Bioactivity and biotechnological production of punicic acid.

Punicic acid (PuA; 18: 3Δ 9cis,11trans,13cis ) is an unusual 18-carbon fatty acid bearing three conjugated double bonds. It has been shown to exhibit a myriad of beneficial bioactivities including anti-cancer, anti-diabetes, anti-obesity, antioxidant, and anti-inflammatory properties. Pomegranate (Punica granatum) seed oil contains approximately 80% PuA and is currently the major natural source of this remarkable fatty acid. While both PuA and pomegranate seed oil have been used as functional ingredients in foods and cosmetics for some time, their value in pharmaceutical/medical and industrial applications are presently under further exploration. Unfortunately, the availability of PuA is severely limited by the low yield and unstable supply of pomegranate seeds. In addition, efforts to produce PuA in transgenic crops have been limited by a relatively low content of PuA in the resulting seed oil. The production of PuA in engineered microorganisms with modern fermentation technology is therefore a promising and emerging method with the potential to resolve this predicament. In this paper, we provide a comprehensive review of this unusual fatty acid, covering topics ranging from its natural sources, biosynthesis, extraction and analysis, bioactivity, health benefits, and industrial applications, to recent efforts and future perspectives on the production of PuA in engineered plants and microorganisms.

Engineering the production of conjugated fatty acids in Arabidopsis thaliana leaves.

The seeds of many nondomesticated plant species synthesize oils containing high amounts of a single unusual fatty acid, many of which have potential usage in industry. Despite the identification of enzymes for unusual oxidized fatty acid synthesis, the production of these fatty acids in engineered seeds remains low and is often hampered by their inefficient exclusion from phospholipids. Recent studies have established the feasibility of increasing triacylglycerol content in plant leaves, which provides a novel approach for increasing energy density of biomass crops. Here, we determined whether the fatty acid composition of leaf oil could be engineered to accumulate unusual fatty acids. Eleostearic acid (ESA) is a conjugated fatty acid produced in seeds of the tung tree (Vernicia fordii) and has both industrial and nutritional end-uses. Arabidopsis thaliana lines with elevated leaf oil were first generated by transforming wild-type, cgi-58 or pxa1 mutants (the latter two of which contain mutations disrupting fatty acid breakdown) with the diacylglycerol acyltransferases (DGAT1 or DGAT2) and/or oleosin genes from tung. High-leaf-oil plant lines were then transformed with tung FADX, which encodes the fatty acid desaturase/conjugase responsible for ESA synthesis. Analysis of lipids in leaves revealed that ESA was efficiently excluded from phospholipids, and co-expression of tung FADX and DGAT2 promoted a synergistic increase in leaf oil content and ESA accumulation. Taken together, these results provide a new approach for increasing leaf oil content that is coupled with accumulation of unusual fatty acids. Implications for production of biofuels, bioproducts, and plant-pest interactions are discussed.

The linin promoter is highly effective in enhancing punicic acid production in Arabidopsis.

KEY MESSAGE: Enhanced levels of punicic acid were produced in the seed oil of Arabidopsis over-expressing pomegranate FATTY ACID CONJUGASE driven by heterologous promoters, among which the linin promoter was the most efficient. Fatty acids with conjugated double bonds play a special role in determining both the nutritional and industrial uses of plant oils. Punicic acid (18:3Δ9cis,11trans,13cis ), a conjugated fatty acid naturally enriched in the pomegranate (Punica granatum) seeds, has gained increasing attention from the biotechnology community toward its production in metabolically engineered oilseed crops because of its significant health benefits. The present study focused on selecting the best heterologous promoter to drive the expression of the P. granatum FATTY ACID CONJUGASE (PgFADX) cDNA as a means of producing punicic acid in Arabidopsis seed oil. Among the four promoters of genes encoding seed storage proteins from different crop species, the linin promoter led to the highest accumulation of punicic acid (13.2% of total fatty acids in the best homozygous line). Analysis of the relative expression level of PgFADX in developing seeds further confirmed that the linin promoter was most efficient in Arabidopsis. In addition, a conserved profile of cis-regulatory elements were identified in four heterologous promoters by bioinformatic analysis, and their possible roles in regulating gene expression during plant development were also discussed based on the results of this study in combination with the literature. This study contributes to metabolic engineering strategies aimed at enhancing the production of bioactive fatty acids in oilseed crops.

Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid.

MAIN CONCLUSION: Arabidopsis was engineered to produce 21.2 % punicic acid in the seed oil. Possible molecular factors limiting further accumulation of the conjugated fatty acid were investigated. Punicic acid (18:3Δ(9cis,11trans,13cis) ) is a conjugated linolenic acid isomer and is a main component of Punica granatum (pomegranate) seed oil. Medical studies have shown that punicic acid is a nutraceutical with anti-cancer and anti-obesity properties. It has been previously demonstrated that the conjugated double bonds in punicic acid are produced via the catalytic action of fatty acid conjugase (FADX), which is a homolog of the oleate desaturase. This enzyme catalyzes the conversion of the Δ(12)-double bond of linoleic acid (18:2Δ(9cis,12cis) ) into conjugated Δ(11trans) and Δ(13cis) -double bonds. Previous attempts to produce punicic acid in transgenic Arabidopsis thaliana seeds overexpressing P. granatum FADX resulted in a limited accumulation of punicic acid of up to 4.4 %, accompanied by increased accumulation of oleic acid (18:1∆(9cis) ), suggesting that production of punicic acid in some way inhibits the activity of oleate desaturase (Iwabuchi et al. 2003). In the current study, we applied a new strategy to enhance the production of punicic acid in a high linoleic acid A. thaliana fad3/fae1 mutant background using the combined expression of P. granatum FADX and FAD2. This approach led to the accumulation of punicic acid at the level of 21 % of total fatty acids and restored the natural proportion of oleic acid observed in the A. thaliana fad3/fae1 mutant. In addition, we provide new insights into the high oleate phenotype and describe factors limiting the production of punicic acid in genetically engineered plants.

Conjugated fatty acid synthesis: residues 111 and 115 influence product partitioning of Momordica charantia conjugase.

Conjugated linolenic acids (CLNs), 18:3 Δ(9,11,13), lack the methylene groups found between the double bonds of linolenic acid (18:3 Δ(9,12,15)). CLNs are produced by conjugase enzymes that are homologs of the oleate desaturases FAD2. The goal of this study was to map the domain(s) within the Momordica charantia conjugase (FADX) responsible for CLN formation. To achieve this, a series of Momordica FADX-Arabidopsis FAD2 chimeras were expressed in the Arabidopsis fad3fae1 mutant, and the transformed seeds were analyzed for the accumulation of CLN. These experiments identified helix 2 and the first histidine box as a determinant of conjugase product partitioning into punicic acid (18:3 Δ(9cis,11trans,13cis)) or α-eleostearic acid (18:3 Δ(9cis,11trans,13trans)). This was confirmed by analysis of a FADX mutant containing six substitutions in which the sequence of helix 2 and first histidine box was converted to that of FAD2. Each of the six FAD2 substitutions was individually converted back to the FADX equivalent identifying residues 111 and 115, adjacent to the first histidine box, as key determinants of conjugase product partitioning. Additionally, expression of FADX G111V and FADX G111V/D115E resulted in an approximate doubling of eleostearic acid accumulation to 20.4% and 21.2%, respectively, compared with 9.9% upon expression of the native Momordica FADX. Like the Momordica conjugase, FADX G111V and FADX D115E produced predominantly α-eleostearic acid and little punicic acid, but the FADX G111V/D115E double mutant produced approximately equal amounts of α-eleostearic acid and its isomer, punicic acid, implicating an interactive effect of residues 111 and 115 in punicic acid formation.

Expression analysis of VfDGAT2 in various tissues of the tung tree and in transgenic yeast.

