Enrichment of γ-linolenic acid from fungal oil by lipase-catalysed reactions

Summary Various lipases have been evaluated as biocatalysts for the enrichment of -linolenic acid from a commercial fungal oil derived from Mucor sp. by selective esterification of the fungal oil fatty acids with n-butanol or by selective hydrolysis of the oil. Lipase from M. miehei (Lipozyme), as compared to lipases from Candida cylindracea, Penicillium cyclopium, and Rhizopus arrhizus, was found to be most effective in the enrichment of -linolenic acid in unesterified fatty acids upon esterification of the fungal oil fatty acids with n-butanol. Thus, the -linolenic acid content could be raised from 10.4% in the starting material to 68.8% in the unesterified fatty acids. Selective hydrolysis of the fungal oil triacyglycerols using Lipozyme resulted in about 1.5-fold enrichment of -linolenic acid in the unhydrolysed acylglycerols. Other lipases tested, such as those from P. cyclopium, C. cylindracea, R. arrhizus, Penicillium sp. (Lipase G), porcine pancreas and Chromobacterium viscosum, were also rather ineffective in the enrichment of -linolenic acid by selective hydrolysis of the fungal oil triacylglycerols. Offprint requests to: K. D. Mukherjee     

Selective enzymic esterification of free fatty acids with n-butanol under microwave irradiation and under classical heating

Selective enzymic esterification of free fatty acids, obtained from blackcurrant oil by chemical saponification, with n-butanol using four immobilized lipases under microwave irradiation and under classical heating was studied. A positive effect of microwave irradiation on chemical yields of the products of the enzymic reactions and specificity of lipases were observed in comparison with a controlled heating in an incubator equipped with shaking (classical heating) applied during the identical enzyme-mediated processes. The maximum quantity of -linolenic acid (30%) was obtained with Lipozyme used as biocatalyst of the reaction under microwave irradiation. The maximum quantity of butyl -linolenate (20%) was obtained by a Pseudomonas cepacia lipase catalyzed esterification under classical heating.     

Substrate specificities of lipases in view of kinetic resolution of unsaturated fatty acids

Several commercially available lipases have been evaluated with regard to their substrate specificity in the esterification of fatty acids having specific positions of cis double bonds, e.g. petroselinic acid (n-12 18:1), alpha-linolenic acid (n-3 18:3), gamma-linolenic acid (n-6 18:3), stearidonic acid (n-3 18:4), dihomogamma-linolenic acid (n-6 20:3), eicosapentaenoic acid (n-3 20:5) and docosahexaenoic acid (n-3 22:6), with n-butanol. A common feature of most lipases, e.g. those from Penicillium cyclopium, Candida cylindracea, Mucor miehei, Rhizopus arrhizus and Penicillium sp. is that fatty acids having the first double bond from the carboxyl end as a cis-4 (n-3 22:6), cis-6 (n-12 18:1, n-6 18:3, n-3 18:4) or a cis-8 (n-6 20:3) double bond are strongly discriminated against compared to the other fatty acids, such as myristic acid (14:0), the reference standard, and n-3 18:3. In the case of the lipase from porcine pancreas, however, the discrimination against the above fatty acids is not as strong as with the other lipases. In contrast, the lipase from Chromobacterium viscosum shows a preference for n-12 18:1, n-6 18:3 and n-3 18:4. The observed substrate specificities can be utilized for enrichment of particular fatty acids by lipase-catalysed kinetic resolution from fatty acid mixtures, derived from naturally occurring fats and other lipids.Dedicated to Prof. David A. Walker, Robert Hill Institute, Department of Animal and Plant Sciences, University of Sheffield, Sheffield, UK, on the occasion of his sixty-fifth birthday on 18 August 1993 Correspondence to: K. D. Mukherjee     

Metabolic diversity in biohydrogenation of polyunsaturated fatty acids by lactic acid bacteria involving conjugated fatty acid production

