Biochimie 86 (2004) 807815

www.elsevier.com/locate/biochi

Fatty acid biosynthesis in microorganisms being used for

Single Cell Oil production
Colin Ratledge *
Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK
Received 1 September 2004; accepted 27 September 2004
Available online 19 October 2004

Abstract
Single cell oils (SCOs) are now produced by various microorganisms as commercial sources of arachidonic acid (ARA) and docosahexaenoic acid (DHA). These oils are now used extensively as dietary supplements in infant formulas. An understanding of the underlying
biochemistry and genetics of oil accumulation in such microorganisms is therefore essential if lipid yields are to be improved. Also an
understanding of the biosynthetic pathways involved in the production of these polyunsaturated fatty acids (PUFAs) is also highly desirable as
a prerequisite to increasing their content in the oils. An account is provided of the biosynthetic machinery that is necessary to achieve oil
accumulation in an oleaginous species where it can account for lipid build up in excess of 70% of the cell biomass. Whilst PUFA production
in most microorganisms uses a conventional fatty acid synthase (FAS) system followed by a series of desaturases and elongases, in
Schizochytrium sp., and probably related thraustochytrid marine protists, PUFA synthesis now appears to be via a polyketide synthase (PKS)
route. This route is discussed. It clearly represents a major departure from conventional fatty acid biosynthesis, possibly as a means of
decreasing the amount of NADPH that is needed in the overall process.
2004 Elsevier SAS. All rights reserved.
Keywords: Fatty acid synthase (FAS); Lipid metabolon; Malic enzyme; Polyketide synthase (PKS); Polyunsaturated fatty acids

1. Introduction
The production of microbial oilsotherwise referred to
as Single Cell Oils (SCO)is now an economic reality.
Although such entities have long been suggested as viable
alternatives to plant oils and fats, the first commercial production of an SCO did not begin until 1985 and this only
lasted for 6 years before it was closed down as no longer
being cost-effective. This oil was produced using Mucor circinelloides and was designed to be rich in the polyunsaturated fatty acid (PUFA), c-linolenic acid (GLA; 18:3, n-6),
for use as an alternative to the then expensive evening primrose oil [1]. The process involved the fungus being grown in
stirred-tank fermenters of 220 m3 capacity with subsequent
harvesting and drying of the biomass, followed by oil extraction and its final refinement and purification. Although the
production did not last many years, it nevertheless established that microorganisms could be realistic commercial

alternatives to some plant oils and that the microbial oil itself
could be extracted and purified using conventional technology without the need for expensive, purpose-built units dealing only with microbial oils. All that remained for SCOs to
re-emerge as economically viable oils in their own right was
to identify an oil, or oils, that were not easy to produce agriculturally and which were intrinsically expensive: the microbial oil had to be saleable at a high price in order that it could
cover the costs of its production. These requirements are now
met by the production of very long chain PUFA which do not
occur to any extent in plant oils and which, until the development of SCO processes could only be obtained from marine
animal sources. These fatty acids are now established as important dietary nutrients for neonatal babies and their mothers and consequently a large market is available to ensure
their worldwide sales [2].

2. Microbial polyunsaturated fatty acids

* Corresponding authors. Tel.: +44-14-8246-5243;
fax: +44-14-8246-5458.
E-mail address: c.ratledge@hull.ac.uk (C. Ratledge).
0300-9084/$ - see front matter 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.biochi.2004.09.017

During the late 1980s and early 1990s, it became clear that
PUFAs were of increasing interest in nutrition and especially

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C. Ratledge / Biochimie 86 (2004) 807815

in the development of newly-born babies. Clear clinical evidence began to accumulate that indicated that docosahexaenoic acid (DHA; 22:6, n-3) and arachidonic acid (ARA;
20:4, n-6) were of particular importance as these fatty acids
formed the greater majority of fatty acids found in brain tissue and were also present in mothers milk but absent from
both cows milk and infant formulas that are frequently used
in place of mothers milk. It was then established that both
these fatty acids were involved in the development of neural
and retinal functions and could thus be of benefit to babies for
them to achieve improved memory and eyesight [37]. The
problem was where one might obtain sufficient quantities of
these PUFAs to offer as nutritional supplements.
Although DHA has long been known to be a major fatty
acid of fish oils, it always occurs along with eicosapentaenoic acid (EPA; 20:5, n-3) which was contra-indicated to
be included in infant diets as it affects the uptake of DHA [8]
which is crucial for neural development. Also, there were,
and still are, severe doubts about the use of fish oils as
supplements because of the presence of environmental manmade pollutants, such as dioxins, PCBs and heavy metals including mercury compounds being taken up by fish and concentrated in the liver and other organs. Indeed some
countries, most conspicuously the USA, have an outright ban
on fish oils being given to infants and young children for

