MOLECULAR SWITCH THAT CONTROLS THE FLUX OF LINOLEIC ACID INTO N-6 OR N-3 POLYUNSATURATED FATTY ACIDS IN MICROORGANISMS

Polyunsaturated Fatty Acids (PUFA) of the n-6 and n -3 series play important roles in nutrition. Microorganisms are important sources of n-6 and n-3 fatty acids; however, most produce either n-6 or n -3 fatty acids as the major PUFAs and very few produce both. This differential production suggests that P UFAs metabolic pathway is strictly controlled in microor ganisms. The major pathway of n-6/n-3 faty acids biosynthesis in lower eukaryotes is composed of ∆12 Desaturase (Des), ω3 Des (∆15, ∆17), ∆6 Des, ∆6 Elongase (Elo), ∆5 Des, ∆5 Elo and ∆4 Des, among which ∆6 Des and ∆15 (ω3) Des, located at the branch point of PUFAs metabolic pathways, are key regulato rs of the flux of linoleic acid (18:2 n-6) into eit her n-6 or n-3 fatty acid metabolic pathways. These latter two enzymes work together as a molecular switch tha t control the production of n-6/n-3 fatty acids. Howe ver the mechanism of the molecular switch is, so fa r, not clear. This review summarizes the recent advancemen t of the molecular base of the differentail product ion of n-6 or n-3 PUFAs in microorganisms.


INTRODUCTION
Poly Unsaturated Fatty Acids (PUFAs) are straight chain fatty acids with chain lengths varying from C18 to C22 and more than two double bonds. Among these, n-6 and n-3 fatty acids are essential to human health and play important but different physiological roles. A balanced ratio of n-6/n-3 fatty acids and the amount of n-3 fatty acids, is regarded as important factors for human health (Harris, 2006;Scott et al., 2007;Candela et al., 2011). However the ratio of n-6/n-3 in current diet oils is commonly around 10:1 to 25:1, which is far from ideal, due to the fact that most of the traditional plant oils, such as soybean oil, sunflower oil, peanut oil, contains high-levels of n-6 fatty acid, linoleic acid (LA; 18:2 n-6)), with the exception of a few plant oils, such as Linum usitatissimum and Perilla frutescens, that contain very high amounts of n-3 fatty acid, Alpha-Linoleic Acid (ALA; 18:3 n-3). Furthermore, most plant species do not usually produce ∆6-desaturated fatty acids, with a few exceptions such as Boraginaceae, Oenothera spp. and Ribes spp. (Gunstone, 1992). In addition, some members of the Primulaceae family accumulate γ-linolenic acid (GLA; 18:3 n-6) and SteariDonic Acid (SDA; 18:4 n-3) (Sayanova et al., 1999;2006). Primula species can produce either n-6 or n-3 ∆6-desaturated fatty acids (GLA or SDA) depending on the substrate preference of ∆6 Desaturase (∆6 Des) but the overall production of n-3 fatty acids is much higher than n-6 fatty acids suggesting a high activity of ω-3 Desaturase (ω-3 Des) is present in these organisms (Sayanova et al., 1999;2006). However, in general, higher plants does not produce PUFAs with carbon chain length beyond C18 and the few that do, do not produce fatty acids with more than two double bonds.

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Very long chain PUFAs (VL-PUFAs, with carbon chain length ≧20) commonly occur in many microorganisms including microalgae (Lenihan-Geels et al., 2013), filamentous fungi (Shimizu et al., 1988), marine protists Schizochytrium and Thraustochytrium (traditionally refered to marine fungi) (Nagano et al., 2011) and bacteria (Abd Elrazak et al., 2013). In microalgae and filamentous fungi, the biosynthetic pathway for PUFAs is the desaturase/elongase system, while in marine protsits, the desaturase/elongase system and Polyketide Synthase (PKS) both exist and in marine bacteria only PKS is available for PUFAs biosynthesis (Metz et al., 2001). Therefore in this review we focus on the regulation of PUFAs biosynthesis in microalgae and filamentous fungi.
Most oleaginuous microorganisms, such as algae and a few fungi, produce oils with n-3 fatty acids as dominant PUFAs, some, such as the zygomycete fungi, produce oils with n-6 fatty acids as dominant PUFAs, only very few microorganisms can produce oils with both n-3 and n-6 fatty acids as major PUFAs. Therefore the production of different series of PUFAs is strictly regulated in each individual microorganism, suggesting that the metabolic flux of LA to n-6/n-3 fatty acids is controlled by endogeneous biosynthetic pathways which are regulated by either the property of the enzymes, such as their substrate preference, or environmental stimuli such as low temperature, which has been shown to induce n-3 fatty acid prodution (Shimizu et al., 1988). The main enzymes involved in the biosynthesis of n-6/n-3 PUFAs in eukaryotic microorganisms (both algae and fungi) are: ∆12 Des, ω-3 (∆15, ∆17) Des, ∆6 Des, ∆6 Elo, ∆5 Des, ∆5 Elo and ∆4 Des (Pereira et al., 2003), as shown in Fig. 1.

