Polyunsaturated Fatty Acid Biosynthetic Pathway Research Articles

N-3 PUFA biosynthesis by the copepod Apocyclops royi documented using fatty acid profile analysis and gene expression analysis.

ABSTRACTThe cyclopoid copepod Apocyclops royi (Lindberg 1940) is one of two dominant mesozooplankton species in brackish Taiwanese aquaculture ponds. Periodically low n-3 polyunsaturated fatty acid (PUFA) content in seston could potentially be a limiting factor for zooplankton diversity. Apocyclops royi’s potential ability to biosynthesize n-3 PUFA was investigated through a short-term feeding experiment on four species of microalgae. Furthermore, we analyzed the expression of genes encoding putative fatty acid elongase (ELO) and desaturase (FAD) enzymes in A. royi on long-term diets of the PUFA-poor Dunaliella tertiolecta and the PUFA-rich Isochrysis galbana. The copepods exhibited high contents of docosahexaenoic acid (DHA, C22:6n-3) (>20% of total fatty acid) even when DHA-starved for two generations, and no significant differences were found in absolute DHA content between treatments. Transcripts correlating to the four enzymes Elovl4, Elovl5, Fad Δ5 and Fad Δ6 in the n-3 PUFA biosynthetic pathway were identified. Gene expression analysis revealed a significantly higher expression of two desaturases similar to Fad Δ6 in copepods fed PUFA-lacking algae compared to copepods fed algae with high PUFA content. These findings suggest a highly active n-3 PUFA biosynthesis and capability of DHA production in A. royi when fed low-PUFA diets.

Metabolic engineering to produce γ-linolenic acid in Brassica napus using a Δ6-desaturase from pike eel

We generated γ-linolenic acid (GLA, C18:3Δ6,9,12)-producing transgenic Brassica napus transformed with McD6DES, the Δ6-desaturase gene identified from pike eel (Muraenesox cinereus) under the control of the seed-specific vicilin promoter. Seed-specific expression of McD6DES in B. napus produced up to 8.4% of GLA by creating a double bond at the sixth position from the carboxyl end of linoleic acid (LA, C18:2Δ9,12) in seeds. These results demonstrate that McD6DES expression enables to reconstitute in polyunsaturated fatty acid biosynthetic pathways, highlighting the potential of GLA biosynthesis as a target for metabolic engineering of oilseed crops.

Influence of feeding stearidonic acid (18:4n-3)-enriched soybean oil, as compared to conventional soybean oil, on tissue deposition of very long-chain omega-3 fatty acids in meat-type chickens

In chickens, the desaturation of α-linolenic acid (ALA; 18:3n-3) to stearidonic acid (SDA; 18:4n-3) is considered to be rate-limiting for the hepatic conversion of ALA to very long-chain (VLC; i.e. >20 C) n-3 polyunsaturated fatty acids (PUFAs). Thus, we hypothesized that feeding broilers SDA plus ALA, as compared to ALA alone, would bypass this inefficient metabolic step and enrich meat with greater amounts of VLC n-3 PUFAs. Female Ross×Heritage broilers were fed mash diets containing 50g/kg of conventional soy oil (CON) from hatch until d 28. On d 29, they were divided into two groups and fed diets containing either 50g/kg CON or 50g/kg of SDA-enriched oil derived from the genetic modification of the soybean (SDASOY) until d 42. Final (42 d) body weights, as well as weight gains and feed conversion values from 29 to 35 d and 36 to 42 d, were not different (P>0.05) between treatments. Compared to the CON treatment, dietary SDASOY increased (P<0.01) total VLC n-3 PUFA contents of skinless and boneless breasts, tenders, and thighs by almost 3-fold. However, the SDASOY diet also contained more total n-3 fatty acids (ALA+SDA) than the CON diet (ALA only), and it was estimated that ALA and SDA were metabolized to VLC n-3 PUFAs and deposited into breast, tenders, and thigh meat with equal efficiency. Docosapentaenoic acid (DPA; 22:5n-3) was the predominant VLC n-3 PUFA in all three muscles, suggesting that another control point downstream of the initial hepatic Δ6-desaturase reaction was rate-limiting in the biosynthesis of DHA from ALA. Alternately, since broilers have the capability to convert ALA to DHA in the liver, it is likely that the capacity of the VLC n-3 PUFA biosynthetic pathway is simply not great enough to allow for the deposition of DHA into muscle at levels equal to those attained by direct dietary supplementation. It is also possible that, rather than undergoing elongation and desaturation, some of the ALA and SDA pool underwent β-oxidation in the liver, as suggested by others, while a large portion of each fatty acid was not metabolized and was transported out of the liver to other tissues, such as adipose. However, the relative hepatic expression of genes whose protein products are involved in fatty acid oxidation (as well as in desaturation and elongation or lipogenesis) were not significantly affected by dietary treatment or age.