these very reasons. As the need for DHA in particular became apparent, it was appreciated by one commercial company (Martek Inc., in the USA) that DHA was a major component of some algal lipids and, if a likely organism could be
found that could be grown in large-scale industrial fermentors, then a source of DHA might be realisable [9]. Simultaneously, it was also known that ARA could be obtained from
microbial sources as work carried out in Japan in the late
1980s had shown this PUFA to be a major component of the
oil from Mortierella alpina [10].
To-day, there are at least three different fermentation processes each using a different microorganism for the production of DHA. For the production of ARA, various strains of
M. alpina are used in separate processes in Europe, China
and possibly Japan. These processes, together with the production organisms involved, are summarised in Table 1. For
inclusion in infant formulas, the ARASCO and
DHASCO are mixed together in the ratio of 2:1 and sold
under the trade name of Formulaid. This is incorporated into
formula preparations in over 60 countries worldwide; approximately 700 tons of this oil mixture was produced in
2003 and the expectation is that this will exceed 1000 tons in
2004 [11].
It is of importance to note that the organisms used in the
SCO processes all have very high contents of the desired

Table 1
Fatty acid profiles (given as relative % w/w) of SCOs in current production
14:0
16:0
18:0
18:1
(A) ARA-SCO processes using M. alpina strains
0.4
8
11
14
DSM
process a
0.2
6
2
4
Wuhan
Alking
process b
12:0
14:0
16:0
16:1
(B) DHA-SCO processes
4
20
18
2
Martek
process c
(DHASCO)
13
29
12
Omega-
Tech
process d
(DHASCO-S)

3
30

Nutrinova
process e
(DHActive)

18:2

18:3 (n-6)

20:3 (n-6)

20:4 (n-6)

22:0

24:0

49

70

18:0

18:1

18:2

18:3 (n-3)

20:3 (n-6)

22:5 (n-6)

22:6 (n-3)

0.4

15

0.6

39

12

25

11

46

a
The DSM process is run in Italy using fermenters of about 100 m3 capacity. The oil content of the cells is considered to be over 40% (w/w). It is sold
exclusively to Martek Biosciences Corp. and is mixed at a ratio of 2:1 (v/v) with DHASCO for incorporation into infant formulas under the trade name
Formulaid. Current production (2003) was 480 tons [11].
b
This is run by Wuhan Alking Engineering Co. Ltd., Wuhan, China. The process is run in fermenters of 50100 m3 capacity. Information about the process
and the oil is limited but refer to: http://www.alking.com.cn/english/aboutus.htm. It has, however, been recently (September 2004) announced that the US
agri-food company, Cargill, is to begin marketing this fatty acid probably outside USA and Europe.
c
Uses C. cohnii which produces over 40% of its biomass as oil. The process is run by Martek Biosciences Corp. using a number of fermenters each about
100 m3. The oil, together with the ARA oil, is used exclusively for infant formulas. Current production (2003) was 240 tons [11].
d
Uses Schizochytruim sp. This oil, formerly known as DHAGold is produced by OmegaTech Inc., Boulder, CO, now owned by Martek. The oil content of the
cells is over 40%. The oil is currently aimed at the adult nutritional supplement market although it has been previously used for improvement of poultry and
other animal feeds. Approximately 10 tons of oil were produced in 2003.
e
Uses Ulkenia sp., which is grown in a 80 m3 fermenter. The oil is produced by Nutrinova GmbH, Frankfurt, Germany and is sold under the trade name of
DHActive [23].