Lactobacillus plantarum AKU 1009a effectively transforms linoleic acid to conjugated linoleic acids of cis-9,trans-11-octadecadienoic acid (18:2) and trans-9,trans-11–18:2. The transformation of various polyunsaturated fatty acids by washed cells of L. plantarum AKU 1009a was investigated. Besides linoleic acid, α-linolenic acid [cis-9,cis-12,cis-15-octadecatrienoic acid (18:3)], γ-linolenic acid (cis-6,cis-9,cis-12–18:3), columbinic acid (trans-5,cis-9,cis-12–18:3), and stearidonic acid [cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (18:4)] were found to be transformed. The fatty acids transformed by the strain had the common structure of a C18 fatty acid with the cis-9,cis-12 diene system. Three major fatty acids were produced from α-linolenic acid, which were identified as cis-9,trans-11,cis-15–18:3, trans-9,trans-11,cis-15–18:3, and trans-10,cis-15–18:2. Four major fatty acids were produced from γ-linolenic acid, which were identified as cis-6,cis-9,trans-11–18:3, cis-6,trans-9,trans-11–18:3, cis-6,trans-10–18:2, and trans-10-octadecenoic acid. The strain transformed the cis-9,cis-12 diene system of C18 fatty acids into conjugated diene systems of cis-9,trans-11 and trans-9,trans-11. These conjugated dienes were further saturated into the trans-10 monoene system by the strain. The results provide valuable information for understanding the pathway of biohydrogenation by anaerobic bacteria and for establishing microbial processes for the practical production of conjugated fatty acids, especially those produced from α-linolenic acid and γ-linolenic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enzymatic fractionation of evening primrose oil by rape lipase: Enrichment of gamma-linolenic acid

Summary Rape lipase discriminates strongly against -linolenic acid (allcis-6, 9, 12–183; GLA). GLA in the fatty acids from evening primrose oil was concentrated from 9.5% to 62% with a 95% yield during esterification of these fatty acids with butanol catalyzed by rape lipase. Hydrolysis of evening primrose oil catalyzed by the rape lipase raised the GLA content in unhydrolyzed acylglycerols to 28%.     

Substrate selectivity of various lipases in the esterification of cis- and trans-9-octadecenoic acid

The substrate selectivity of numerous commercially available lipases from microorganisms, plants and animal tissue towards 9-octadecenoic acids with respect to the cis/trans configuration of the CC double bond was examined by the esterification of cis- and trans-9-octadecanoic acid (oleic and elaidic acid respectively) with n-butanol in n-hexane. A great number of lipases studied, e.g. those from Pseudomonas sp., porcine pancreas or Carica papaya, were unable to discriminate between the isomeric 9-octadecenoic acids. However, lipases from Candida cylindracea and Mucor miehei catalysed the esterification of oleic acid 3–4 times faster than the corresponding reaction of elaidic acid and therefore have a high preference for the cis isomer. Of all biocatalysts examined, only recombinant lipases from Candidaantarctica favoured elaidic acid as substrate. While the preference of Candida antarctica lipase B for the trans isomer was quite low, Candida antarctica lipase A had an extraordinary substrate selectivity and its immobilized enzyme preparation [Chirazyme L-5 (3) from Boehringer] esterified elaidic acid about 15 times faster than oleic acid. Received: 29 October 1998 / Received revision: 18 December 1998 / Accepted: 21 December 1998     

