PUFA desaturases

Elongases (marked s on Fig. 1.1) and PUFA desaturases (marked A6 and A5 on Fig. 1.1) are not usually classified as lipogenic enzymes. However, they play a critical role in the synthesis of a variety of fatty acids either by acting on endogenously produced fatty acids (from lipogenesis) or on exogenous (dietary) fatty acids. Therefore, we chose to briefly introduce these enzymes. 

Mutant Mut49 was considered to be defective in A6 desaturase because of a high 18 2co6 level and low levels of 18 3o>6, DHGA and A A in its mycelia. In addition to the accumulation of 18 2 6, two nonmethylene-interrupted PUFAs, i.e., 5,11,14-cis-eicosatrienoic acid (20 3A5) and 5,11,14,17-cis-eicosatetraenoic acid (20 4A5), were found [158], These PUFAs are considered to be converted from 18 2co6 or 18 3ca3, respectively, by three subsequent reactions, i.e., A6 desaturation, elongation and A5 desaturation (Fig. 15). 

Three major families of unsaturated fatty acids are seen in warm-blooded animals, that is, the n-9, monounsaturated fatty acids (e.g. oleic acid, OA), and the n-6 and n-3, both polyunsaturated fatty acids (PUFAs). However, only the n-6 and n-3 families, derived from LA and ALA, respectively, are EFA. These must be obtained from the diet since mammals lack the desaturase enzymes necessary for the insertion of a double bond in the n-6 and n-3 positions of the fatty acid carbon chain. Fatty acid nomenclature is as follows The first number denotes the number of carbon atoms in the acyl chain and the second refers to the number of unsaturated (double) bonds. This is followed by a symbol n or co and a number that denotes the number of carbon atoms from the methyl terminal of the molecule to the first double bond. Hence, LA is 18 2(n-6), while the more unsaturated ALA is denoted as 18 3(n-3) (Figure 26.1). These fatty acids must be metabolized to their longer chain derivatives before carrying out many of their activities. 

DHA, will be able to achieve this is, at least to the present authors, far from clear. It is not beyond the bounds of possibility that such genetically modified plants may produce much less oil than the normal plant as the energy and reducing power needed (by the desaturase and elongase reactions) to produce the PUFAs must come from the same sources that are being used to synthesize the normal fatty acids. The situation in plants may parallel what was found with the production of GLA in Mucor spp. (see Section 2.2.1) You can either find strains that produce a lot of oil but have little GLA, or you can find strains that produce a lot of GLA but have little oil, but you cannot have both occurring simultaneously. It may then take additional genetic manipulations to correct this imbalance, but distortion of one metabolic pathway can only be achieved at the expense of another so this is not likely to be a trivial task. 

Apart from the above mentioned enzymes, there also exist a few enzymes involved in PUFA metabolic pathways that have recently been found. One of them is a novel n-3 fatty acid desaturase, isolated from an EPA-rich fungus, Saprolegnia diclina [248]. The gene was isolated by PCR amplification from a fungus cDNA library and then, expressed in S. cerevisiae, which was cultured in the presence of several FA as substrates. The study showed that the recombinant protein could exclusively desaturase C20 n-6 PUFAs, with preference for AA which was converted into EPA. This represents a completely novel and different activity from any organism previously described, and its potential for use in EPA production in transgenic oilseed crops has been outlined. 

In another attempt, a A5-desaturase from C. elegans and a A6-desaturase from B. officinalis were coexpressed with CEELOl, an elongase from C. elegans in S. cerevisiae [239] to reconstitute the PUFA biosynthetic pathway. Expression of the A6-desaturase and CEELOl was confirmed in the presence of LA and ALA, whilst the A5-desaturase and the elongase were expressed only when LA was... 

The relationship between substrate and product for A-9-desaturase is reflected by the desaturase index, defined as [RA a- (RA + VA)]. Various approaches to calculating desaturase index in milk fat are discussed by Kelsey et al. (2003). In the study by Corl et al. (2001), the desaturase index was 0.23 for the hay and concentrate diet and 0.20 when the diet was supplemented with PH VO. Kay et al. (2004) reported a desaturase index of 0.25 for the pasture diet and 0.22 when the diet was supplemented with sunflower oil. Piperova et al. (2002) observed desaturase indices for highland low-fiber diets of 0.40 and 0.35, respectively. Shingfield et al. (2003) reported desaturase indices of 0.18 and 0.15 with a grass silage diet without or supplemented with fish oil these values are probably lower than others because the analytical methods accounted for minor CLA isomers that typically co-elute with RA or because of inhibition of A-9-desaturase by the long-chain PUFA from the fish oil supplement. The desaturase index, as defined earlier, should approximate the proportion of VA desaturated in the tissues. A summary of endogenous RA synthesis estimates and the proportion of VA desaturated in the tissues is in Table III. 

Mammals are dependent on dietary sources of essential fatty acids as they lack the desaturase enzymes necessary to synthesize them. Kao et al. (2006) engineered transgenic mice expressing the ffl-3 fatty acid desaturase enzyme from the nematode Caenorhabditis elegans, which synthesizes a wide range of PUFA and possesses the only known example of an ffl-3 desaturase enzyme in the animal kingdom. The milk from these mice had more ffl-3 and less ffl-6 PUFA, and hence had showed an overall decrease in the ffl-6 ffl-3 PUFA ratio in the milk. The milk phospholipids from the transgenic mice had an ffl-6 ffl-3 ratio of 1.78 as compared to 9.82 in the control animals. The authors anticipate that this maybe a suitable method to improve the nutritional profile of dairy-based diets. 

---