Field trial evaluation of the accumulation of omega-3 long chain polyunsaturated fatty acids in transgenic Camelina sativa: Making fish oil substitutes in plants

The global consumption of fish oils currently exceeds one million tonnes, with the natural de novo source of these important fatty acids forming the base of marine foodwebs. Here we describe the first field-based evaluation of a terrestrial source of these essential nutrients, synthesised in the seeds of transgenic Camelina sativa plants via the heterologous reconstitution of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway. Our data demonstrate the robust nature of this novel trait, and the feasibility of making fish oils in genetically modified crops. Moreover, to our knowledge, this is the most complex example of plant genetic engineering to undergo environmental release and field evaluation.

The Generation of Transgenic Mice with Fat1 and Fad2 Genes that have their own Polyunsaturated Fatty Acid Biosynthetic Pathway

Background: Microorganisms and higher plants possess their own omega-3 and omega-6 polyunsaturated fatty acid (PUFAs) biosynthetic pathways. The n-6 fatty acid desaturase gene fad-2 codes for the n-6 desaturase enzyme that coverts oleic acid (OA 18:1 n-9) into linoleic acid (LA 18:2 n-6). The n-3 fatty acid desaturase gene fat-1 codes for the n-3 desaturase enzyme that converts n-6 PUFAs into n-3 PUFAs. Mammals lack n-3 and n-6 desaturase enzymes; therefore, they must obtain their omega-3 and omega-6 fatty acids by consuming plants or seafood. The beneficial effects of n-3 and n-6 PUFAs on human development and cardiovascular health have been well documented. Methods: Here, we generated fat-1 and fad-2 transgenic mice by introducing mammal expression vectors containing the fat-1 and fad-2 genes via microinjection. Results: Seven transgenic mice were obtained that expressed functional n-3 and n-6 desaturase enzymes. Analysis of the fatty acid contents of transgenic mouse livers revealed that n-6 and n-3 PUFA levels were greatly increased in the transgenic mice compared to wild-type mice. The use ratios of n-9 PUFAs (18:1 n-9) and n-6 PUFAs were both greater in the transgenic mice than in the wild-type controls. Conclusion: These transgenic mice were capable of producing their own omega-3 and omega-6 fatty acids. They have the same fatty acid metabolic pathways as higher plants and microbes.

PMO-248 Mucosal upregulation of arachidonic acid production in active ulcerative colitis: delivery of pro-inflammatory eicosanoid precursors

IntroductionUlcerative colitis (UC) is a colonic inflammatory disorder of unconfirmed aetiology. Eicosanoids, inflammatory mediators involved in UC pathogenesis, are enzymatically converted from dietary polyunsaturated fatty acids (PUFA), arachidonic acid (AA)...

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described.

Active ulcerative colitis is associated with altered fatty acid precursor availability for the eicosanoid synthetic pathway

IntroductionThe polyunsaturated fatty acids (PUFA) arachidonic acid (AA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) are generated from dietary essential fatty acids linoleic acid (LA) and α-linolenic...

Towards the production of high levels of eicosapentaenoic acid in transgenic plants: the effects of different host species, genes and promoters

Eicosapentaenoic acid (EPA, 20:5n-3) plays an important role in many aspects of human health. In our efforts towards producing high levels of EPA in plants, we investigated the effects of different host species, genes and promoters on EPA biosynthesis. Zero-erucic acid Brassica carinata appeared to be an outstanding host species for EPA production, with EPA levels in transgenic seed of this line reaching up to 25%. Two novel genes, an 18-carbon omega3 desaturase (CpDesX) from Claviceps purpurea and a 20-carbon omega3 desaturase (Pir-omega3) from Pythium irregulare, proved to be very effective in increasing EPA levels in high-erucic acid B. carinata. The conlinin1 promoter from flax functioned reasonably well in B. carinata, and can serve as an alternative to the napin promoter from B. napus. In summary, the judicious selection of host species and promoters, together with the inclusion of genes that enhance the basic very long chain polyunsaturated fatty acid biosynthetic pathway, can greatly influence the production of EPA in plants.

