Vernonia DGATs increase accumulation of epoxy fatty acids in oil

Vernolic acid (cis‐12‐epoxy‐octadeca‐cis‐9‐enoic acid) is valuable as a renewable chemical feedstock. This fatty acid can accumulate to high levels in the seed oil of some plant species such as Vernonia galamensis and Stokesia laevis which are unsuitable for large‐scale production. A cost‐effective alternative for production of epoxy fatty acids is to genetically engineer its biosynthesis in commercial oilseeds. An epoxygenase cDNA (SlEPX) responsible for vernolic acid synthesis and two acyl‐CoA : diacylglycerol acyltransferase cDNAs (VgDGAT1 and VgDGAT2) catalysing triacylglycerol (TAG) formation were cloned from developing seeds of S. laevis and V. galamensis. Co‐expression of SlEPX and VgDGAT1 or VgDGAT2 greatly increases accumulation of vernolic acid both in petunia leaves and soybean somatic embryos. Seed‐specific expression of VgDGAT1 and VgDGAT2 in SlEPX mature soybean seeds results in vernolic acid levels of ～15% and 26%. Both DGAT1 and DGAT2 increase epoxy fatty acid accumulation with DGAT2 having much greater impact.     

Vernonia DGATs can complement the disrupted oil and protein metabolism in epoxygenase-expressing soybean seeds

Plant oils can be useful chemical feedstocks such as a source of epoxy fatty acids. High seed-specific expression of a Stokesia laevis epoxygenase (SlEPX) in soybeans only results in 3-7% epoxide levels. SlEPX-transgenic soybean seeds also exhibited other phenotypic alterations, such as altered seed fatty acid profiles, reduced oil accumulation, and variable protein levels. SlEPX-transgenic seeds showed a 2-5% reduction in total oil content and protein levels of 30.9-51.4%. To address these pleiotrophic effects of SlEPX expression on other traits, transgenic soybeans were developed to co-express SlEPX and DGAT (diacylglycerol acyltransferase) genes (VgDGAT1 & 2) isolated from Vernonia galamensis, a high accumulator of epoxy fatty acids. These side effects of SlEPX expression were largely overcome in the DGAT co-expressing soybeans. Total oil and protein contents were restored to the levels in non-transgenic soybeans, indicating that both VgDGAT1 and VgDGAT2 could complement the disrupted phenotypes caused by over-expression of an epoxygenase in soybean seeds.     

Distribution of distinct arachidonoyl-specific and non-specific isoenzymes of diacylglycerol kinase in baboon (Papio cynocephalus) tissues.

We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-2-arachidonoyl diacylglycerol and sn-1,2-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly 'preferred' the substrate sn-1-stearoyl-2-arachidonoylglycerol, the two other species preferred sn-1,2-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-2-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa.     

Quantitative deuterium isotopic profiling at natural abundance indicates mechanistic differences for delta 12-epoxidase and delta 12-desaturase in Vernonia galamensis

Quantitative (2)H NMR spectroscopy can determine the natural abundance ((2)H/(1)H) ratio at each site of a molecule. In natural products, variation in these values is related to the reaction mechanisms in the pertinent biosynthetic pathway. For the first time, this novel approach has been exploited to probe for mechanistic differences in the introduction of different functionalities into a long-chain fatty acid. Vernolic acid, a major component of the seed oil of Vernonia galamensis, contains both an epoxide and a desaturation. The site-specific isotopic distribution ((2)H/(1)H)(i) has been determined for both vernolic acid and linoleic acid isolated from the same V. galamensis oil. It is found that the ((2)H/(1)H) ratio of vernolic acid shows a pattern along the entire length of the chain, consistent with linoleic acid being its immediate precursor. Notably, the C13 relates to the C13 of linoleic acid but not to the C13 of oleic acid. Furthermore, the C12 and C13 positions in vernolic acid are less depleted, consistent with a change in hybridization state from sp(2) to sp(3). However, the C11 position shows a marked relative enrichment in the vernolic acid, implying that it plays a role in the epoxidase but not the desaturase mechanism. Thus, although it can be concluded that the catalytic mechanisms for the epoxidase and desaturase activities are similar, marked differences in the residual ((2)H/(1)H) patterns indicate that the reaction mechanisms are not identical.     

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.     

Transgenic production of epoxy fatty acids by expression of a cytochrome P450 enzyme from Euphorbia lagascae seed

Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Delta(12)-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Delta(12)-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developing E. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to the CYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Delta(9,15)) in cultures supplied with alpha-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Delta(9,15). Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Delta(12)-epoxy group of vernolic acid.     

Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content

Top-down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content.     