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In this article, we summarize the recent findings about n-6/n-3 fatty acid production in microorganisms that contain both ∆6 Des and ω-3 Des. We also give a summary of the biochemical characterization of ∆6 Des and ω-3 Des and provide a rational correlation between the LA flux and the substrate preference and activity of the molecular switch.

MICROORGANISMS THAT CONTAIN BOTH ∆6 DES AND Ω-3 DES GENES
Not all PUFAs-producing microorganisms with desaturase systems contain both ∆6 Des and ω-3 Des genes. For example, the filamentous fungi Mucor circinelloides that produces significant amounts of GLA (Kennedy et al., 1993), but not ALA or SDA, has the ∆6 Des gene but not that coding for ∆15 (ω-3) Des in its genome as revealed by genomic analysis (http://genome.jgi-psf.org/Mucci2/Mucci2.home.html). In contrast, the green microalga, Chlamydomonas reinhardtii, that produces 80% of its total fatty acids as n-3 fatty acid ALA (18:3 n-6) has ω-3 Des gene but not ∆6 Des gene in its genome (Nguyen et al., 2013). Neither of these microorganisms has both ∆6 Des and ω-3 Des and clearly no switching between the PUFA pathways is possible. Therefore, these and similar oleaginous microorganisms that only produce PUFAs of a single series will not be further discussed in this paper.
Although genomic sequences of many fungi and microalgae are available, the desaturases of those organisms have yet to be identified. Those organisms that have both ∆6 and ω-3 Des, based on the PUFAs profile of the microorganisms, including some where molecular work has been carried out on their desaturases, are listed in Table 1. Most microorganisms that have both ∆6 Des and ω-3 Des are filamentous fungi and microalgae. In general, yeast species have ∆15 (ω-3) Des but not ∆6 Des in their genome; however, one yeast, Candida diddensiae, may have both ∆6 and ω-3 Des based on its fatty acid profile (Rateldge, 1997). This, however, is from a single report and the constituent desaturases were not identified. Interestingly, cyanobacteria species show highly diversified desaturase systems (Dyal and Narine, 2005): Group 1 have only ∆9 Des and, as a consequence, they produce only monounsaturated fatty acids; group 2 have ∆9 Des, ∆12 Des and ∆15 (ω-3) Des and, accordingly, these organisms can produce LA, that is further converted to n-3 fatty acid ALA (18:3 n-3); group 3 contain ∆9 Des, ∆12 Des and ∆6 Des and produce LA, that is further converted to GLA (18:3 n-6); group 4 contain ∆9 Des, ∆12 Des, ∆15 (ω-3) Des and ∆6 Des, therefore the LA produced in this group of cyanobacteria can be converted into both n-6 GLA and n-3 ALA.

