of bacterial phyla [15]. An example of HGT of the pfa
gene cluster is reported to be present in one strain of Vibrio spp. but absent in many closely related
Vibrio genomes [15]. Comparing the pfa gene region of Vibrio splendidus 12B01 with corresponding and
flanking regions of the genomes of other Vibrio species, it was concluded that the pfa gene cluster had
clearly been inserted into a genome island [15]. Polz et al. pointed out that the horizontal acquisition
of genes in bacteria is frequently observed within particular functional categories, which include
many surface structures such as O-antigen or membrane-spanning transporters as well as secondary
metabolite production/modification [106]. They hypothesized that the horizontally-acquired genes
might ensure resistance to rapidly changing biological factors such as immune defenses or viral
and protozoan predation and confer competition interference and predation resistance [106]. Since
PKS systems are regarded as secondary-metabolite gene clusters [107], it is likely that some bacteria
harboring the pfa-like genes have acquired them by HGT. A prototype of the pfa-like and ole-like genes,
which might have been similar to the contemporary pfa-like and ole-like genes, might have existed in
primeval anaerobic bacteria that are assumed to have synthesized substances similar to LC-PUFAs and
LC-HCs. Some facultative and strict anaerobic bacteria such as Colwellia psychrerythraea, S. oneidensis,
and G. bemidjiensis BemT might have continued to maintain the pfa-like gene prototype, which might
have rendered them a slight advantage over other bacteria in their environmental niche; for example,
conferring a protective role against oxidative damage as the atmospheric O2 concentration increased.
On the other hand, some strict and facultative anaerobic and aerobic bacteria might not have acquired
them from the start or might have lost them during the course of evolution (Figure 4b). It is possible
that the pfa genes found in Clostridium thermocellum, Desulfobacterium autotrophicum, and Rhodococcus
erythropolis might be remnant genes [12]. Furthermore, given that no aerobic bacteria that synthesize
LC-PUFAs and/or LC-HCs by the Pfa and Ole systems have yet been reported, almost all aerobic
bacteria might have lost the activity to synthesize these compounds via the anaerobic pathway.
Some cyanobacteria, which are oxygenic and harbor a pfa-like gene, hglE [108,109], might
instead have started to synthesize other specialized substances such as heterocyst glycolipids at
the commencement of nitrogen fixation. Heterocyst glycolipids serve to protect the oxygen-sensitive
nitrogenase enzyme in such specialized cells, termed heterocysts [110]. The losses of the pfa-like genes
and the ability to synthesize LC-PUFA and/or LC-HC might be frequently observed in aerobic bacteria
as they likely were substituted for other types of oxidative stress defense mechanisms such as catalase
and superoxide dismutase.

5. Conclusions and Perspectives

The pfa genes with their diverse cluster organization and domain structure are widely distributed
among bacteria and eukaryotic algae. These bacteria include those categorized as marine and terrestrial
bacteria, gram-negative and gram-positive bacteria, strictly and facultatively anaerobic, and strictly
aerobic bacteria. In marine environments, the pfa genes have been found in algae that belong to two
different heterokont and haptophyte groups (phyla). The pfa genes are considered to encode the PUFA
synthase responsible for LC-PUFA biosynthesis via the so-called anaerobic pathway in bacteria and
algae. However, the production of LC-PUFAs has been confirmed only in limited numbers of isolated
bacteria and algae and the synthesized LC-PUFA molecular species appear to depend on the type of pfa
cluster. For example, in most bacteria, the type A, type B, and type D cluster organizations are involved
in the synthesis of EPA, DHA, and ARA, respectively; with the exception of some myxobacterial species
that exhibit type L organization yet also have the ability to produce ARA, EPA, and DHA. Notably, the
Mar. Drugs 2016, 14, 94 17 of 23