The tung tree (Vernicia fordii Hemsl.; Vf) has great potential as an industrial crop owning to its seed oil that has multiple uses. Diacylglycerol acyltransferases (DGATs) catalyze the last and most committed step of triacylglycerol (TAG) biosynthesis. In order to examine the physiological role of the VfDGAT2 gene in the tung tree, we characterized its expression profiles in different tung tissues/organs and seeds at different developmental stages. Oil content and α-eleostearic acid production during seed development were also examined. Expression studies showed that VfDGAT2 was expressed in all tissues tested, with the highest expression in developing seeds where the expression was about 19-fold more than that in leaves. VfDGAT2 showed temporal-specific expression during seed development and maturation. Notably, the expression of VfDGAT2 in developing seeds was found to be consistent with tung oil accumulation and α-eleostearic acid production. The expression level of VfDGAT2 was lower in the early stages of oil accumulation and α-eleostearic acid biosynthesis, rapidly increased during the peak periods of fatty acid synthesis in August, and then decreased during completion of the accumulation period at the end of September. When the VfDGAT2 gene was transferred to the oleaginous yeast Rhodotorula glutinis, its expression was detected along with fatty acid products. The results showed that VfDGAT2 was highly expressed in transgenic yeast clones, and the total fatty acid content in one of these clones, VfDGAT2-3, was 7.8-fold more than that in the control, indicating that VfDGAT2 contributed to fatty acid accumulation into TAG and might be a target gene for improving tung oil composition through genetic engineering.

Parasitism by Cuscuta pentagona sequentially induces JA and SA defence pathways in tomato.

While plant responses to herbivores and pathogens are well characterized, responses to attack by other plants remain largely unexplored. We measured phytohormones and C(18) fatty acids in tomato attacked by the parasitic plant Cuscuta pentagona, and used transgenic and mutant plants to explore the roles of the defence-related phytohormones salicylic acid (SA) and jasmonic acid (JA). Parasite attachment to 10-day-old tomato plants elicited few biochemical changes, but a second attachment 10 d later elicited a 60-fold increase in JA, a 30-fold increase in SA and a hypersensitive-like response (HLR). Host age also influenced the response: neither Cuscuta seedlings nor established vines elicited a HLR in 10-day-old hosts, but both did in 20-day-old hosts. Parasites grew larger on hosts deficient in SA (NahG) or insensitive to JA [jasmonic acid-insensitive1 (jai1)], suggesting that both phytohormones mediate effective defences. Moreover, amounts of JA peaked 12 h before SA, indicating that defences may be coordinated via sequential induction of these hormones. Parasitism also induced increases in free linolenic and linoleic acids and abscisic acid. These findings provide the first documentation of plant hormonal signalling induced by a parasitic plant and show that tomato responses to C. pentagona display characteristics similar to both herbivore- and pathogen-induced responses.

Comparative kinetics of fatty acid-amino acid conjugate elicitor biosynthesis by midgut tissue microsomes of Lepidopterous caterpillar larvae.

N-Linolenoyl-L-glutamine is one of several structurally similar fatty acid-amino acid conjugate (FAC) elicitors found in the oral secretions of Lepidopterous caterpillars and its biosynthesis is catalyzed by membrane-associated alimentary tissue enzyme(s). FAC elicitors comprise 17-hydroxylated or non-hydroxylated linolenic acid coupled with L-glutamine or L-glutamate by an amide bond. We demonstrate in vitro biosynthesis of N-linolenoyl-L-glutamine by Manduca sexta, Heliothis virescens, and Helicoverpa zea tissue microsomes. Comparison of N-linolenoyl-L-glutamine biosynthesis kinetics for these species suggests that concurrent biosynthesis and hydrolysis contribute to proportions of FAC elicitors found in their oral secretions. The apparent K(m) values for coupling of sodium linolenate were 8.75±0.79, 14.3±3.7 and 20.7±3.4 mM and V(max) values were 2.92±0.14, 6.81±1.2 and 4.95±0.55 nmol/min/mg protein for H. zea, H. virescens and M. sexta, respectively. The K(m) values for coupling of L-glutamine were 10.5±0.26, 22.3±2.0 and 18.9±2.4 mM and V(max) values were 1.78±0.21, 3.71±0.50 and 2.49±0.41 nmol/min/mg of protein for H. zea, H. virescens and M. sexta, respectively.

Generation of Fad2 and Fad3 transgenic mice that produce n-6 and n-3 polyunsaturated fatty acids.