Diverse effects of essential (n−6 andn−3) fatty acids on cultured cells

Fatty acids (FAs) have long been recognized for their nutritional value in the absence of glucose, and as necessary components of cell membranes. However, FAs have other effects on cells that may be less familiar. Polyunsaturated FAs of dietary origin (n–6 andn–3) cannot be synthesized by mammals, and are termed essential because they are required for the optimal biologic function of specialized cells and tissues. However, they do not appear to be necessary for normal growth and metabolism of a variety of cells in culture. The essential fatty acids (EFAs) have received increased attention in recent years due to their presumed involvement in cardiovascular disorders and in cancers of the breast, pancreas, colon and prostate. Manyin vitro systems have emerged which either examine the role of EFAs in human disease directly, or utilize EFAs to mimic thein vivo cellular environment. The effects of EFAs on cells are both direct and indirect. As components of membrane phospholipids, and due to their varying structural and physical properties, EFAs can alter membrane fluidity, at least in the local environment, and affect any process that is mediated via the membrane. EFAs containing 20 carbons and at least three double bonds can be enzymatically converted to eicosanoid hormones, which play important roles in a variety of physiological and pathological processes. Alternatively, EFAs released into cells from phospholipids can act as second messengers that activate protein kinase C. Furthermore, susceptibility to oxidative damage increases with the degree of unsaturation, a complication that merits consideration because lipid peroxidation can lead to a variety of substances with toxic and mutagenic properties. The effects of EFAs on cultured cells are illustrated using the responses of normal and tumor human mammary epithelial cells. A thorough evaluation of EFA effects on commercially important cells could be used to advantage in the biotechnology industry by identifying EFA supplements that lead to improved cell growth and/or productivity.Abbreviations AA arachidonic acid (20 carbons: 4 double bonds,n–6) - BHA butylated hydroxyanisole - BHT butylated hydroxytoluene - cAMP cyclic adenosine monophosphate - CHO Chinese hamster ovary - DAG diacylglycerol - DGLNA dihomo--linolenic acid (203,n–6) - DHA docosahexaenoic acid (226,n–3) - EFA essential fatty acid - EGF epidermal growth factor - EGFR epidermal growth factor receptor - EPA eicosapentaenoic acid (205,n–3) - FA fatty acid - FBS fetal bovine serum - GLNA -linolenic acid (183,n–6) - LA linoleic acid (182,n–6) - LNA -linolenic acid (183,n–3) - LT leukotriene - MDA malondialdehyde - NAD nicotinamide adenine dinucleotide - NDGA nordihydroguaiaretic acid - OA oleic acid (181,n–9) - PG prostaglandin - PKC protein kinase C - PUFA polyunsaturated fatty acid - SFM serum-free medium - TX thromboxane     

Synthesis of flavor and fragrance esters using <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsaturated (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10%. Similar results were observed in a study of the alcohol specificity of the enzyme for esterification of acetic and butyric acids with four alcohols: n-butyl, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral -substituted or -substituted carboxylic acids and n-butyl alcohol was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an organic solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids.     

Production of arachidonic acid by Mortierella fungi

Summary Various Mortierella fungi were assayed for their productivity of arachidonic acid (ARA). Only strains belonging to the subgenus Mortierella accumulated detectable amounts of ARA together with dihomo--linolenic acid. None of the strains belonging to the subgenus Micromucor tested accumulated these C-20 fatty acids, although they produced a C-18 fatty acid, -linolenic enic acid. A soil isolate, M. alpina 1S-4, was found to grow well in a liquid medium containing glucose and yeast extract as carbon and nitrogen sources, respectively. Addition of several natural oils such as olive and soybean oils to the medium increased the accumulation of ARA. Under optimal culture conditions in a 5-1 bench-scale fermentor, the fungus produced 3.6 g/l of ARA in 7 days. On cultivation for 10 days at 28°C in a 2000-1 fermentor, the same fungus produced 22.5 kg/kl mycelia (dry weight) containing 9.9 kg lipids, in which ARA comprised 31.0% of the total fatty acids. On standing the harvested mycelia for a further 6 days, major mycelial fatty acids (i.e. palmitic acid, oleic acid, linoleic acid, etc.) other than ARA rapidly decomposed and the ARA content of the total fatty acids reached nearly 70%.     

Effect of diets containing γ-linolenic acid on n-6 highly unsaturated fatty acid content of rotifer (Brachionus plicatilis)

The effect of various diets containing linoleic and/or -linolenic acids was studied on n-6 fatty acid composition of the rotifer Brachionus plicatilis. The rotifer's abilities for transformations of n-6 fatty acids were evaluated. Diets containing only linolenic acid as n-6 fatty acid induced low levels of other n-6 fatty acids in rotifers while a diet containing also -linolenic acid led to substantial amounts of di homo -linolenic acid in the rotifers through elongation. Desaturation of -linoleic acid to gamma linolenic appears to be the limiting factor of n-6 highly unsaturated fatty acid biosynthesis by the rotifer. Two sets of experiments were compared using different techniques and different sources of -linolenic acid: Spirulina in inert food or borage oil in emulsion with baker's yeast. Rotifers fed with inert diet with Spirulina contained arachidonic acid while those fed with borage oil had very low arachidonic content. High level of n-3 fatty acids incorporated into the diets seemed to exert inhibitory effects on n-6 transformation rate.     