C. Ratledge / Biochimie 86 (2004) 807815

fatty acid as the main, if not sole, PUFA. For example,

Crypthecodinium cohnii, which is used in the Martek process, produces a triacylglycerol oil in which DHA can reach
between 40% and 50% of the total fatty acids and, moreover,
occurs as the sole PUFA; indeed the only other unsaturated
fatty acid present in this oil is a relatively small percentage of
oleic acid (18:1). These oils therefore have a unique characteristic and are completely unlike oils obtained from plants
or animals where there is always a range of both saturated
and unsaturated fatty acids. Where the dietary requirement is
for a single PUFA, this can only be met by a microbial oil.
In the space of 20 years, we have therefore gone from
SCOs being academic curiosities to them being major suppliers of key PUFAs for infant nutrition. There is also great
interest in using these materials as dietary supplements for
adults, and especially in the use of DHA-rich oils, to help
prevent coronary heart problems. There have also been recent suggestions that DHA may also be useful as a dietary
supplement to prevent the onset of such degenerative disorders as Alzheimers disease [12] and even to be useful for the
treatment of attention deficit syndrome in children [13]. In
all cases, the need is for a DHA-only oil and this therefore
means using an SCO.
Details concerning the various SCO processes as outlined
in Table 1 have been recently assembled in a monograph on
the topic [2] and readers wishing more information on any of
these issues are therefore directed to this monograph as the
most recent and comprehensive account of this subject.

3. The biochemistry of oil-accumulation

Of crucial importance in the further development of the
SCO processes is the understanding of how microorganisms
synthesise their fatty acids and how they are able to accumulate so much oil. In some microorganisms, the content of oil
in a microorganism can exceed 70% of the biomass. Moreover, the accumulated oil is almost invariably in the form of
triacylglycerols, which is exactly the same form in which
plant oils occur. From this information, it should be a logical
step to identify the genes coding for the key enzymes that
contribute to fatty acid synthesis and accumulation. This
should lead to the manipulation of the microorganisms to increase their lipid levels and/or to increase the content of the
major PUFAs within the lipid. This review is then aimed at
providing a short synopsis of where we stand with this information. It is though appreciated by all those involved with
microbial oils that ultimately the cheapest way of providing
them will be via plant crops. However, as stated above, no
plant produces any PUFA longer than C18 and, therefore, to
achieve such production genetic manipulation of plants will
be necessary. However, the current public outcry against
genetically-engineered plants might indicate that it could be
many years before such GM oils, assuming that they could
be created, are allowed to be sold to the general public sale,
particularly as the majority of the oils would be destined for

809

inclusion in infant formulas. A discussion of these issues

though, is beyond the scope of this review.
Although all living organisms must synthesise a minimum amount of lipid for their membranes and other structural and functional roles, only a relatively small number of
microorganisms are able to accumulate lipid above about
20% of their cell mass where it functions as a reserve storage
material. Bacteria, in general, do not produce triacylglycerols but, instead, produce poly-b-hydroxy-butyrates and
-alkanaoates as storage polymers. Oil accumulation is therefore only found in some yeasts and fungi together with a
smaller number of algae. These are termed as the oleaginous
species.
At first inspection, the fatty acid biosynthetic pathway in
most oleaginous microorganisms is the same as is found in
non-oleaginous species as exampled by Saccharomyces cerevisiae. There is, however, a crucial difference between the
oleaginous and non-oleaginous organism.
In order to achieve lipid accumulation in a microorganism, it needs to be grown in a medium with an excess of carbon substrate and a limiting amount of nitrogen (although
other nutrients can be made limiting, nitrogen is the usual
one that is used in this context). Thus when the organism
grows, it quickly exhausts the supply of nitrogen but it continues to assimilate the carbon source (usually glucose or an
alternative carbohydrate). This is then channelled directly
into lipid synthesis with the resulting build up of triacylglycerols within the cell as discrete oil droplets. Oil accumulation
may reach over 70% of the cell biomass but not in every oleaginous species. Non-oleaginous microorganisms, by definition, do not accumulate lipid. When placed in the same
nitrogen-limiting growth medium, non-oleaginous microorganisms either tend to cease further cell proliferation or, if
they continue to assimilate the available carbohydrate substrate, then this is diverted into various polysaccharides, including glycogen and various glucans, mannans, etc. Oil accumulation, beyond a very small level (usually less than 10%
of the biomass), does not occur.
Therefore, it can be concluded that the ability of an organism to accumulate large quantities of oil must lie outside the
immediate area of fatty acid biosynthesis, as this biosynthetic machinery is common to all microorganisms. The reasons for oleaginicity would appear to be twofold:
The ability to produce a continuous supply of acetyl-CoA
directly in the cytosol of the cell as a necessary precursor
for fatty acid synthetase (FAS), and,
the ability to produce a sufficient supply of NADPH as the
essential reductant used in fatty acid biosynthesis.
The formation of acetyl-CoA in oleaginous microorganisms has been attributed to the presence of ATP:citrate lyase
(ACL, reaction no. 1) which does not appear to occur in the
majority of non-oleaginous species:
Citrate + CoA + ATP acetyl CoA +
oxaloacetate + ADP + Pi