Linoleic acid (18 : 2, n-6) and α-linolenic acid (18 : 3, n-3) are polyunsaturated fatty acids (PUFAs), which are essential for mammalian health, development and growth. However, the majority of mammals, including humans, are incapable of synthesizing n-6 and n-3 PUFAs. Mammals must obtain n-6 and n-3 PUFAs from their diet. Fatty acid desaturase (Fad) plays a critical role in plant PUFA biosynthesis. Therefore, we generated plant-derived Fad3 single and Fad2-Fad3 double transgenic mice. Compared with wild-type mice, we found that PUFA levels were greatly increased in the single and double transgenic mice by measuring PUFA levels. Moreover, the concentration of n-6 and n-3 PUFAs in the Fad2-Fad3 double transgenic mice were greater than in the Fad3 single transgenic mice. These results demonstrate that the plant-derived Fad2 and Fad3 genes can be expressed in mammals. To clarify the mechanism for Fad2 and Fad3 genes in transgenic mice, we measured the PUFAs synthesis-related genes. Compared with wild-type mice, these Fad transgenic mice have their own n-3 and n-6 PUFAs biosynthetic pathways. Thus, we have established a simple and efficient method for in vivo synthesis of PUFAs.

Microbial Production of Conjugated Linoleic Acid and Conjugated Linolenic Acid Relies on a Multienzymatic System.

Conjugated linoleic acids (CLAs) and conjugated linolenic acids (CLNAs) have gained significant attention due to their anticarcinogenic and lipid/energy metabolism-modulatory effects. However, their concentration in foodstuffs is insufficient for any therapeutic application to be implemented. From a biotechnological standpoint, microbial production of these conjugated fatty acids (CFAs) has been explored as an alternative, and strains of the genera Propionibacterium, Lactobacillus, and Bifidobacterium have shown promising producing capacities. Current screening research works are generally based on direct analytical determination of production capacity (e.g., trial and error), representing an important bottleneck in these studies. This review aims to summarize the available information regarding identified genes and proteins involved in CLA/CLNA production by these groups of bacteria and, consequently, the possible enzymatic reactions behind such metabolic processes. Linoleate isomerase (LAI) was the first enzyme to be described to be involved in the microbiological transformation of linoleic acids (LAs) and linolenic acids (LNAs) into CFA isomers. Thus, the availability of lai gene sequences has allowed the development of genetic screening tools. Nevertheless, several studies have reported that LAIs have significant homology with myosin-cross-reactive antigen (MCRA) proteins, which are involved in the synthesis of hydroxy fatty acids, as shown by hydratase activity. Furthermore, it has been suggested that CLA and/or CLNA production results from a stress response performed by the activation of more than one gene in a multiple-step reaction. Studies on CFA biochemical pathways are essential to understand and characterize the metabolic mechanism behind this process, unraveling all the gene products that may be involved. As some of these bacteria have shown modulation of lipid metabolism in vivo, further research to be focused on this topic may help us to understand the role of the gut microbiota in human health.

Biosynthesis of gamma-linolenic acid in developing seeds of borage (Borago officinalis L.).

delta 6-desaturation of [14C]linoleoyl-CoA or [14C]oleoyl-CoA leading to the synthesis of gamma-linolenic acid was studied in vitro with microsomal fractions from developing seeds of Borago officinalis. Time course of the reaction, effects of protein and precursor concentrations and nucleotide requirements were examined. These parameters allowed us to improve the in vitro delta 6-desaturation assay. We observed that the precursors were acylated mainly in phosphatidylcholine, diacylglycerol and triacylglycerol, and then desaturated. NADH was absolutely required when [14C]oleoyl-CoA was the precursor, but not when [14C]linoleoyl-CoA was the precursor although it stimulated the reaction. The in vitro delta 6-desaturase activity was found mainly in phosphatidylcholine, associated with enriched endoplasmic reticulum membranes (ER) from embryos. No activity was observed in ER from seed coat or seedling. During maturation of the seeds, delta 6-desaturase reached its highest activity 14 to 16 days after pollination.

Differential biosynthesis of polyunsaturated fatty acids by Tetrahymena supplemented with ergosterol.

Tetrahymena grown with foreign sterols such as ergosterol incorporate them into cellular membranes at the expense of the native compound, tetrahymanol. It is shown that cells grown with ergosterol have a lessened capacity to produce the polyunsaturated linoleic and gamma-linolenic acids from [14C]oleic acid. However, the same cells have normal capacities to introduce double bonds at C-6 into linoleate, alpha-linolenate, or cis-vaccenate. Thus, a presumed 12-desaturase is inhibited in the presence of ergosterol, while desaturation at C-6 is unaffected.