Substrate selectivity of lipases in the esterification of cis/trans-isomers and positional isomers of conjugated linoleic acid (CLA)

The substrate selectivity of several microbial lipases has been examined in the esterification of the conjugated linoleic acid (CLA) isomers cis-9,trans-11-, cis-9,cis-11-, trans-9,trans-11- and trans-10,cis-12-octadecadienoic acid with n-butanol in n-hexane. Lipases from Candida cylindracea and Mucor miehei had a preference for the cis-9,trans-11-octadecadienoic acid, while Chirazyme L-5, a Candida antarctica lipase A, accepted the trans-9,trans-11-fatty acid with a high selectivity. Moreover, lipase from Candida cylindracea and Chirazyme L-5 catalysed the esterification of the cis-9,trans-11-octadecadienoic acid with n-butanol faster than the corresponding reaction of the trans-10,cis-12-fatty acid.     

Trapping of 2,7-Dichlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-Dioxin in Aqueous Solution by Enzymatic Reaction of Fungal Manganese Peroxidase in the Presence of Polyunsaturated Fatty Acids

Abstract:In the presence of polyunsaturated fatty acids, including cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), 2,7-dichlorodibenzo-p-dioxin (DCDD) was treated with manganese peroxidase (MnP) from white rot basidiomycete Phanerochaete sordida YK-624. After incubation with MnP, DCDD could not be extracted from the reaction mixture with n-hexane and was trapped in the water layer. DCDD was released by alkalification of the water layer. DCDD was also trapped after treatment with lipoxidase, which produces hydroperoxides from unsaturated lipids. The amounts of thiobarbituric acid-reactive substances produced in the MnP reactions with three highly unsaturated fatty acids were higher than the amounts produced with three fatty acids with a lower degree of unsaturation. These results suggest that a DCDD-trapping compound may be produced by peroxidation of the polyunsaturated fatty acids.Received: 22 October 2002 / Accepted: 6 December 2002     

Enrichment of very-long-chain mono-unsaturated fatty acids by lipase-catalysed hydrolysis and transesterification

Partial hydrolysis of triacylglycerols of high-erucic-acid seed oils from white mustard (Sinapis alba), oriental mustard (Brassica juncea) and honesty (Lunaria annua), catalysed by lipases from Candida cylindracea and Geotrichum candidum, leads to enrichment of erucic acid and other very-long-chain mono-unsaturated fatty acids (VLCMFA) in the acylglycerols (mono-, di- and triacylglycerol) while the C₁₈ fatty acids (oleic, linoleic and linolenic) are enriched in the fatty acid fraction. Partial hydrolysis of the high-erucic-acid triacylglycerols, catalysed by lipases from porcine pancreas, Chromobacterium viscosum, Rhizopus arrhizus and Rhizomucor miehei yields fatty acids with substantially higher levels of VLCMFA, as compared to the starting material, while the C₁₈ fatty acids are enriched in the acylglycerol fraction. Lipases from Penicillium sp. and Candida antarctica are ineffective for the fractionation of either group of fatty acids. Transesterification of the high-erucic-acid triacylglycerols with ethyl, propyl or butyl acetate or with n-butanol, catalysed by the lipase from R. miehei, leads to enrichment of VLCMFA in the alkyl (ethyl, propyl or butyl) esters, whereas the C₁₈ fatty acids are enriched in the acetylacylglycerols and acylglycerols.     

Is strain DS5 hydratase a C-10 positional specific enzyme? Identification of bioconversion products from α- and γ-linolenic acids byFlavobacterium sp. DS5

Summary Previously, we reported the isolation of a new microbial strain,Flavobacterium sp. DS5 (NRRL B-14859) which converted oleic and linoleic acids to their corresponding 10-keto- and 10--ydroxy-fatty acids. The hydration enzyme seemed to be specific to the C-10 position. Now we have identified, by GC/MS, NMR, and FTIR, the bioconversion products from -linolenic acid as 10-hydroxy-12(Z), 15(Z)-octadecadienoic acid and from -linolenic acid as 10-hydroxy-6(Z), 12(Z)-octadecadienoic acid. Products from 9(E)-unsaturated fatty acids were also identified as their corresponding 10-hydroxy or 10-keto fatty acids. From these results, it is concluded that strain DS5 hydratase is indeed a C-10 positional-specific enzyme and prefers an 18-carbon mono-unsaturated fatty acid. Among the C18 unsaturated fatty acids, an additional double bond on either side of the C-9 position lowers the enzyme hydration activity.     

