Update on GM canola crops as novel sources of omega-3 fish oils

There is considerable interest in new sources of omega‐3 long‐chain (here defined as fatty acids ≥C20) polyunsaturated fatty acids (LC‐PUFA), specifically eicosapentaenoic acid (EPA; 20:5n‐3) and docosahexaenoic acid (DHA; 22:6n‐3), commonly known as omega‐3 fish oils, to supplement the limited supplies of oceanic fish oil (Tocher, 2015). These alternative sources include extraction of other diverse marine organisms (e.g. krill, plankton etc.), algal fermentation, and the genetic engineering of microbes such as yeasts. Another approach is the synthesis of omega‐3 fish oils in transgenic plants (reviewed in Napier et al., 2015), and this short article will discuss the recent results obtained by two major industry collaborations

Functional characterization of the \u3ci\u3eArabidopsis thaliana\u3c/i\u3e orthologue of Tsc13p, the enoyl reductase of the yeast microsomal fatty acid elongating system

The protein encoded by the Arabidopsis At3g55360 gene was selected as a candidate for the enoyl reductase of the microsomal elongase system based on its homology to the Tsc13p protein of S. cerevisiae. The studies presented here demonstrate that heterologous expression of At3g55360 functionally complements the temperature-sensitive phenotype of a yeast tsc13 mutant that is deficient in enoyl reductase activity. Furthermore, AtTSC13 is shown to interact physically with the Elo2p and Elo3p components of the yeast elongase complex. At3g55360 apparently encodes the sole enoyl reductase activity associated with microsomal fatty acid elongation in Arabidopsis. Consistent with this conclusion, AtTSC13 is ubiquitously expressed in Arabidopsis

The supply of fish oil to aquaculture: a role for transgenic oilseed crops?

The importance of an alternative and sustainable supply of omega-3 long chain polyunsaturated fatty acids (LC omega-3) has long been established. As these biologically active fatty acids have a role in nutrition and health, there is an ever increasing demand for oils containing LC omega-3 e.g. eicosapentaenoic (EPA) and docosahexaenoic acid (DHA). These fatty acids are produced by micoroganisms and enter our diet through the consumption of fish. However, in order that the nutritional requirements of fish in aquaculture are met and sufficient levels are deposited to meet the requirements of human consumers, EPA and DHA must be supplied in excess. Given the importance of the aquaculture industry in delivering healthy foodstuff, the question of how to resource the supply of LC omega-3 then arises; traditional sources of EPA and DHA (fish oil) are challenged, whilst vegetable oils do not contain EPA or DHA. Therefore research efforts have focused on the successful reconstitution of LC omega-3 biosynthesis in oilseed crops. The production of EPA and DHA in the seed oil of agricultural crops has the capacity to deliver large volumes of these fatty acids. The expression of optimised combinations of the genes required to produce these fatty acids in the seed of the crop Camelina sativa has been achieved and the utility of this approach demonstrated. This represents a significant breakthrough – the provision of an effective alternative to the use of omega-3 fish oil by the global aquaculture industry

Identification of a cDNA encoding a novel C18-Δ9 polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana11The nucleotide sequence reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession number AF390174.

AbstractIsochrysis galbana, a marine prymnesiophyte microalga, is rich in long chain polyunsaturated fatty acids such as docosahexaenoic acid (C22:6n-3, Δ4,7,10,13,16,19). We used a polymerase chain reaction-based strategy to isolate a cDNA, designated IgASE1, encoding a polyunsaturated fatty acid-elongating activity from I. galbana. The coding region of 263 amino acids predicts a protein of 30 kDa that shares only limited homology to animal and fungal proteins with elongating activity. Functional analysis of IgASE1, by expression in Saccharomyces cerevisiae, was used to determine its activity and substrate specificity. Transformed yeast cells specifically elongated the C18-Δ9 polyunsaturated fatty acids, linoleic acid (C18:2n-6, Δ9,12) and α-linolenic acid (C18:3n-3, Δ9,12,15), to eicosadienoic acid (C20:2n-6, Δ11,14) and eicosatrienoic acid (C20:3n-3, Δ11,14,17), respectively. To our knowledge this is the first time such an elongating activity has been functionally characterised. The results also suggest that a major route for eicosapentaenoic acid (C20:5n-3, Δ5,8,11,14,17) and docosahexaenoic acid syntheses in I. galbana may involve a Δ8 desaturation pathway