The novel pathway for ketodiene oxylipin biosynthesis in Jerusalem artichoke (Helianthus tuberosus) tubers.

The new route of the plant lipoxygenase pathway, directed specifically towards the ketodiene formation, was detected during in vitro experiments with Jerusalem artichoke (Helianthus tuberosus) tubers. Through this pathway (9Z,11E,13S)-13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD) is reduced to corresponding 13-hydroxy acid (13-HOD), which is in turn dehydrogenated into ketodiene (9Z,11E,13S)-13-oxo-9,11-octadecadienoic acid (13-KOD). Dehydrogenation of 13-HOD into 13-KOD was not dependent on the presence of either NAD or NADP, but was strongly dependent on the presence of oxygen. Under anoxic conditions, 13-HOD dehydrogenation was blocked, but addition of 2,6-dichlorophenolindophenol restored it. Sulfite addition fully suppressed the aerobic dehydrogenation of 13-HOD. Hydrogen peroxide is a by-product formed by the enzyme along with 13-KOD. These data suggest that the ketodiene biosynthesis in H. tuberosus tubers is catalyzed by flavin dehydrogenase. (9S,10E,12Z)-9-Hydroxy-10,12-octadecadienoic acid (9-HOD) is dehydrogenated by this enzyme as effectively as 13-HOD, while alpha-ketol, (9Z)-12-oxo-13-hydroxy-9-octadecenoic acid, and ricinoleic acid did not act as substrates for dehydrogenase. The enzyme was soluble and possessed a pH optimum at pH 7.0-9.0. The only 13-HOD dehydrogenase known so far was detected in rat colon. However, unlike the H. tuberosus enzyme, the rat dehydrogenase is NAD-dependent.

Effect of dietary fats on arachidonic acid and eicosapentaenoic acid biosynthesis and conversion to C22 fatty acids in isolated rat liver cells.

The desaturation, chain elongation and esterification of [1-14C]eicosapentaenoic acid, [1-14C]arachidonic acid, [1-14C]eicosatrienoic acid, [1-14C]linolenic acid and [1-14C]linoleic acid were studied in isolated liver cells. Rats fed diets with either 15% hydrogenated coconut oil or 15% partially hydrogenated marine oil, both deficient in essential fatty acids, 15% soybean oil or standard pellet diet with 6% fat, were used. The delta 4-desaturation of 22:5(n - 3) and 22:4(n - 6) as well as the delta 6-desaturase activity was distinctly higher in cells from animals fed coconut or marine oil than with soybean oil or standard pellet. The rate of delta 5-desaturation of 20:3(n - 6) and 20:4(n - 3) was nearly the same in cells from rats fed coconut, marine and soybean oils and higher than with standard pellet. The chain elongation of 20:5(n - 3) to 22:5(n - 3) was distinctly more pronounced than the elongation of 20:4(n - 6) with all four diets. 20:5(n - 3) was mainly esterified in the phospholipids with marine and coconut oils, and mainly in triacylglycerol with standard pellet and soybean oils. The proportion of [1-14C]20:4(n - 6) in the phospholipids to that in triacylglycerol decreased in the order marine oil greater than coconut oil greater than standard pellet greater than soybean oil. The different endogenous arachidonic acid content in the phospholipids induced by the different diets increased in the same order. 20:5(n - 3) was rapidly esterified in triacylglycerol and phospholipids, then liberated especially from the triacylglycerol fraction, chain elongated to 22:5(n - 3) and reesterified.

Production of gamma-linolenic acid from the marine green alga Chlorella sp. NKG 042401.

gamma-Linolenic acid (GLA) production using a high GLA producing marine green alga, Chlorella sp. NKG 042401, was studied. GLA was presented in the galactolipid fraction (37.9%/total fatty acids). The effects of growth conditions on GLA production were studied. Optimum salinity for GLA production was 5 g l-1, at which salinity the highest cell concentration was achieved, resulting in a 1.6-fold increase in GLA productivity. Total fatty acid, however, was not drastically affected by change of salinity. Nitrogen starvation decreased the ratio of unsaturated fatty acids, and consequently GLA ratio in total fatty acid decreased. The urea adduct method was used to concentrate GLA from crude extract. As a result, after 5 sequential concentration procedures, GLA was concentrated 5-fold with a yield of 49%.