Molecular Cloning and Functional Characterization of Fatty Acyl Desaturase and Elongase cDNAs Involved in the Production of Eicosapentaenoic and Docosahexaenoic Acids from <Emphasis Type="Bold">α</Emphasis>-Linolenic Acid in Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

Fish are the only major dietary source for humans of -3 highly unsaturated fatty acids (HUFAs) and with declining fisheries farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term and will constrain continuing growth of aquaculture activities. Greater understanding of how fish metabolize and biosynthesize HUFA may lead to more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase complementary DNA, SalDes, was isolated that include an open reading frame of 1362 bp specifying a protein of 454 amino acids. The protein sequence includes all the characteristics of microsomal fatty acid desaturases, including 3 histidine boxes, 2 transmembrane regions, and an N-terminal cytochrome b₅ domain containing a heme-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast Saccharomyces cerevisiae showed SalDes is predominantly an -3 5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from -linolenic acid (18:3n-3). The desaturase showed only low levels of 6 activity toward C₁₈ polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that included an open reading frame of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo included characteristics of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C₁₈ > C₂₀ > C₂₂. Thus this one polypeptide product displays all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3.     

Modification of fatty acid selectivity of <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase A by error-prone PCR

Objective

To generate Candida antarctica lipase A (CAL-A) mutants with modified fatty acid selectivities and improved lipolytic activities using error-prone PCR (epPCR).

Results

A Candida antarctica lipase A mutant was obtained in three rounds of epPCR. This mutant showed a 14 times higher ability to hydrolyze triacylglycerols containing conjugated linoleic acids, and was 12 and 14 times more selective towards cis-9, trans-11 and trans-10, cis-12 isomers respectively, compared to native lipase. Lipolytic activities towards fatty acid esters were markedly improved, in particular towards butyric, lauric, stearic and palmitic esters.

Conclusion

Directed molecular evolution is an efficient method to generate lipases with desirable selectivity towards CLA isomers and improved lipolytic activities towards esters of fatty acids.

     

High content of polyunsaturated fatty acids in muscle phospholipids of a fast runner,the European brown hare (Lepus europaeus)

To investigate both seasonal changes and possible intracorporal gradients of phospholipid fatty acid composition, skeletal muscles (n=124), hearts (n=27), and livers (n=34) from free-living brown hares (Lepus europaeus) were analyzed. Phospholipids from both skeletal muscles and heart had a high degree of unsaturation with 66.8±0.63% and 65.7±0.5% polyunsaturated fatty acids, respectively. This is the highest proportion of polyunsaturated fatty acids reported in any mammalian tissue. Polyunsaturated fatty acid content in skeletal muscles was 2.3% greater in winter compared to summer (F_1,106=17.7; P=0.0001), which may reflect thermoregulatory adjustments. Arachidonate (C20:4n-6) showed the greatest seasonal increase (+2.5%; F=7.95; P=0.0057). However, there were no pronounced differences in polyunsaturated fatty acid content between skeletal muscles from different locations in the body (m. iliopsoas, m. longissimus dorsi and m. vastus). Total muscle phospholipid polyunsaturated fatty acid content was correlated with polyunsaturated fatty acid content in triacyglycerols from perirenal white adipose tissue depots (r²=0.61; P=0.004). Polyunsaturated fatty acids were enriched in muscle phospholipids (56.8–73.6%), compared to white adipose tissue lipids (20.9–61.2%), and liver phospholipids (25.1–54.2%). We suggest that the high degree of muscle membrane unsaturation is related to hare-specific traits, such as a high maximum running speed.Abbreviations BMR basal metabolic rate - DPA docosapentaenoic acid - DHA docosahexaenoic acid - FA fatty acid - MUFA monounsaturated fatty acid - PC principal component - PUFA polyunsaturated fatty acid - SFA saturated fatty acid - UI unsaturation index - WAT white adipose tissueCommunicated by: G. Heldmaier     