Modifying the lipid content and composition of plant seeds: engineering the production of LC-PUFA

Omega-3 fatty acids are characterized by a double bond at the third carbon atom from the end of the carbon chain. Latterly, long chain polyunsaturated omega-3 fatty acids such as eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and docosahexanoic acid (DHA; 22:6 Δ4,7,10,13,16,19), which typically only enter the human diet via the consumption of oily fish, have attracted much attention. The health benefits of the omega-3 LC-PUFAs EPA and DHA are now well established. Given the desire for a sustainable supply of omega-LC-PUFA, efforts have focused on enhancing the composition of vegetable oils to include these important fatty acids. Specifically, EPA and DHA have been the focus of much study, with the ultimate goal of producing a terrestrial plant-based source of these so-called fish oils. Over the last decade, many genes encoding the primary LC-PUFA biosynthetic activities have been identified and characterized. This has allowed the reconstitution of the LC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate omega-3 LC-PUFA to levels similar to that found in fish oil. In this review, we will describe the most recent developments in this field and the challenges of overwriting endogenous seed lipid metabolism to maximize the accumulation of these important fatty acids

Modulation of lipid biosynthesis by stress in diatoms

Diatoms are responsible for up to 40% of the carbon fixation in our oceans. The fixed carbon is moved through carbon metabolism towards the synthesis of organic molecules that are consumed through interlocking foodwebs, and this process is strongly impacted by the abiotic environment. However, it has become evident that diatoms can be used as ‘platform’ organisms for the production of high valuable bio-products such as lipids, pigments and carbohydrates where stress conditions can be used to direct carbon metabolism towards the commercial production of these compounds. In the first section of this review, some aspects of carbon metabolism in diatoms and how it is impacted by environmental factors are briefly described. The second section is focused on the biosynthesis of lipids and in particular omega-3 long-chain polyunsaturated fatty acids and how low temperature stress impacts on the production of these compounds. In a third section, we review the recent advances in bioengineering for lipid production. Finally, we discuss new perspectives for designing strains for the sustainable production of high-value lipids

Genetic manipulation of gamma-linolenic acid (GLA) synthesis in a commercial variety of evening primrose (Oenothera sp.)

A robust Agrobacterium-mediated transformation procedure was developed for Rigel, a commercial cultivar of evening primrose, and used to deliver a cDNA encoding a Delta(6)-desaturase from borage under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the transformed plants demonstrated an altered profile of polyunsaturated fatty acids, with an increase in gamma-linolenic acid and octadecatetraenoic acid in leaf tissues when compared with control lines

A nutritionally-enhanced oil from transgenic Camelina sativa effectively replaces fish oil as a source of eicosapentaenoic acid for fish

For humans a daily intake of up to 500mg omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) is recommended, amounting to an annual requirement of 1.25 million metric tonnes (mt) for a population of 7 billion people. The annual global supply of n-3 LC-PUFA cannot meet this level of requirement and so there is a large gap between supply and demand. The dietary source of n-3 LC-PUFA, fish and seafood, is increasingly provided by aquaculture but using fish oil in feeds to supply n-3 LC-PUFA is unsustainable. Therefore, new sources of n-3 LC-PUFA are required to supply the demand from aquaculture and direct human consumption. One approach is metabolically engineering oilseed crops to synthesize n-3 LC-PUFA in seeds. Transgenic Camelina sativa expressing algal genes was used to produce an oil containing n-3 LC-PUFA to replace fish oil in salmon feeds. The oil had no detrimental effects on fish performance, metabolic responses or the nutritional quality of the fillets of the farmed fish

Nutritional evaluation of an EPA-DHA oil from transgenic Camelina sativa in feeds for post-smolt Atlantic salmon (Salmo salar L.)