(1)

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C. Ratledge / Biochimie 86 (2004) 807815

to operate efficiently, its substrate, namely citric acid must

itself be made readily available and, moreover, to be available in the cytosol of the cell where fatty acid sythesis occurs.
Citric acid is, of course, synthesised as part of the tricarboxylic acid (TCA) cycle within the mitochondrion of the
cell. (As stated above, all oleaginous microorganisms are eukaryotes and so have mitochondria.) The feature that is
unique to the oleaginous microorganisms, and which allows
citric acid accumulation, is that the activity of isocitrate dehydrogenase as a component of the TCA cycle is dependent
on the presence of AMP; no such dependency occurs with
the enzyme from non-oleaginous microorganisms. The concentration of AMP itself is regulated by the activity of AMP
deaminase (reaction no. 2):

AMP inosine 5 monophosphate + NH3

(2)

It is this enzyme whose activity is up-regulated at the onset of nitrogen limitation in the growth medium of the oleaginous microorganism possibly as a means of trying to scavenge additional ammonium ions from intracellular materials.
Nitrogen limitation in the cultivation of an oleaginous microorganism induces a cascade of reactions leading to the
formation of acetyl-CoA:
At the onset of nitrogen exhaustion, oleaginous cells show
an increased activity of AMP deaminase, which is up to
fivefold greater than in cells before N limitation.
The increased activity of AMP deaminase decreases the
cellular content of AMP, including its content in the mitochondrion.

The diminished content of AMP in the mitochondrion

stops isocitrate dehydrogenase from working as in oleaginous cells, this enzyme is strictly dependent on AMP for
activity.
As a result, isocitrate cannot be metabolised; it thus accumulates and is then readily equilibrated with citric acid
(via aconitase).
Citrate therefore accumulates in the mitochondrion.
An efficient citrate efflux system exists in the mitochondrial membrane for the export of citrate (in exchange for
malate, see below).
Citrate enters the cytosol and is cleaved by ACL to give
acetyl-CoA and oxaloacetate.
The acetyl-CoA is used for fatty acid biosynthesis.
The oxaloacetate is converted via malate dehydrogenase
to malate, which is then used as the counterion in the citrate efflux system.
This sequence of events is shown diagrammatically in
Fig. 1.
Although this metabolism of glucose to acetyl-CoA is
able to account for the flux of the carbon substrate into fatty
acid biosynthesis under nitrogen-limited conditions, it is not
the complete story. Some microorganisms have been found
in which ACL activity is present though without the cells being able to accumulate lipid; however, the corollary is not
true: no oil-accumulating microrganism has yet been reported that does not have ACL activity. Some other enzyme
must be needed, therefore, to ensure lipid accumulation.
Fatty acids, it must be remembered, are highly reduced
materials and to achieve their synthesis as a ready supply of

Fig. 1. A scheme to show how the citrate/malate cycle and the cytosolic transhydrogenase cycle could provide sufficient precursors of acetyl-CoA and
NADPH for lipogenesis in oleaginous microorganisms (from Refs. [14,15]). Enzymes: 1, pyruvate decarboxylase; 2, malate dehydrogenase; 3, malic enzyme;
4, pyruvate dehydrogenase; 5, citrate synthase; 6, ATP:citrate lyase; 7, citrate/malate translocase. Net carbon balance: pyruvate acetyl-CoA + CO2. Net
reaction for NADPH production: NADH + NADP+ + ATP NAD+ + NADPH + Pi. The transhydrogenase cycle can operate independently of the carbon flux
from citrate in the mitochondrion to acetyl-CoA in the cytosol and consequently can provide all the NADPH needed for both fatty acid biosynthesis and fatty
acid desaturation and elongation reactions.