AGPAT functional domain is accommodated only in the type L pfa gene cluster (pfa3, see Figure 1) of
myxobacteria. Eukaryotic algae that carry the type E organization principally produce DHA.
The full set of pfa genes encoding the PUFA synthase is believed to be responsible primarily for the
synthesis of LC-PUFAs, the mechanism of which can be relatively easily deduced from the functional
domain structure of the individual Pfa proteins as these domain structures are quite similar to those
of the known type I and type II FASs and those of the various types of PKSs. In this article, we first
proposed mechanisms for the chain-termination of LC-PUFA biosynthesis in bacteria [111]. The most
probable chain-termination in the LC-PUFA synthesis process likely occurs by their release from the
ACPs accommodated in the PfaA protein by the thioesterase encoded by the newly designated orf6
gene, which is present proximately upstream the pfaA gene of the type A and type B clusters in some
bacteria. In addition, in the Schizochytrium system a thioesterase domain integrated within the PUFA
synthase has been reported to participate in its LC-PUFA synthesis [111]. Further characterization of
the Orf6 protein is required to specify its involvement in LC-PUFA synthesis.
The in situ role of the pfa genes is still unknown in almost all organisms. One of the primary and
common functions of the pfa genes might be the synthesis of medium chain PUFAs, which can be
used as a substrate of the Ole proteins to generate LC-HCs such as C31:9. This is suggested because
almost all isolated bacteria that synthesize LC-PUFAs produce C31:9 whereas some bacteria including
strict anaerobic bacteria are capable of producing C31:9 but not LC-PUFAs. Therefore, the pfa genes
are likely to primarily synthesize C31:9 or other types of polyunsaturated HCs. Detailed analysis of
the products generated by the Pfa proteins in pfa-carrying bacteria could be expected to allow a more
thorough understanding of the relationship between the pfa genes and their in situ products. Similarly,
the recent analysis of the structure of the tandem ACP domains existing within PfaA by the small-angle
X-ray scattering method suggested that the structural flexibility of the tandem ACP contributes in an
additive and parallel manner toward LC-PUFA production. The continued application of additional
new methods using purified PUFA synthase and individual Pfa proteins is necessary to clarify their
in vivo functions.
The global but patchy ecological distributions of LC-PUFA-producing bacteria and eukaryotes
that carry pfa genes are both interesting and informative. In bacteria, pfa genes but not LC-PUFAs are
commonly distributed in both strict and anaerobic bacteria and in aerobic bacteria as well. In the latter,
no LC-PUFA products generated by the pfa genes have been reported, whereas some strictly anaerobic
bacteria produce C31:9 using both the Pfa and Ole protein systems. Considering these findings and
the antioxidative function of LC-PUFAs in bacteria, we assumed the evolutionary movement of pfa
genes via HGT from hypothetical ancestral anaerobic bacteria to the existing strictly and facultatively
anaerobic bacteria and aerobic bacteria that carry pfa genes. Of these, the aerobic bacteria would likely
not be required to produce LC-PUFAs as a membrane phospholipid component or as medium chain
PUFAs as a precursor of C31:9 or other polyunsaturated HCs via Pfa and Ole to protect themselves
against ambient oxygen or exogenous ROS. This is because oxic challenges to aerobic bacteria always
exceed their capacity to reduce the toxicity of oxygen by LC-PUFAs or LC-HCs and they are capable of
synthesizing any PUFAs via the aerobic pathway (by aerobic desaturation and elongation of the fatty
acids) even if these compounds are needed.
A recently obtained realization of the physiological role of LC-PUFAs in bacteria is that of their
participation in specific membrane functions rather than in the adjustment of the physical fluidity of the
whole cell membrane. The newly-understood functions of LC-PUFAs, which have been specified using
EPA-producing bacteria and their EPA-deficient mutants as well as EPA-producing E. coli recombinants
carrying pfa genes, include an antioxidative activity against exogenous ROS, membrane efflux activity
toward antibiotics, and microdomain formation responsible for cell division at low temperatures
and/or high pressures. In addition to the techniques describe above, artificial membrane systems
reconstituted with selected purified membrane proteins and phospholipids containing LC-PUFAs as
their acyl components might also serve to further elucidate the physiological roles of LC-PUFAs.
Mar. Drugs 2016, 14, 94 18 of 23

In addition to their functions in bacteria and eukaryotic microorganisms, LC-PUFAs have

considerable nutritional and pharmaceutical value as well. A better understanding of the specifics of
LC-PUFA production and function in specific microorganisms will likely facilitate the development of
cost-effective and sustainable commercial production protocols of LC-PUFAs, as well as providing
clues toward the potential pharmacologic utilization of these compounds in novel areas as further
potentially unexpected functions are discovered.

Acknowledgments: The S. pneumatophori SCRC-2738-derived pfa genes and S. marinintestina IK-1 used in this
study were provided by the Sagami Chemical Research Center, Japan and by Yutaka Yano of Hokkaido National
Fisheries Research Institute, Fisheries Research Agency, Japan, respectively. This study was partly supported by
the National Institute of Polar Research through General Collaboration Projects No. 27-23 to Kiyohito Yoshida,
Mikako Hashimoto, and Naoki Morita, and No. 27-24 to Yoshitake Orikasa and Kiyohito Yoshida.
Conflicts of Interest: The authors declare no conflicts of interest.

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