Fatty Acid Desaturases, Polyunsaturated Fatty Acid Regulation, and Biotechnological Advances

Polyunsaturated fatty acids (PUFAs) are considered to be critical nutrients to regulate human health and development, and numerous fatty acid desaturases play key roles in synthesizing PUFAs. Given the lack of delta-12 and -15 desaturases and the low levels of conversion to PUFAs, humans must consume some omega-3 and omega-6 fatty acids in their diet. Many studies on fatty acid desaturases as well as PUFAs have shown that fatty acid desaturase genes are closely related to different human physiological conditions. Since the first front-end desaturases from cyanobacteria were cloned, numerous desaturase genes have been identified and animals and plants have been genetically engineered to produce PUFAs such as eicosapentaenoic acid and docosahexaenoic acid. Recently, a biotechnological approach has been used to develop clinical treatments for human physiological conditions, including cancers and neurogenetic disorders. Thus, understanding the functions and regulation of PUFAs associated with human health and development by using biotechnology may facilitate the engineering of more advanced PUFA production and provide new insights into the complexity of fatty acid metabolism.


Introduction
Polyunsaturated fatty acids (PUFAs) consist of more than two double bonds and are a group of critical nutrients that modulate brain development and cognition as well as many diseases such as cardiovascular disease, cancers, and diabetes [1,2]. Twenty-carbon PUFAs are precursors of eicosanoids that regulate inflammatory and immune responses through pro-and anti-inflammatory activities; docosahexaenoic acid (DHA, 22:6n-3) is a precursor of anti-inflammatory docosanoids [3,4]. In humans, PUFAs are synthesized by fatty acid desaturases (FADSs), which are encoded by three genes on the human chromosome 11 [5] and are regulated by PUFA consumption after ingestion of linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), which are dietary essential fatty acids in humans. However, only a small proportion of fatty acids is converted to PUFAs consisting of more than 20 carbons [6].
Alternative transcripts (AT) of FADS1, FADS2, and FADS3 have been reported to be expressed in different baboon tissues and human neuroblastoma, SK-N-SH [35][36][37]. They have specific structural characteristics; for example, FADS2AT1 includes only three histidine boxes except in the cytochrome b5 domain, suggesting that FADS2AT1 is related to non-methylene interrupted desaturation [36]. FADS3AT1 and FADS3AT3 contain whole conserved structural domains of cytochrome b5 and three histidine boxes, whereas FADS3AT5 retains intron 5 and other FADS3ATs have different structures. FADS3ATs are expressed in a tissue-specific manner and their expression is regulated according to neuronal cellular differentiation [35]. In addition, the alternative transcripts of FADS1AT1 were identified; FADS1AT1 modulated the delta-6 and delta-8 desaturation functions of FADS2, which was the first report of the function of FADSAT and of the one gene's splice variant role to regulate the other genes [37]. Unlike FADS1 and FADS2, the function of FADS3 in omega fatty acid synthesis and regulation has not been reported. However, FADS3 may catalyze an unexpected ∆13-desaturation of trans-vaccenate [38].
The diverse roles of FADS impacting human health have been evaluated using genetic and genomics approaches. Activation of fatty acid desaturation leads to pro-inflammatory conditions such as coronary artery diseases among the population consuming excessive meat and few vegetables, such as in the westernized diet [39]. A genome-wide association study showed that a single-nucleotide polymorphism (SNP) near FADS1 was significantly associated with the plasma concentrations of EDA (eicosadienoic acid, 20:2n-6), EPA, and ARA [40]. FADS and breastfeeding are associated with differences in the IQs of children [41], and FADS1 and FADS2 clusters in human chromosome 11 alter the fatty acid composition in pregnant and lactating women [42]. An SNP in FADS2 was found to be significantly associated with the occurrence of attention-deficit/hyperactivity disorder, and dietary consumption of essential fatty acids was found to be related to the dopamine pathway in attention-deficit/hyperactivity disorder in patients [43]. Microarray analysis of insulin-resistant and insulin-sensitive individuals showed that FADS1 was differentially regulated in both adipose and muscle of insulin-resistant individuals [44], possibly through an association between neighboring SNPs and fasting glucose homeostasis [45]. Homeostasis model assessment of insulin resistance was associated with the FADS gene cluster as well as with fatty acid composition in serum phospholipids [44,46]; moreover SNPs mapped to the FADS locus were strongly associated with ARA, EPA, and DGLA in European-Americans [47]. FADS3 was shown to be critical in hyperlipidemia [48] and implantation sites [49]. Figure 1 shows the PUFA synthetic pathways to 22-carbon fatty acids from oleic acid in eukaryotic systems via diverse fatty acid desaturases. Delta-12 desaturase and delta-15 desaturase have been identified in lower eukaryotes [50,51], plants [52,53], and animals [54][55][56][57] except mammals. These species can produce the omega-3 and omega-6 fatty acids. However, mammals, including humans, cannot synthesize omega fatty acids de novo and must consume them in a diet or in other nutritional supplements. Therefore, LA and ALA are known as dietary essential fatty acids for humans. Generally, after the synthesis of 18-carbon saturated fatty acid in cells, stearic acid (SA, 18:0) is desaturated to oleic acid (OA, 18:1n-9) by stearoyl-CoA desaturases [30]. LA is synthesized by delta-12 desaturase from OA and converted into ALA by delta-15 desaturase [58]. However, palmitic acid (PA, 16:0) is converted to palmitoleic acid (16:1n-7) by stearoyl-CoA desaturases [30] or into 16:1n-10 fatty acid by FADS2 as well [59]. Omega-3 and omega-6 fatty acids are competitive toward desaturases and elongases for the addition of carbons or double bonds in their chains, respectively. LA and ALA are converted to GLA and stearidonic acid (SA, 18:4n-3), respectively, by delta-6 desaturase. By two-carbon elongation, GLA and SA are metabolized to DGLA and eicosatetraenoic acid (ETA, 20:4n-3), respectively. These fatty acids are changed to ARA and EPA, respectively. ARA and EPA are elongated to adrenic acid (ADA, 22:4n-6) and omega-3 docosapentaenoic acid (ω-3 DPA, 22:5n-3), respectively. In mammals, it has been thought that ADA and ω-3 DPA are elongated to omega-6 tetracosatetraenoic acid (24:4n-6) and omega-3 tetracosapentaenoic acid (24:5n-3) and that delta-6 desaturated to omega-6 tetracosapentaenoic acid (24:5n-6), and omega-3 tetracosahexaenoic acid (24:5n-6) and then betaoxidized to produce omega-6 docosapentaenoic acid (ω-6 DPA, 22:5n-6) and DHA (22:6n-3) in peroxisomes [60]. However, ω-6 DPA and DHA were found to be generated by delta-4 desaturation in primates [33], marine vertebrate [61], and lower eukaryotes [62,63]. Moreover, omega-3 desaturases, which convert omega-6 fatty acids to omega-3 fatty acids, were identified in cyanobacteria [64], some plants [52,53], lower eukaryotes [50,51], and animals such as nematodes [56].