Fatty acid metabolism in the calanoid copepod Paracalanus parvus: 1. Polyunsaturated fatty acids.

The metabolic fate of radioactive linoleate and alpha-linolenate administered to the South Atlantic copepod Paracalanus parvus was studied. The wild copepod was able to incorporate the labeled acids dissolved in seawater. The radioactive linoleate was elongated to 20:2omega6 and 22:2omega6 and desaturated by a delta6 desaturase to 18:3omega6. alpha-Linolenate was also desaturated by a delta6 desaturase to 18:4omega3 and elongated to 20:3omega3. The copepod was able to convert alpha-18:3 to 20:5omega3 and 22:6omega3.

Biosynthesis of long-chain polyenoic acids from arachidonic acid in cultures of enriched spermatocytes and spermatids from mouse testis.

Primary spermatocytes (PS), round spermatids (RS) and condensing spermatids (CS) from mouse testes were enriched on Sta-Put 1 X g density gradients and cultured for 22 or 44 hr in the presence of [1-14C]arachidonate. Mass and radioactivity were measured by gas radiochromatography of constituent fatty acids of the various complex lipid classes fractionated by thin layer chromatography. Patterns and levels of incorporation were compared with those of whole testis, both in vitro and in vivo. The 20:4, 22:4, 22:5, 24:4 and 24:5 of the germinal cells contained levels of radioactivity in each lipid class which were consistent with an important role for the germinal cells in long-chain polyenoic acid (LCPA) metabolism. Cells which represented later stages of spermatogenesis (RS, CS) incorporated much higher percentages and absolute amounts of radioactivity into the fatty acids derived from 20:4 by elongation-desaturation pathways than did PS or whole testis in vitro. These differences were most pronounced in triacylglycerol of CS. Distributions of mass and radioactivity among lipid classes suggest synthesis of triacylglycerol by CS with a high degree of specificity for 22 or 24 carbon LCPA at the sn-3 position.

Oxidative desaturation of alpha-linoleic, linoleic, and stearic acids by human liver microsomes.

The desaturation of stearic, linoleic, and alpha-linolenic acids by human liver microsomes were studied. The microsomes were isolated from liver biopsies obtained during operation. It was shown that human liver microsomes are able to desaturate 1-14-C-alpha-linoleic acid to octadeca-6,9,12,15,-telraenoic acid: 1-15-C-linoleic acid to gammalinolenic acid; and 1-14-C-stearic acid to oleic acid in the same system described in the rat. However, the desaturation activity obtained was low compared to other mammals. This effect was attributed to fasting, pre-medication, or the anaesthesia.

Selection of Rhizopus strains for L(+)-lactic acid and gamma-linolenic acid production.

The production of L(+)-lactic acid and formation of gamma-linolenic acid by 50 Rhizopus strains growing on saccharidic substrates were investigated. Formation of acids was observed on solid cultivation media but mainly during submerged fermentation. Strains with the highest selectivity of both L(+)-lactic acid production and gamma-linolenic acid formation were tested in a laboratory fermenter. The best producer was treated by UV irradiation to increase the fatty acid content in the biomass, especially that of gamma-linolenic acid. The conversion of 10% saccharidic substrate by this newly prepared strain Rhizopus arrhizus CCM 8109 results in more than 95% of theoretical yield of L(+)-lactic acid and permits a volume productivity of 0.4 g gamma-linolenic acid per liter.

Oligounsaturated fatty acid production by selected strains of micromycetes.

Fifteen strains of filamentous fungi from the Culture Collection of Fungi (Charles University, Prague) were tested for their lipid production, fatty acid composition with emphasis on accumulation of oligounsaturated fatty acids. All cultures contained palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2) and gamma-linolenic (18:3) acid (GLA). The mycelium of Cunninghamella elegans, Rhizopus arrhizus, Mortierella parvispora, M. elongata and M. alpina contained arachidonic acid (ARA) in the range of 2.3-33.5% of the total fatty acids. The strains used in our experiment were capable to accumulate a relatively high amount of intracellular lipid (9.6-20.1% in dry biomass). The highest content of GLA (22.3 mg/g) was found in Mucor circinelloides. The strain of M. alpina containing 47.1 mg/g of ARA could be considered as the best producer of ARA.