C. Ratledge / Biochimie 86 (2004) 807815

811

Fig. 2. A diagram to show the organisation of a hypothesised lipogenic metabolon. The flux of carbon from the mitochondrion, via citrate efflux and acetyl-CoA
formation in the cytosol, thence into fatty acids and finally into long chain PUFAs (LCPUFA) occurring in the membranes of the endoplasmic reticulum, is
shown by the continuous lines. The system uses pyruvate (from glycolysis) as the provider of intramitochondrial acetyl-CoA and for citric acid production as is
shown in Fig. 1 but which is not repeated here for clarity. OAA: oxaloacetate; AC-CoA: acetyl-CoA; Mal-CoA: malonyl-CoA; FAS: fatty acid synthase; ACL:
ATP:citrate lyase; ACC: acetyl-CoA carboxylase; CMT: citrate/malate translocase; ME: malic enzyme; PC: pyruvate carboxylase; MDH: malate dehydrogenase. The yellow enzymes form the citrate/malate cycle while the purple enzymes represent the cytosolic transhydrogenase cycle (see also Fig. 1). (From
Ref. [14].)

reductant as NADPH is essential. The synthesis of 1 mol of a

C18 fatty acid requires 16 mols NADPH to be provided as
2 mols NADPH are needed to reduce each 3-keto-fattyacyl
group arising after every condensation reaction of acetylCoA with malonyl-CoA as part of the standard fatty acid
synthetase complex into the saturated fatty acyl chain, which
then undergoes a further cycle of chain lengthening.
The major supplier of NADPH for fatty acid biosynthesis
is now considered to be malic enzyme (reaction no. 3):
+

Malate + NADP pyruvate + CO2 + NADPH

with the fatty acid metabolon (Fig. 2) see Ref. [14]. It is

the opinion of the author and his colleagues that in oleaginous microorganisms there is no such thing as a general pool
of NADPH which can be produced by a number of enzymes;
instead, for fatty acid biosynthesis we propose that there is an
integration of the system for producing NADPH (e.g. malic
enzyme) with the fatty acid synthesising machinery. Only in
this way can a concerted accumulation of triacylglycerols be
achieved [14,15].

(3)

Malic enzyme activity has been found in most oleaginous

microorganisms where it is proposed to form an integrated
metabolon complex that combines with ACL and fatty acid
synthase (FAS) to ensure a direct channelling of acetyl-CoA
into fatty acids, which are finally esterified with glycerol into
triacylglycerols and incorporated via the endoplasmic
recticulum into fatty acid droplets (see Fig. 2).
A full account of the underlying biochemistry of lipid accumulation in oleaginous microorganisms has been recently
presented elsewhere [14] and the reader is referred to this
review for full details of the schemes described above and in
the figures.
Malic enzyme activity does not, however, appear to be
ubiquitous amongst oleaginous microorganisms and may be
absent in some oleaginous yeasts, including Lipomyces sp.
and some Candida sp. [unpublished work]. Here, it is probable that an alternative NADPH-generating enzyme, such as
a cytosolic NADPH-dependent isocitrate dehydrogenase,
though other enzymes are possible, will be found that will be
dedicated to fatty acid biosynthesis much in the same way as
malic enzyme is considered to be functionally associated

4. Formation of polyunsaturated fatty acids

Fatty acid biosynthesis in almost all organisms culminates
in the formation of either C16 or C18 saturated fatty acids.
These fatty acids are then modified through a sequence of
desaturases and elongases so that an extended range of unsaturated and PUFAs are producedsee Fig. 3. The fatty acids
which are produced in greatest abundance depend, of course,
on the genetic make-up of individual species. In oleaginous
yeasts, the range of fatty acids is somewhat limited: oleic
(18:1) and linoleic (18:2) acids together with palmitic (16:0)
or palmitoleic (16:1) acids are the most frequently found
fatty acids. When it occurs, 18:3 as the n-3 isomer, is usually
less than 10% [16]. It is only in the fungi and microalgae that
PUFAs appear at levels of over 20% of the total fatty acids.
Commercial interest (see above) has therefore concentrated
on those fungi and microalgae that produce the highest levels
of desirable PUFAs coupled with the highest contents of triacylglycerols.
The oils under current production using microbial technology have been listed in Table 1. In all cases, the oils containing the various PUFAs are in the form of triacylglycerols