Characterization of Both Polyunsaturated Fatty Acid Biosynthetic Pathways in Schizochytrium sp.

Schizochytrium produces long chain polyunsaturated fatty acids (PUFAs) via a PUFA synthase. Targeted mutagenesis of one gene of this synthase was conducted to confirm PUFA synthase function and determine its metabolic necessity. The resulting mutants were auxotrophic and required supplementation with PUFAs. In vivo labeling experiments with radioactive fatty acids demonstrated the presence of several elongase and desaturase activities associated with the standard pathway of PUFA synthesis. However, this system was missing a critical Delta12 desaturase activity and was therefore not capable of synthesizing PUFAs from the 16- or 18-carbon saturated fatty acid products of the fatty acid synthase. Because Schizochytrium uses a PUFA synthase system for the production of PUFAs, the existence of a partial desaturase-elongase system (if not a simple vestige) is suggested to be either a scavenging mechanism for intermediate fatty acids prematurely released by the PUFA synthase or for PUFAs found in the organism's native environment.

α-Linolenic acid, Δ6-desaturase gene polymorphism, and the risk of nonfatal myocardial infarction

Background:Δ6-Desaturase (FADS2) is the rate-limiting step in the polyunsaturated fatty acid (PUFA) biosynthetic pathway.Objective:The aim was to test whether the common deletion [T/-] in the promoter of FADS2affects the PUFA biosynthetic pathway and consequently modifies the effect of α-linolenic acid (ALA) on myocardial infarction (MI).Design:Case subjects (n=1694) with a first nonfatal acute MI were matched by age, sex, and area of residence to 1694 population-based control subjects in Costa Rica. PUFAs were quantified by gas-liquid chromatography from plasma and adipose tissue samples. Least-squares means from generalized linear models and odds ratios (ORs) and 95% CIs from multiple conditional logistic regression models were estimated.Results:The prevalence of the variant T/- allele was 48%. Eicosapentaenoic acid, γ-linolenic acid, and arachidonic acid decreased in adipose tissue and plasma with increasing number of copies of the variant allele with a monotonic trend (P< 0.05 for all). Fasting plasma triacylglycerols by genotype were 2.08 mmol/L for TT, 2.16 mmol/L for T-, and 2.26 mmol/L for - - [ie, homozygous for the variant (deletion) allele] (P= 0.03). The FADS2deletion was not associated with MI and did not significantly modify the association between adipose tissue ALA and the risk of MI.Conclusions:The FADS2deletion may prevent the conversion of ALA into very-long-chain PUFAs. However, this metabolic effect is not translated into an attenuated risk between ALA and MI among carriers of the variant. It is possible that, at current intakes of ALA, any potential defect in the transcription of the gene is masked by the availability of substrate. Further research in populations deficient in ALA intake is warranted.

Functional characterization of front‐end desaturases from trypanosomatids depicts the first polyunsaturated fatty acid biosynthetic pathway from a parasitic protozoan

A survey of the three kinetoplastid genome projects revealed the presence of three putative front-end desaturase genes in Leishmania major, one in Trypanosoma brucei and two highly identical ones (98%) in T. cruzi. The encoded gene products were tentatively annotated as Delta8, Delta5 and Delta6 desaturases for L. major, and Delta6 desaturase for both trypanosomes. After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative Delta6 desaturases could have Delta4 desaturase activity, based mainly on the conserved HX(3)HH motif for the second histidine box, when compared with Delta4 desaturases from Thraustochytrium, Euglena gracilis and the microalga, Pavlova lutheri, which are more than 30% identical to the trypanosomatid enzymes. After cloning and expression in Saccharomyces cerevisiae, it was possible to functionally characterize each of the front-end desaturases present in L. major and T. brucei. Our prediction about the presence of Delta4 desaturase activity in the three kinetoplastids was corroborated. In the same way, Delta5 desaturase activity was confirmed to be present in L. major. Interestingly, the putative Delta8 desaturase turned out to be a functional Delta6 desaturase, being 35% and 31% identical to Rhizopus oryzae and Pythium irregulareDelta6 desaturases, respectively. Our results indicate that no conclusive predictions can be made about the function of this class of enzymes merely on the basis of sequence homology. Moreover, they indicate that a complete pathway for very-long-chain polyunsaturated fatty acid biosynthesis is functional in L. major using Delta6, Delta5 and Delta4 desaturases. In trypanosomes, only Delta4 desaturases are present. The putative algal origin of the pathway in kinetoplastids is discussed.

Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants.

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the omega3/6 Delta8 -desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Delta9 -specific elongating activity from Isochrysis galbana, a Delta8 -desaturase from Euglena gracilis and a Delta5 -desaturase from Mortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Delta9 -elongating activity, which may bypass rate-limiting steps present in the conventional Delta6 -desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.

Genetic dissection of polyunsaturated fatty acid synthesis in Caenorhabditis elegans.

Polyunsaturated fatty acids (PUFAs) are important membrane components and precursors of signaling molecules. To investigate the roles of these fatty acids in growth, development, and neurological function in an animal system, we isolated Caenorhabditis elegans mutants deficient in PUFA synthesis by direct analysis of fatty acid composition. C. elegans possesses all the desaturase and elongase activities to synthesize arachidonic acid and eicosapentaenoic acid from saturated fatty acid precursors. In our screen we identified mutants with defects in each fatty acid desaturation and elongation step of the PUFA biosynthetic pathway. The fatty acid compositions of the mutants reveal the substrate preferences of the desaturase and elongase enzymes and clearly demarcate the steps of this pathway. The mutants show that C. elegans does not require n3 or Delta5-unsaturated PUFAs for normal development under laboratory conditions. However, mutants with more severe PUFA deficiencies display growth and neurological defects. The mutants provide tools for investigating the roles of PUFAs in membrane biology and cell function in this animal model.

Production of C20 polyunsaturated fatty acids (PUFAs) by pathway engineering: identification of a PUFA elongase component from Caenorhabditis elegans

Using a combination of database-mining and functional characterization, we have identified a component of the polyunsaturated fatty acid (PUFA) elongase. Co-expression of this elongating activitiy with fatty acid desaturases has allowed us to heterologously reconstitute the PUFA biosynthetic pathway. Both these enzymes (desaturases and elongase components) have undergone gene-duplication events which provide a paradigm for the diverged nature of PUFA biosynthetic activities.

Production of C20 polyunsaturated fatty acids (PUFAs) by pathway engineering: identification of a PUFA elongase component from Caenorhabditis elegans

Using a combination of database-mining and functional characterization, we have identified a component of the polyunsaturated fatty acid (PUFA) elongase. Co-expression of this elongating activity with fatty acid desaturases has allowed us to heterologously reconstitute the PUFA biosynthetic pathway. Both these enzymes (desaturases and elongase components) have undergone gene-duplication events which provide a paradigm for the diverged nature of PUFA biosynthetic activities.

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway.

A Caenorhabditis elegans ORF encoding the presumptive condensing enzyme activity of a fatty acid elongase has been characterized functionally by heterologous expression in yeast. This ORF (F56H11. 4) shows low similarity to Saccharomyces cerevisiae genes involved in fatty acid elongation. The substrate specificity of the C. elegans enzyme indicated a preference for Delta(6)-desaturated C18 polyunsaturated fatty acids. Coexpression of this activity with fatty acid desaturases required for the synthesis of C20 polyunsaturated fatty acids resulted in the accumulation of arachidonic acid from linoleic acid and eicosapentaenoic acid from alpha-linolenic acid. These results demonstrate the reconstitution of the n-3 and n-6 polyunsaturated fatty acid biosynthetic pathways. The C. elegans ORF is likely to interact with endogenous components of a yeast elongation system, with the heterologous nematode condensing enzyme F56H11.4 causing a redirection of enzymatic activity toward polyunsaturated C18 fatty acid substrates.