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Biochimie 86 (2004) 807815 www.elsevier.

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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 rst 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 nal renement and purication. Although the production did not last many years, it nevertheless established that microorganisms could be realistic commercial
* 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

alternatives to some plant oils and that the microbial oil itself could be extracted and puried 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 During the late 1980s and early 1990s, it became clear that PUFAs were of increasing interest in nutrition and especially

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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 benet to babies for them to achieve improved memory and eyesight [37]. The problem was where one might obtain sufcient quantities of these PUFAs to offer as nutritional supplements. Although DHA has long been known to be a major fatty acid of sh 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 sh 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 sh and concentrated in the liver and other organs. Indeed some countries, most conspicuously the USA, have an outright ban on sh oils being given to infants and young children for
Table 1 Fatty acid proles (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 7 4

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

18:3 (n-6) 4 2

20:3 (n-6) 4

20:4 (n-6) 49 70

22:0 3

24:0 1 5

18:0 0.4

18:1 15

18:2 0.6

18:3 (n-3)

20:3 (n-6)

22:5 (n-6)

22:6 (n-3) 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].

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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 decit 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

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 rst 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 denition, 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 sufcient 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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to operate efciently, 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 vefold 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 efcient citrate efux 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 efux 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 ux 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 sufcient 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 ux 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.

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Fig. 2. A diagram to show the organisation of a hypothesised lipogenic metabolon. The ux of carbon from the mitochondrion, via citrate efux and acetyl-CoA formation in the cytosol, thence into fatty acids and nally 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]. 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 modied 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

(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 nally esteried 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 gures. 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

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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 nal 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 nal 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 difculties 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

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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 identication 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, identied at least 11 regions within the ve 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 identied 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 nally 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 nal 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 nding [25] a D4-desaturase in a Thraustochytrium sp. (which is closely related to Schizochytrium and Ulkenia) that DHA is

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 nally 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 conguration and would moreover be at the 2,3position. An isomerase is therefore conjectured which will convert the bond into the cis conguration 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 nally DHA at 22:6(4,7,10,13,16,19). To accommodate the presence of the D4-desaturase identied by Qui et al. [25] one would have to speculate that 20:5(n-3), the penultimate intermediate would be converted rst to 22:5(7,10,13,16,19) and that this would be the substrate for this nal desaturase. However, to create 22:5(n-6), which is found in the thraustochytrids, an entirely different sequence to the one given in the gure 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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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 proles 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 identied 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 nal 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 difcult 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: rst 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 insignicant 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 sunower, 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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