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C. Ratledge / Biochimie 86 (2004) 807815

Fig. 3. Pathways for the formation of PUFA in microorganisms using the conventional FAS route. Fatty acids are synthesised from acetyl-CoA and malonylCoA (which is itself formed from acetyl-CoA via acetyl-CoA carboxylasesee Fig. 2) using the FAS complex of enzymes. The saturated fatty acid, stearic acid
is then successively desaturated and elongated through a series of reactions leading to the formation of various PUFAs. PUFAs fall into two categories, the
n-3 and n-6 series, depending on the position of the final double bond nearest the terminal methyl group. In M. alpina, which is used for the production of ARA,
there is a D17 (n-3) desaturase that can then form EPA under certain conditions. It is possible that a similar n-3 desaturase may occur in some organisms that
produce DPA(n-6), which would then be converted by this enzyme into DHA (see text for further discussion).

which are formed from the fatty acids via phosphatic acid
and its subsequent dephosphorylation to a diacylglycerol followed by a final acylation step ([14], and see also [17] for a
detailed review of this subject). In some photosynthesising
microalgae, not under current consideration for commercial
production of PUFA-rich oils, triacylglycerols are not the
major lipid component and, instead, there are numerous lipid
types present that are involved in the make-up of the photosynthesising membrane systems. This complexity of lipids in
these organisms is disadvantageous to them being used commercially. Also these organisms, being obligate phototrophs,
pose severe difficulties for their large-scale cultivation which
have not yet been overcome.
As is shown in Table 1, a key feature of the range of fatty
acids found in Schizochytrium sp. and Ulkenia sp., both of
which are heterotrophic eukaryotic organisms belonging to
the order of Thraustochytriales within the phylum of Labyrinthulomycota, is the occurrence of a second PUFA, docosapentaenoic acid (DPA, 22:5) along with DHA. This fatty acid
occurs, not as a member of the expected n-3 series (i.e. like
DHA), but as the n-6 isomersee Fig. 3. Although DPA is
unusual fatty acid, it also occurs in small amounts in animal
lipids and extensive animal trials in the safety of this oil
[18,19] have established that DPA is not detrimental to the
dietary usefulness of DHA. Although it does not act as a

pro-DHA fatty acid, DPA is not an anti-DHA fatty acid as

has been found with EPA (20:5, n-3) which is contraindicated for inclusion in infant formulas as it diminishes the
assimilation of DHA into brain tissue [8].
The reasons for the unusual occurrence of DPA in the lipids of this group of organisms is unknown. It is possible that
DPA might be converted into DHA by an n-3 desaturase
similar to the one found in M. alpina which, under certain
conditions, can covert ARA into EPA (see Fig. 3) but, whatever reason is found, it will almost certainly be connected
with the manner in which these very long chain PUFAs are
synthesised. Unlike other eukaryotic microorganisms, the
entire pathway of DHA formation in these thraustochytrid
organisms would appear to be via a bacterial-like polyketide
synthase (PKS) route and not by the conventional FAS route.
This is described below.
Until recently, the pathway of fatty acid formation in all
microorganisms was considered to be by the routes shown in
Fig. 3 involving a FAS complex to provide the starting
C16 and C18 fatty acids for subsequent desaturation and
elongation reactions. However, an examination of the genetic make-up of fatty acid biosynthesis in Schizochytrium
sp. [20] has shown that it uses a PKS-like system that still
involves acetyl-CoA and malonyl-CoA being the essential
building blocks but which does not involve in situ reduction