Fatty acid composition of vitellogenin from four teleost species

The fatty acid compositions of vitellogenin and liver from cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus) and wolffish (Anarhichas lupus) were determined. Vitellogenin was isolated from plasma of estradiol-17-treated fish by precipitation with EDTA-Mg²⁺ and distilled water or by high-performance ion-exchange chromatography. In all investigated species, vitellogenin contained 16–18% (w/w) lipid, in which polyunsaturated fatty acids, predominantly 20:5 (n-3) and 22:6 (n-3), comprised about 50% of the total fatty acids. The proportions of saturated, monounsaturated, polyunsaturated and (n-3) fatty acids in vitellogenin of the different species were generally similar, although the relative content of specific fatty acids was distinctive for each species. The distribution of fatty acids in total lipids of vitellogenin was highly consistent among individual females of each species. In contrast, liver fatty acid composition varied considerably, both within and between species. Altogether, the differences in the fatty acid composition of vitellogenin and liver from each species indicate that a specific selection of fatty acids occurs during the lipidation of vitellogenin.Abbreviations BHT butylated hydroxytoluene - E-17 estradiol-17 - EDTA ethylenedinitrilo tetra-acetic acid disodium salt dihydrate - FA fatty acids - FAME fatty acid methyl esters - HDL high density lipoproteins - PUFA polyunsaturated fatty acids - SD standard deviation - TLC thin-layer chromatography - VHDL very high density lipoproteins - VLDL very low density lipoproteins - v/v volume per volume - w/v weight per volume - w/w weight per weight     

Identification of Primula “front-end” desaturases with distinct n−6 or n−3 substrate preferences

cDNA clones encoding cytochrome b₅ fusion desaturases were isolated from Primula cortusoides L. and Primula luteola Ruprecht, species previously shown to preferentially accumulate either n−6 or n−3 Δ6-desaturated fatty acids, respectively. Functional characterisation of these desaturases in yeast revealed that the recombinant Primula enzymes displayed substrate preferences, resulting in the predominant synthesis of either γ-linolenic acid (n−6) or stearidonic acid (n−3). Independent expression of the two Primula desaturases in transgenic Arabidopsis thaliana confirmed these results, with γ-linolenic acid and stearidonic acid accumulating in both leaf and seed tissues to different levels, depending on the substrate specificity of the desaturase. Targeted lipid analysis of transgenic Arabidopsis lines revealed the presence of Δ6-desaturated fatty acids in the acyl-CoA pools of leaf but not seed tissue. The implications for the transgenic synthesis of C₂₀ polyunsaturated fatty acids via the elongation of Δ6-desaturated fatty acids are discussed, as is the potential of using Primula desaturases in the synthesis of C₁₈ n−3 polyunsaturated fatty acids such as stearidonic acid.     

Functional characterisation of two cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>5</Subscript>-fusion desaturases from<Emphasis Type="Italic"> Anemone leveillei</Emphasis>: the unexpected identification of a fatty acid Δ<Superscript>6</Superscript>-desaturase

The Ranunculaceae are known to accumulate a wide range of unusual fatty acids in their seed lipids, and this variability has been advocated as a taxonomic marker. The Anemone species, Anemone leveillei L. and Anemone rivularis Buch.-Ham., have previously been reported to accumulate ⁵-desaturated fatty acids in their seed tissue [K. Aitzetmüller (1995) Plant Syst Evol 9:229–240]. Two cDNAs, AL1 and AL2, with similarity to plant cytochrome b₅-fusion "front-end" desaturases were isolated from developing seeds of A. leveillei and their function identified by expression in Saccharomyces cerevisiae. AL2 was characterised as a sphingolipid long-chain-base ⁸-desaturase, while AL1 acted as a fatty acid desaturase. However, AL1 did not produce ⁵-desaturated fatty acids as expected; instead, when expressed in transgenic S. cerevisiae or Arabidopsis thaliana this enzyme was functionally characterised as a ⁶-desaturase. Northern analysis confirmed the expression of this gene in seed tissue and leaf tissue of A. leveillei, though ⁶-desaturated fatty acids were found to accumulate only in the leaf tissue. The unexpected characterisation of a ⁶-desaturase in A. leveillei has implications for the use of fatty acids in chemotaxonomic studies. This is also the first report of a higher-plant ⁶-desaturase from a family other than the Boraginaceae.Abbreviations ALA -linolenic acid - DMOX 4,4-dimethyloxazoline - EDA eicosadienoic acid - FAME fatty acid methyl ester - GLA -linolenic acid - LA linoleic acid - LCB long chain base - ORF open reading frame - OTA octadecatetraenoic acid