MOLECULAR SWITCH OF MICROORGANISMS THAT PRODUCE N-6 FATTY ACIDS AS MAJOR LIPIDS
Species belonging to the genus Mortierella can produce large amounts of Arachidonic Acid (ARA; 20:4 n-6) but very little Eicosapentaenoic Acid (EPA; 20:5 n-3) (Murata and Wada, 1995). Mortierella alpina is currently used to produce ARA commercially. Genomic analysis has shown that this fungus contains ∆15 (ω-3) Des, ∆6 Des, ∆6 Elo and ∆5 Des fatty acid desaturation/elongation systems for the production of series PUFAs . Although it contains a complete set of desaturase genes for the biosynthesis of both ARA (n-6) and EPA (n-3), at physiological growth temperatures, it produces ARA as the only major VL-PUFA. This regulation of PUFA metabolic flux is partly due to the substrate specificity of the ∆6 Des Table 2, Sakuradani and Shimizu, 2003;Sakuradani et al., 2005;Zhu et al., 2002) which prefers n-6 fatty acid LA to n-3 ALA as substrate and its ω-3 Des must be repressed at physiological growth temperature (Shimizu et al., 1998). Although the ω-3 Des have been cloned and biochemically studied in a few microorganisms Table 5, (Gellerman and Schlenk, 1979;Pereira et al., 2004;Sakamoto et al., 1994;Wada and Murata, 1990), its expression level in oleaginous microorganisms has not been determined so far. However over-expression of ω-3 Des in Mt. alpina by genetic engineering increased the production of EPA from 5 to 35% of the total FAs and ARA decreased from 60 to 30% of total FAs, while the amount of total fatty acids did not change significantly (Ando et al., 2009). These results suggested that the ∆6 Des and ω-3 Des work together as a switch, which is regulated by their substrate preference and activity, controlling the flux of LA into n-6 and/or n-3 fatty acid pathways. Furthermore, at low temperatures (12-14°C) some species of Mt. alpina can produce significant amount of EPA (2% w/w of TFA for strain ATCC 32222; 15% w/w of TFA for strain IS-4). These results suggested that ω-3 Des, which converts LA to ALA, is regulated by temperature. Whereas cold-induced expression of ω-3 Des gene at mRNA level have been found in algae and many plants (Wada and Murata 1990;Shi et al., 2011;Zhang et al., Science Publications AJBB 2011; Takemura et al., 2012) and it is most sensitive to changes in temperature compared to other desaturases such as ∆9, ∆12 and ∆6 Des gene (Sakamoto et al., 1994). Therefore, it is suggested that ω-3 Des gene in Mt. alpina is repressed at physiological temperature of 25-28°C, leading to flux of LA to ARA and at low temperature, it is induced, leading to the partial flux of LA to ALA, which is further metabolized to EPA.
Recent work has found that a phototrophic alga, Parietochloris incisa, can produce ARA up to 60% of the total FA, but with very little EPA Table 4, (Iskandarov et al., 2010), although the ∆6 Des of this organism can convert LA and ALA at similar activities. This suggests that its ∆15 or ω-3 Des gene must be repressed at physiological temperatures, which resulting in the switch-off of LA flux to ALA and therefore leading to diminished flux of ALA to EPA. Similarly, other organisms such as Synechocystis sp. PCC 6803 and Conidiobolus obscurus that can produce more n-6 fatty acids than n-3 fatty acids, even though their ∆6 Des works equally with either n-6 or n-3 fatty acid Table 3, (Reddy et al., 1993;Tan et al., 2011).
Pythium irregulare, is a rare example of a microorganism that can produce both n-6 ARA and n-3 EPA as its major PUFAs Table 4, (O'Brien et al., 1993;Hong et al., 2002). The ∆6 Des of this fungus can catalyze the desaturation of both LA and ALA at high conversion yields, albeit with slight substrate preference towards LA. These results indicate that a ω-3 Des is actively expressed in this organism. Furthermore, the fungus produced much more EPA than ARA (1.4-fold increase) when grow at low temperature state, likely due to the induction of ω-3 Des gene by low temperature.