C. Ratledge / Biochimie 86 (2004) 807815

of the intermediates. It is therefore an anaerobic system as

opposed to the aerobic system used in other eukaryotic cells.
(The term anaerobic route does not imply that the pathway
only occurs if the organism is grown anaerobically (all these
organisms are in fact obligate aerobes) but that O2 is not involved in the synthesis of double bonds as occurs with conventional desaturases involved in producing double bonds in
fatty acids. Thus, O2 is not needed for the synthesis of DHA
in these eukaryotic marine protists).
Simultaneously with its identification in Schizochytrium,
the PKS-fatty acid synthesising system was also found to occur in the bacteria Shewanella and Moritella marina (formerly known as Vibrio marinus) [20] which produce EPA
(20:5, n-3) and DHA, respectively. Earlier work [21,22] had
suggested from an analysis of the ORFs in Shewanella and V.
marinus that these were likely to yield protein domains that
were homologous with FAS enzymes found in Escherichia
coli and other bacteria, and that formation of these very long
chain PUFAs in Shewanella and Vibrio (Moritella) was via a
conventional FAS system followed by successive elongation
and desaturation, as shown in Fig. 3. Metz et al. [20], however, identified at least 11 regions within the five ORFs of the
Shewanella genetic fragment as putative enzyme domains
and, of these 11 regions eight were strongly related to PKS
proteins though the remaining three regions did appear to be
homologues of bacterial FAS proteins. The eight PKS domains leading to PUFA biosynthesis were found in
Schizochytrium sp. as well as in Shewanella, though in a different genetic sequence. They are: 3-ketoacyl synthase
(KS)also sometimes known as the condensing enzyme,
malonyl-CoA:ACP acyltransferase (MAT), acyl carrier protein (ACP), 3-ketoacyl-ACP reductase (KR), acyltransferase
(AT), chain length factor (CLF), enoyl reducatase (ER) and a
dehydrase/isomerase (DH). A similar PUFAPKS system
has also been identified in an Ulkenia sp. (see Table 1) [23].
Thus, in the DHA-synthesising thraustochytrid organisms, long chain fatty acid biosynthesis appears to occur by a
novel, bacterial-like PKS system in which the growing fatty
acyl chain must not be reduced into completely saturated
fatty acids, as occurs with a conventional eukaryotic FAS
system but involves fatty acyl intermediates that remain unsaturated as the chain continues to be lengthenedsee
Fig. 4.
The exact biosynthetic mechanism has a long way to go
before it is finally unravelled and there is much speculation
as to what are the intermediates that might be involved. For
example, as described above, it is not yet clear why
DPA(n-6) should be incorporated into the lipids of
Schizochytrium sp. and Ulkenia sp. Does this indicate that
the PKS systems builds a series of n-6 fatty acyl intermediates with the final step then being a n-3 desaturase as is indicated in Fig. 3? Or are DHA and DPA produced by a common mechanism with some branch-point along the pathway?
Qiu [24], however, has suggested on the basis of finding
[25] a D4-desaturase in a Thraustochytrium sp. (which is
closely related to Schizochytrium and Ulkenia) that DHA is

813

Fig. 4. A suggested scheme to account for the formation of DHA by the PKS
route of synthesis in Schizochytrium sp. and probably other thraustochyrid
organisms. (See text for further information concerning the genetic organisation of the PKS in Schizochytrium sp.) The sequence would begin with the
transfer of both acetyl-CoA and malonyl-CoA into their corresponding esters with ACP. These are then condensed via KSalso sometimes known as
the condensing enzymeto give the 3-ketobutyryl intermediate, which is
then converted into butyryl-ACP using KR, followed by a DH and finally by
an ER. This is then the equivalent of what occurs in a conventional FAS
mediated system. However, in successive additions of malonyl-CoA to the
fatty acyl chain, the double bond introduced by the DH can be retained. If
the sequence follows the accepted route of fatty acid biosynthesis in bacteria, and to which this pathway is related, then the double bond would initially be in the trans configuration and would moreover be at the 2,3position. An isomerase is therefore conjectured which will convert the bond
into the cis configuration and simultaneously move it to the 3,4-position as
shown, for example, in the conversion of 6:1(n-4) to 6:1(n-3). Successive
additions of malonyl-CoA will lead to a lengthening chain in which the double bonds are retained if they are in the right position. It is suggested that in
the diagram after the formation of 8:1(cis-5), the sequence will proceed to
10:2(cis-4,cis-7), then to 12:3(c3,c6,c9) followed by 14:3(5,8,11),
16:4(4,7,10,13), 18:5(3,6,9,12,15), 20:5(5,8,11,14,17) and finally DHA at
22:6(4,7,10,13,16,19). To accommodate the presence of the D4-desaturase
identified by Qui et al. [25] one would have to speculate that 20:5(n-3), the
penultimate intermediate would be converted first to 22:5(7,10,13,16,19)
and that this would be the substrate for this final desaturase. However, to
create 22:5(n-6), which is found in the thraustochytrids, an entirely different
sequence to the one given in the figure and indicated above would have to be
followed in which a n-6 series of fatty acids [beginning with 10:1(cis-4)]
were synthesised. Such a sequence would not involve the participation of a
D4-desaturase. (A similar PKS route to DHA biosynthesis also occurs in
Ulkenia sp.see Table 1 and also text.)