Vegetable oils (VO) are possible substitutes for fish oil in aquafeeds but their use is limited by their lack of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA). However, oilseed crops can be modified to produce n-3 LC-PUFA such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, representing a potential option to fill the gap between supply and demand of these important nutrients. Camelina sativa was metabolically engineered to produce a seed oil with around 15 % total n-3 LC-PUFA to potentially substitute for fish oil in salmon feeds. Post-smolt Atlantic salmon (Salmo salar) were fed for 11-weeks with one of three experimental diets containing either fish oil (FO), wild-type Camelina oil (WCO) or transgenic Camelina oil (DCO) as added lipid source to evaluate fish performance, nutrient digestibility, tissue n-3 LC-PUFA, and metabolic impact determined by liver transcriptome analysis. The DCO diet did not affect any of the performance or health parameters studied and enhanced apparent digestibility of EPA and DHA compared to the WCO diet. The level of total n-3 LC-PUFA was higher in all the tissues of DCO-fed fish than in WCO-fed fish with levels in liver similar to those in fish fed FO. Endogenous LC-PUFA biosynthetic activity was observed in fish fed both the Camelina oil diets as indicated by the liver transcriptome and levels of intermediate metabolites such as docosapentaenoic acid, with data suggesting that the dietary combination of EPA and DHA inhibited desaturation and elongation activities. Expression of genes involved in phospholipid and triacylglycerol metabolism followed a similar pattern in fish fed DCO and WCO despite the difference in n-3 LC-PUFA contents

Functional Characterization of the Arabidopsis β-Ketoacyl-Coenzyme A Reductase Candidates of the Fatty Acid Elongase

In plants, very-long-chain fatty acids (VLCFAs; \u3e18 carbon) are precursors of sphingolipids, triacylglycerols, cuticular waxes, and suberin. VLCFAs are synthesized by a multiprotein membrane-bound fatty acid elongation system that catalyzes four successive enzymatic reactions: condensation, reduction, dehydration, and a second reduction. A bioinformatics survey of the Arabidopsis (Arabidopsis thaliana) genome has revealed two sequences homologous to YBR159w encoding a Saccharomyces cerevisiae β-ketoacyl reductase (KCR), which catalyzes the first reduction during VLCFA elongation. Expression analyses showed that both AtKCR1 and AtKCR2 genes were transcribed in siliques, flowers, inflorescence stems, leaves, as well as developing embryos, but only AtKCR1 transcript was detected in roots. Fluorescent protein-tagged AtKCR1 and AtKCR2 were localized to the endoplasmic reticulum, the site of fatty acid elongation. Complementation of the yeast ybr159Δ mutant demonstrated that the two KCR proteins are divergent and that only AtKCR1 can restore heterologous elongase activity similar to the native yeast KCR gene. Analyses of insertional mutants in AtKCR1 and AtKCR2 revealed that loss of AtKCR1 function results in embryo lethality, which cannot be rescued by AtKCR2 expression using the AtKCR1 promoter. In contrast, a disruption of the AtKCR2 gene had no obvious phenotypic effect. Taken together, these results indicate that only AtKCR1 is a functional KCR isoform involved in microsomal fatty acid elongation. To investigate the roles of AtKCR1 in postembryonic development, transgenic lines expressing RNA interference and overexpression constructs targeted against AtKCR1 were generated. Morphological and biochemical characterization of these lines confirmed that suppressed KCR activity results in a reduction of cuticular wax load and affects VLCFA composition of sphingolipids, seed triacylglycerols, and root glycerolipids, demonstrating in planta that KCR is involved in elongation reactions supplying VLCFA for all these diverse classes of lipids