MOLECULAR SWITCH OF MICROORGANISMS THAT PRODUCE N-3 FATTY ACID AS MAJOR LIPIDS
Many oleaginous microalgae can produce n-3 VL-PUFAs and the ∆6 Des from several n-3 fatty acid producing organisms have been characterized biochemically Table 3, (Dunstan et al., 1992;Domergue et al., 2005;Petrie et al., 2010a;2010b;Wagner et al., 2010;Ahmann et al., 2011). In general, the substrate preference of ∆6 Des correlates with the ratio of n-3/n-6 fatty acids. Marine microalgae are one of the primary producers of n-3 fatty acids, such as EPA and DHA and the ratio of n-3/n-6 fatty acids in these organisms are usually very high. In terms of the substrate specificities of the ∆6 Des in these organisms, as shown in Table 3, they usually prefer to use n-3 fatty acids ALA. For example, ∆6 Des from Ostreococcus lucimarinus showed strong preference for ALA, 39% of fed ALA was converted to SDA, while only 6.6% of fed LA was converted to GLA. ∆6 Des from Micromona pusilla also shows strong preference for n-3 fatty acids, the conversion yield of ALA is ten times higher than that of LA.
These data suggests that the substrate preference of ∆6 Des play a major role in directing the flux of LA into n-3/n-6 fatty acid pathway. However, it is noteworthy that ∆6 Des from microalgae Phaeodactylum ricornutum, Glossomastix chrysoplasta and Thalassiosira pseudonana have no significant substrate preference ( Table 4), but these organisms can still produce n-3 fatty acids as major PUFAs, indicating that an active ω-3 Des gene is highly expressed in these organisms, although the ω-3 Des from these organisms have not been characterized so far. Taken together, the n-3 fatty acid preference of ∆6 Des and the potential high-level expression of ω-3 Des gene probably lead to the high production of n-3 fatty acids and thus high ratio of n-3/n-6 fatty acids.

POTENTIAL REGULATORY MECHANISM OF THE MOLECULAR SWITCH
The molecular switch of n-6/n-3 PUFA production is controlled by the substrate preference of ∆6 Des and the activity of ω-3 Des. Although many studies have been carried out to determine the substrate preference of heterogeneous expressed ∆6 Des, no structure-function studies have been achieved so far, not to mention how this substrate preference of ∆6 Des may be regulated by nutritional or environmental conditions. The molecular mechanism for the sensing of lowtemperature and induced expression of desaturase gene in eukaryotic organisms is unknown, however it has been investigated in cyanobacteria and Bacillus subtilis (Suzuki and Murata, 2000;Aguilar et al., 2001) and well reviewed (Sakamoto and Murata, 2002;Shivaji and Prakash, 2010). It was suggested that the primary signal perceived by a bacterium when exposed to low-temperature is the rigidification of the membrane. This causes the activation of a membrane-associated sensor, which in turn, activates a cytosolic response regulator. Besides, this activated response regulator binds to the promoter of the desaturase and as a consequence the desaturase is induced. In cyanobacteria the Hik 33 (sensor)-Rer 26 (response regulator), while in Bacillus the DesK (sensor)-DesR (response regulator), the two component system for lowtemperature signaling has been identified (Suzuki and Murata, 2000;Aguilar et al., 2001).

SUMMARY
Most fungi, microalgae and some bacteria produce PUFAs through oxidative desaturation of fatty acids but only some of them can produce both GLA and ALA and their derived fatty acids, these organisms contain both ∆6 and ω-3 Des. The substrate preference of ∆6 Des of microorganisms plays a major role in directing the metabolic flux of LA into either n-6 or n-3 PUFAs. It produces n-6 or n-3 PUFAs as major product according to its substrate preference of n-6 or n-3 fatty acids. However when the substrate preference of ∆6 Des for LA or ALA is comparable, then the activity of ω-3 Des, which is controlled by its genetic background, but also regulated by environmental stimuli such as temperature, determines the metabolic flux of LA into n-6 or n-3 fatty acids. When the activity of ω-3 Des is very low, the microorganism produces n-6 fatty acids as major lipids; when the activity of ω-3 Des is very high, then it produces n-3 fatty acids as major lipids and at certain activity level, it may produce equal amount of n-6 and n-3 fatty acids. The substrate preference of ∆6 Des is controlled by its protein structure which is primary determined by the amino acid sequence of the protein,

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whether and how this substrate preference may be regulated by other nutritional or environmental conditions is, so far, not clear. The molecular mechanism of low temperature induced expression of fatty acid desaturase in bacteria has been investigated, the two component system for the sensing of the low temperature signal and the transduction of this signal to the induction of the expression of fatty acid desaturase has been identified in some bacteria. However the mechanism for the low temperature induced expression of ω-3 Des in eukaryotic microorganisms is completely unknown. Research into this area should be exciting and provide insights to the understanding of the biochemistry of n-6/n-3 PUFA biosynthesis in oleaginous microorganisms. Importance of a balanced omega 6/omega 3 ratio for the maintenance of health. Nutritional recommendations. Importancia del equilibrio del índice omega-6/omega-3 en el mantenimiento de un