Fatty Acid Desaturases for Synthesis of PUFA
Desaturases catalyze the introduction of double bonds between the carboxylic end of a molecule and a preexisting double bond to introduce further unsaturation into existing PUFAs or make PUFAs de novo in mammals deprived of dietary PUFA. These animals contain two types of desaturases to produce PUFA: front-end desaturases and methyl-end desaturases [65]. Front-end desaturases such as delta-4, delta-5, and delta-6 desaturases help to introduce double bonds between the carboxylic end of a molecule and a pre-existing double bond to generate PUFA. Based on regioselectivity, the positions of double bonds in fatty acids are referred to as delta-or omega-; desaturases are also named in this manner. The front-end desaturase was first identified in cyanobacteria [66], and subsequently identified in diverse species including plants, animals, algae, and fungi. Unlike front-end desaturases, methyl-end desaturases such as delta-12 and delta-15 desaturases (ω3 desaturases) assist in adding a double bond between pre-existing double bond and methyl end in fatty acids [30]. However, mammals, including humans, do not contain these methyl-end desaturases for producing essential fatty acids. Therefore, mammals must acquire essential fatty acids such as LA and ALA from foods or nutritional supplements.
Delta-6 desaturases catalyze the addition of a double bond at the 6th carbon-carbon bond position from the carboxylic acid end in fatty acids. Generally, delta-6 desaturases have common structural features of the N-terminal cytochrome b5-domain and histidine motifs, whereas the desaturase from Synechocystis sp. does not include the cytochrome b5 domain region [65]. Delta-6 desaturase is a rate-limiting enzyme in the synthesis of PUFA for generating GLA and SA from LA and ALA in mammals and humans, respectively. In addition to delta-6 desaturation activity, these enzymes showed multiple desaturase activities in mammals to produce 16:1n-10 fatty acid from a palmitic acid (16:0) [32] and to generate DGLA and ETA from eicosadienoic acid (EDA, 20:2n-6) and eicosatrienoic acid (ETE, 20:3n-3), respectively, as a delta-8 desaturase [31].
Delta-5 desaturases add a double bond at the 5th carbon-carbon bond from the carboxylic acid end in fatty acids. These desaturases were first identified in a fungus, Mortierella alpina [67,68], and subsequently have been found in many organisms. The desaturases in marine invertebrates showed bifunctional activities with delta-5 and delta-6 desaturation [69,70]. Delta-5 desaturase generally synthesizes ARA and EPA using DGLA and ETA as substrates. However, some delta-5 desaturases produce non-methylene-interrupted fatty acids such as 18:3-5, 9, and 12 and 20:4-5, 11, 14, and 17 [71,72]. Similarly, delta-5 desaturase, coded for by the FADS1 gene in primates, showed desaturation activities on EDA and ETE in breast cancer cells, suggesting that delta-5 desaturase may play a role in replacing delta-6 desaturase in a delta-6 desaturation-deficient system [73].
Delta-4 desaturases catalyze the addition of a double bond at the 4th carbon-carbon bond from the carboxylic acid end in fatty acids. Delta-4 desaturase was first identified in a lower eukaryote, Thraustochyrium sp. [62], and subsequently in microalgae [63], followed by the marine vertebrate delta-4 desaturase [61]. FADS2, known as a delta-6 and delta-8 desaturase, has delta-4 desaturase activity to produce ω-6 DPA and DHA from the substrates ADA and ω-3 DPA, respectively, in human cells [33]. FADS2 may be used to produce DHA in marine products using transgenic approaches.