The involvement of phospholipid:diacylglycerol acyltransferases in triacylglycerol production

We have characterized three CoA-independent types of enzyme, phospholipases, phospholipid:diacylglycerol acyltransferases (PDATs) and cholinephosphotransferases, responsible for the removal of unusual fatty acids from phosphatidylcholine (PC) in microsomal preparations from developing oil seeds. The metabolism of sn-2-[(14)C]acyl-PC was monitored in microsomal preparations from various oilseeds having either medium-chain, acetylenic, epoxy or hydroxy fatty acids as their major fatty acids in the oil. The results indicate that PDAT plays a major role in removing ricinoleic acid and vernolic acid from phospholipids in Ricinus communis and Crepis palaestina seeds, respectively. However, vernolic, crepenynic and capric acids are primarily removed from phospholipids by phospholipases in Euphorbia lagascae, Crepis rubra and elm seeds, respectively. Further, we show that significant PDAT activity is also present in vegetative tissues of Arabidopsis thaliana.     

Characterization of microsomal diacylglycerol acyltransferase activity from bovine adipose and muscle tissue

The activity of the triacylglycerol bioassembly enzyme, diacylglycerol acyltransferase (DGAT), was characterized in microsomal fractions prepared from bovine subcutaneous (SC) adipose, intramuscular (IM) adipose, and muscle (pars costalis diaphragmatis) tissue. The activity of DGAT was generally higher from SC adipose tissue than from IM adipose or muscle tissue. The characteristics of DGAT activity from the three bovine tissues resembled the activity characteristics observed in previous studies from various other organisms and tissues; the pH optimum was near neutrality, the activity was almost completely inhibited by pre-incubation with N-ethylmaleimide (NEM), and the enzyme accepted a broad range of acyl-CoAs and sn-1,2-diacylglycerols. In some aspects, the SC adipose tissue DGAT activity was different from the DGAT activity from the other two tissues. The SC adipose tissue DGAT activity was not as susceptible to inhibition by NEM as the enzymes from the two other tissue sources, and it exhibited increased specificity for substrates containing oleoyl moieties. The differences in DGAT properties between the three bovine tissues may account to some extent for the differences in the relative fatty acid composition and the positional distribution of fatty acids in triacylglycerol between bovine tissues. The observed differences in enzymatic properties also support recent biochemical and molecular genetic observations that imply the existence of multiple DGAT genes and/or isoforms.     

Isolation of a gene encoding a 1,2-diacylglycerol-sn-acetyl-CoA acetyltransferase from developing seeds of Euonymus alatus

1,2-Diacyl-3-acetyl-sn-glycerols (ac-TAG) are unusual triacylglycerols that constitute the major storage lipid in the seeds of Euonymus alatus (Burning Bush). These ac-TAGs have long-chain acyl groups esterified at both the sn-1 and sn-2 positions of glycerol. Cell-free extracts of developing seeds of E. alatus contain both long-chain acyl-CoA and acetyl-CoA sn-1,2-diacylglycerol acyltransferase (DGAT) activity. We have isolated a gene from developing seeds of Euonymus alatus that shows a very high sequence similarity to the members of the DGAT1 gene family (i.e. related to acyl-CoA:cholesterol acyltransferases). This Euonymus DGAT1 gene, when expressed in wild type yeast, results in a 5-fold enhancement of long-chain triacylglycerol (lc-TAG) accumulation, as well as the appearance of low levels of ac-TAG. Hydrogenated ac-TAG molecular species were identified by gas chromatography-mass spectrometry. Microsomes isolated from this transformed yeast show diacylglycerol:acetyl-CoA acetyltransferase activity, which is about 40-fold higher than that measured in microsomes prepared from yeast transformed with the empty vector or with the Arabidopsis thaliana DGAT1 gene. The specific activity of this microsomal acetyltransferase activity is of the same order of magnitude as the microsomal long-chain DGAT activities measured for yeast lines transformed with the empty vector or either the Arabidopsis or Euonymus DGAT1 genes. Despite this, ac-TAG accumulation in yeast transformed with the Euonymus DGAT1 gene was very low (0.26% of lc-TAG), whereas lc-TAG accumulation was enhanced. Possible reasons for this anomaly are discussed. Expression of the Euonymus DGAT1-like gene in yeast lines where endogenous TAG synthesis has been deleted confirmed that the gene product has both long-chain acyl- and acetyltransferase activity.     

Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.     

sn-1,2-diacylglycerol kinase of Escherichia coli. Diacylglycerol analogues define specificity and mechanism

A detailed structure/function analysis of the substrate specificity of Escherichia coli sn-1,2-diacylglycerol kinase was performed with three goals in mind: (a) to define the substrate specificity; (b) to discover inhibitors; and (c) to elucidate the specificity of diacylglycerol-dependent inactivation. Forty-seven structural analogues of sn-1,2-diacylglycerol were prepared and examined as substrates, inhibitors, and irreversible inactivators of the enzyme using mixed micellar assay methods. Modification of the acyl chains or the sn-2 ester affected the apparent Km but had only small effects on Vm; modifications of the sn-1 ester, sn-3 methylene, or sn-3 hydroxyl had large effects on the apparent Vm and smaller effects on Km. Consistent with these observations, diacylglycerol analogues modified only in the acyl chains or sn-2 ester were not diacylglycerol kinase inhibitors, whereas analogues with substitutions of the sn-1 ester or sn-3 hydroxyl frequently caused inhibition. A hydrogen bond-donating group was required for an analogue to be a diacylglycerol kinase inhibitor. Studies of diacylglycerol kinase inactivation by the various analogues were consistent with the previous conclusion that this process involves an interaction of diacylglycerols with an enzyme conformation different from that active in catalysis (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 15062-15069). Studies with a water-soluble diacylglycerol, sn-1,2-dibutyrylglycerol, allowed direct comparison of diacylglycerol kinase activity in mixed micelles with that in native membranes. The results are discussed in relation to the structural requirements of other diacylglycerol-dependent enzymes.     

Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin

The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3^cisΔ9,12,15) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.     

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.     

Expression and characterization of diacylglycerol acyltransferase from Arabidopsis thaliana in insect cell cultures

Diacylglycerol acyltransferase (DGAT) catalyses the acylation of the sn-3 hydroxy group of sn-1,2-diacylglycerol using acyl-CoA. The gene encoding DGAT from Arabidopsis thaliana has been cloned and the function of the enzyme proved by expression of the coding sequence using a bacculovirus expression system in insect cell cultures. The expressed protein catalysed the synthesis of [(14)C]triacylglycerol from [(14)C]diacylglycerol and oleoyl-CoA. The heterologously expressed DGAT activity was found mostly associated with the 100000 g pellet. The optimum activity was achieved at a neutral pH, in the presence of Mg2+, and at an optimum oleoyl-CoA concentration of 20 microM. The DGAT used the substrates palmitoyl-CoA and oleoyl-CoA equally effectively. In these experiments, the inclusion of recombinant acyl-CoA binding protein had a relatively small effect upon DGAT activity.     

Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content

SUMMARY: A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.     

Enzyme activities of intestinal triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.)

The substitution of fish oil with plant-derived oil in diets for carnivorous fish, such as Atlantic salmon, has previously revealed the potentially deleterious supranuclear accumulation of lipid droplets in intestinal cells (enterocytes) which may compromise gut integrity, and consequently, fish health. This suggests that unfamiliar dietary lipid sources may have a significant impact on intestinal lipid metabolism, however, the mode of lipid resynthesis is largely unknown in teleost fish intestine. The present study aimed at characterising three key lipogenic enzymes involved in the biosynthesis of triacylglycerol (TAG) and phosphatidylcholine (PC) in Atlantic salmon enterocytes: monoacylglycerol acyltransferase (MGAT), diacylglycerol acyltransferase (DGAT), and diacylglycerol cholinephosphotransferase (CPT). Furthermore, to investigate the dietary effect of plant oils on these enzymes, two experimental groups of fish were fed a diet with either capelin (fish oil) or vegetable oil (rapeseed oil:palm oil:linseed oil, 55:30:15 w/w) as the lipid source. The monoacylglycerol (MAG) pathway was highly active in the intestinal mucosa of Atlantic salmon as demonstrated by MGAT activity (7 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)) and DGAT activity (4 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)), with MGAT appearing to also provide adequate production of sn-1,2-diacylglycerol for potential utilisation in PC synthesis via CPT activity (0.4 nmol CDP-[(14)C]choline incorporated min(-1) mg protein(-1)). Both DGAT and CPT specific activity values were comparable to reported mammalian equivalents, although MGAT activity was lower. Nevertheless, MGAT appeared not to be the rate-limiting step in salmon intestinal TAG synthesis. The homology between piscine and mammalian enzymes was established by similar stimulation and inhibition profiles by a variety of tested cofactors and isomeric substrates. The low dietary n-3/n-6 PUFA ratio presented in the vegetable oil diet did not significantly affect the activities of MGAT, DGAT, or CPT under optimised assay conditions, or in vivo intestinal mucosa lipid class composition, when compared to a standard fish oil diet.