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C. Ratledge / Biochimie 86 (2004) 807815

possibly synthesised in this particular species by a conventional series of elongases and desaturases similar to that
shown above in Fig. 3. This, however, ignores the close taxonomic similarity between Schizochytrium and Thraustochytrium and also ignores that the profiles of the fatty acids
from each organism are somewhat similar [26] with both organisms having DPA(n-6) rather than DPA(n-3). It would
therefore seem reasonable to suggest that pathways of fatty
acid biosynthesis in these two organisms should be the same.
No genetic analysis, though, was carried out on the Thraustochytrium sp. examined by Qiu et al. [25] and thus some
caution should be exercised before assuming that this species
does not have the PKS system as identified in the
Schizochytrium sp. by Metz et al. [20]. However, the presence of the D4 desaturase would appear incontrovertible but
no evidence has yet been provided for any other fatty acid
desaturase being detected, either at the genetic or protein
level in any of the DHA-synthesising microorganisms mentioned in Table 1.
With regard to the synthesis of the C14 to C18 fatty acids
which occur in all the DHA-synthesising microorganisms, it
is not clear if there is a separate conventional FAS system
that operates alongside the PKS system or whether these
fatty acids are also produced by the latter pathway. Analysis
of appropriate genetic mutants would clearly provide an unambiguous answer to this and other outstanding questions
concerning PUFA biosynthesis.
The operation of a PKS system helps to explain why many
of the DHA-producing organisms do not have intermediate
chain-length fatty acyl groups in their lipids; the PKS system
would seem to be like a drain-pipe; the building units (acetyl
and malonyl-CoA) go in at one end and the final product
emerges at the other. Intermediates are apparently too tightly
bound to be released as free CoA esters and to be available
for triacylglycerol biosynthesis. Although no work has yet
been reported on the mechanism of DHA biosynthesis in C.
cohnii that would satisfactorily account for DHA being the
sole PUFA being produced by this organism, it will be of
clear interest to determine whether it uses a similar PKS system to the Schizochytrium one; if this is found to be the case
then it would also help to explain why it has always been
difficult to identify conventional, eukaryotic-like desaturases
in C. cohnii (C. Ishiwaka, J.P.Wynn and C. Ratledge, unpublished work).
A PKS system would also serve to decrease the overall
requirement for NADPH being needed both for the reduction
of the growing fatty acid acyl chain (mainly at the level of the
reduction of the 2,3-enoyl intermediate to leave an unsaturated fatty acyl chain for the next condensation reactionsee
Fig. 4) and for the operation of desaturases used in the formation of unsaturated fatty acids in all other systems. As
these desaturases are also linked to O2, the PKS system of
PUFA production is therefore, like the bacterial FAS system
an anaerobic (non-oxygen requiring) system. From an evolutionary point of view, it would seem sensible to avoid having to use NADPH in two separate sequences: first in produc-

ing a saturated fatty acid and then in reducing it to a PUFA. If

the double bond in the growing fatty acyl chain can be retained (see Fig. 4) then this dispenses with the need for any
desaturase to operate on the acyl chain. A clear economy of
reducing power is thereby achieved!
Finally, and not of insignificant implications, the occurrence of a gene cluster coding for the entire pathway of
PUFA biosynthesis in Schizochytrium, as well as in PUFAproducing bacteria opens up the prospects of being able to
clone this contiguous gene sequence into various plants in
order to achieve PUFA production in an agronomically important crop such as sunflower, soybean or rapeseed. Consequently, interest in the biochemistry and molecular biology
of PUFA production in microorganisms is likely to be a continuing focus of activity for some years to come.

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