PUFA Regulation and Biotechnology
Higher plants generally cannot synthesize PUFAs containing more than 20-carbons, except a few species that produce GLA and stearidonic acid (SDA, 18:4n-3) [6]. However, because of environmental concerns regarding the use of fish oils, transgenic plant oil production has been developed even though PUFA production in transgenic species has not been completely accomplished because of metabolic bottlenecks in the complex pathway associated with a variety of genes [89,99]. In 1996, tobacco was the first transgenic plant reported; it used cyanobacterial delta-6 desaturase to accumulate GLA [86]. Borage delta-6 desaturase transgenic tobacco produced a high level of delta-6 desaturated fatty acids [10], and transgenic B. juncea yielded GLA using delta-6 desaturase from P. irregulare [87]. In 2004, different desaturases from different species, delta-9 desaturase from I. galbana, delta-8 desaturase from E. gracilis, and delta-5 desaturase from M. alpina, were cloned into A. thaliana to produce PUFAs [88]. Additional genes as well as ω3 desaturase have been cloned into plants [14,89,90]. Moreover, seed-specific expression of desaturases in tobacco and linseed as well as soybean seed was reported to produce seeds that accumulate PUFAs [6]. Recent reports showed that high levels of PUFAs not only accumulate in plant seeds [91,92], but also that ω-3 desaturase was cloned into rice seed to produce high levels of ALA [13].
Except for nematodes, animals including humans have not been reported to contain ω3 desaturase genes to change the ratio of omega-3 and omega-6 fatty acids. Transgenic animals were first engineered by introducing fat-1 from C. elegans into mice; these animals produced higher levels of ω-3 fatty acids than ω-6 fatty acids in their tissues and organs [11]. Transgenic pigs containing cloned hfat-1, a humanized fat-1, were generated to produce high levels of ω-3 fatty acids, increasing the levels of these fatty acids from approximately 2% to approximately 8% [12]. It may be nutritionally beneficial to produce pork rich in ω-3 fatty acids without altering the quality of pork.
Moreover, transgenic systems using desaturases have been studied for clinical treatments or disease and physiological problem recovery. FADS2 knock-out mice with PUFA deficiency were found to have uncommon conditions involving reproduction, the skin, and the intestine [93]. FADS1 knock-out mice lacking ARA were also generated [94]. These mice could not survive for more than 12 weeks; however, supplementation with ARA helped to extend their life spans. Recently, a report showed that FADS1 compensated for the functions of FADS2 on twenty-carbon fatty acids under FADS2-deficient conditions such as MCF-7 breast cancer cell conditions [73]. FADS1 expression was found to be regulated by FADS2 polymorphisms, in which insertion-deletions were observed to result from differences in major and minor alleles. Additionally, different sensitivity toward simvastatin, a lipid-lowering drug, as well as GW3965, a LXR agonist, was observed [95]. In addition to PUFA regulation of human physiological systems associated with FADS genes, fat-1 gene transfer prohibited neuronal apoptosis in rat cortical neurons [96]. Colitis-associated colon cancer and prostate cancer were reduced in transgenic fat-1 mice [97,98]. These results suggest that the ratio of omega-3 and omega-6 fatty acids is very important for treating neuronal function and cancer development. Thus, gene therapy using desaturases may help in the treatment or delay of many diseases as well as cancers.

Conclusions
PUFAs are important nutrient and structural components in cell membranes and are associated with human health and development. PUFAs containing 20 or more carbons are also precursors of eicosanoids or docosanoids, which are signaling molecules critical in the regulation of inflammatory and immune responses. Once humans consume LA and ALA, which are dietary essential fatty acids, diverse PUFAs are synthesized by FADS, which are known to be related to many diseases and physiological conditions. However, humans must acquire PUFAs via foods or nutritional supplements because they do not contain delta-12 desaturases and convert molecules to PUFAs very slowly. Biotechnology methods have been used to clone and introduce numerous desaturases into diverse species such as animals, plants, and microorganisms to yield the critical PUFAs EPA and DHA. In conclusion, understanding the function and regulation of PUFAs linked to human health as well as recent PUFA biotechnology techniques may facilitate the engineering of more efficient PUFAs and aid in advanced PUFA production in new transgenic species. Novel therapeutic techniques may be developed for the clinical treatment of associated diseases. Moreover, these methods may also assist in obtaining new insights into the metabolic complexity of PUFAs regulated by fatty acid desaturases.