Biosynthesis of polyunsaturated fatty acids (PUFAs)

In contrast to higher plants, in which the number of double bonds in FAs does not comprises up to three, algae and animals can introduce up to eight double bonds into FAs (Linko and Karinkanta 1970; Mansour et al. 1999).

The introduction of double bonds into the acyl chain is catalysed by desaturases, which show different substrate specificities. The plastid-localised soluble desaturase acts on acyl chains bound to ACP and is termed acyl-ACP-desaturase. The stearoyl-ACP Δ⁹ -desaturase introduces a double bond into stearic acid resulting in 18:1^Δ9-ACP, as described in the previous section (Shanklin and Cahoon 1998). Except from the soluble acyl-ACP desaturase family, all other desaturases are integral membrane proteins with either lipid substrates or CoA substrates. In plants and cyanobacteria acyl-lipid-desaturases introduce double bonds into FAs whereas in some yeast and animal cells acyl-CoA-desaturases catalyse the introduction of double bonds into FAs (Somerville et al. 2000). Recently the first acyl-CoA-dependent desaturase was identified from the microalga Ostreococcus tauri (Domergue et al. 2005).

The initial substrate for the PUFA biosynthetic pathway is 18:1^9Z after its incorporation into PC. Then a Δ¹²-desaturase introduces the second double bond, resulting in LA, which may be further desaturated by a Δ¹⁵-desaturases to ALA. These modified FAs are then exchanged by other 18:1 acyl residues and may be released into the cytoplasm as acyl-CoA derivatives. In the cytoplasm the acyl-CoA derivatives are extended to about C-26 or even longer acyl chains by specific elongase complexes. These elongase reactions have several important differences between that of FAS reactions in the plastids: the elongases are membrane-bound and ACP is not involved in the elongation process (Somerville et al. 2000). In plants and mammals it is believed that FA elongation is a four-step process with the condensation of malonyl-CoA with a long-chain acyl-CoA as the initial reaction (Parker-Barnes et al. 2000). The initial condensation in plants is catalysed by a β-ketoacyl-CoA synthase (KCS). Surprisingly, the corresponding condensing enzymes in yeast, the yeast FA elongase system (ELO) (Toke and Martin 1996), do not share any sequence similarities to plant KCS and also other cloned fungal, algal, moss and mammalian PUFA elongases share homology with yeast ELO sequences and not with plant KCS sequences (Zank et al. 2002).

16 Further desaturation steps lead to the synthesis of FAs with more than 3 double bonds.

During the biosynthesis of VLCPUFA a continuous exchange between the biosynthetic steps of CoA-bound and lipid-linked acyl residues takes place (Fig. 1). This lipid/CoA pathway requires an efficient acyl exchange between phospholipids and the acyl-CoA pool and is supposed to be the rate limiting step in the VLCPUFA synthesis in plants. In contrast, the CoA pathway does not require a shuttling of acyl residues between acyl-CoA pool and phospholipids. Acyl-CoA specific desaturases allow a subsequent desaturation and elongation of FA, which exclusively takes place in the acyl-CoA pool.

The most important VLCPUFAs are arachidonic acid (AA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), and docosahexaenoic acid (DHA, 22:6n-3). The pathway shown in Fig. 1 is regarded as the biosynthetic pathway of VLCPUFAs found in all eukaryotes.

According to the position of the closest double bond to the omega end of the PUFAs, two pathways are distinguishable: the ω3 and ω6-pathway. In addition,some algae can produce EPA using another pathway, termed the Δ8-alternative pathway, in which LA is converted to 20:3(8Z,11Z,14Z) (Qi et al. 2002). First, LA is elongated to 20:2^(11Z,14Z) by a Δ9-elongase and then, a Δ8-desaturase introduces the third double bond, resulting in 20:3(8Z,11Z,14Z), which can sequentially be converted to AA and then EPA (Fig. 1).

Another alternative pathway of PUFAs is the Sprecher pathway which was found for mammalian cells (Sprecher 2000) and is also considered to be present also in some species of the class Dinophyceae (Henderson 1999; Bergé and Barnathan 2005). In this pathway DHA is not directly obtained by elongation and desaturation of EPA, but formed via β-oxidation of 24:6(6Z,9Z,12Z,15Z,18Z,21Z), which is synthesised by elongation and desaturation of 22:5(7Z,10Z,13Z,16Z,19Z) at the ER and then transported to the peroxisomes (Fig. 1).

17

18:3 ^6Z,9Z,12Z 18:4 6Z,9Z,12Z,15Z

20:3 ^8Z,11Z,14Z 20:4 8Z,11Z,14Z,17Z Δ5-desaturase

24:6 6Z, 9Z,12Z,15Z,18Z,21Z Peroxisomal β-oxidation

18:3 ^6Z,9Z,12Z 18:4 6Z,9Z,12Z,15Z

20:3 ^8Z,11Z,14Z 20:4 8Z,11Z,14Z,17Z Δ5-desaturase

24:6 6Z, 9Z,12Z,15Z,18Z,21Z Peroxisomal β-oxidation

Figure 1. Schematic representation of VLCPUFA biosynthesis via the ω6- and ω3-pathway. Additional route for the synthesis of AA is the Δ8-alternative pathway.

Additional route for the synthesis of DHA is the mammalian Sprecher pathway.

Desaturase-catalysed steps are coloured in green, elongase catalysed steps in red (Napier 2007).

Besides the standard FA biosynthetic pathway consisting of oxygen dependent desaturation and elongation reactions VLCPUFAs can be synthesised via an anaerobic pathway catalysed by polyketide synthases (PKS) (Metz et al. 2001). PKSs carry out similar reactions as FAS and use acyl carrier protein (ACP) as covalent attachment site for the growing chain. In contrast to the standard VLCPUFA synthesis which requires approximately 30 enzyme activities and almost 70 reactions, the PKSs consists of a single, multidomain enzyme, carrying out the complete cycle of reduction dehydration and reduction (Bentley and Bennett 1999). Often this cycle is abbreviated resulting in highly derivatised carbon chains, such as aflatoxins and antibiotics. But some marine organisms, like Schizochytrium, produce EPA and DHA via the PKS pathway (Metz et al. 2001).

18 Over the last years, a multitude of interesting PUFAs from microalgae were identified.

One of these PUFAs is 18:5n-3, which was first found in the dinophyte Prorocentrum minimum (Joseph 1975). Until now the biosynthesis of 18:5n-3 is not fully understood.

Joseph et al. proposed two ways: 18:5n-3 could either be synthesised by desaturation (Δ³-desaturase) of 18:4n-4 or by β-oxidation of EPA (Fig. 1). Upon a following elongation step (Δ³-elongase) of 18:5n-3, EPA is obtained. This pathway may be a fourth alternative to synthesise EPA.