Development of a Brassica napus (Canola) Crop Containing Fish Oil-Like Levels of DHA in the Seed Oil.

Plant seeds have long been promoted as a production platform for novel fatty acids such as the ω3 long-chain (≥ C20) polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) commonly found in fish oil. In this article we describe the creation of a canola (Brassica napus) variety producing fish oil-like levels of DHA in the seed. This was achieved by the introduction of a microalgal/yeast transgenic pathway of seven consecutive enzymatic steps which converted the native substrate oleic acid to α-linolenic acid and, subsequently, to EPA, docosapentaenoic acid (DPA) and DHA. This paper describes construct design and evaluation, plant transformation, event selection, field testing in a wide range of environments, and oil profile stability of the transgenic seed. The stable, high-performing event NS-B50027-4 produced fish oil-like levels of DHA (9-11%) in open field trials of T3 to T7 generation plants in several locations in Australia and Canada. This study also describes the highest seed DHA levels reported thus far and is one of the first examples of a deregulated genetically modified crop with clear health benefits to the consumer.

Metabolic engineering for enhanced oil in biomass.

The world is hungry for energy. Plant oils in the form of triacylglycerol (TAG) are one of the most reduced storage forms of carbon found in nature and hence represent an excellent source of energy. The myriad of applications for plant oils range across foods, feeds, biofuels, and chemical feedstocks as a unique substitute for petroleum derivatives. Traditionally, plant oils are sourced either from oilseeds or tissues surrounding the seed (mesocarp). Most vegetative tissues, such as leaves and stems, however, accumulate relatively low levels of TAG. Since non-seed tissues constitute the majority of the plant biomass, metabolic engineering to improve their low-intrinsic TAG-biosynthetic capacity has recently attracted significant attention as a novel, sustainable and potentially high-yielding oil production platform. While initial attempts predominantly targeted single genes, recent combinatorial metabolic engineering strategies have focused on the simultaneous optimization of oil synthesis, packaging and degradation pathways (i.e., 'push, pull, package and protect'). This holistic approach has resulted in dramatic, seed-like TAG levels in vegetative tissues. With the first proof of concept hurdle addressed, new challenges and opportunities emerge, including engineering fatty acid profile, translation into agronomic crops, extraction, and downstream processing to deliver accessible and sustainable bioenergy.

Seed-specific RNAi in safflower generates a superhigh oleic oil with extended oxidative stability.

Vegetable oils extracted from oilseeds are an important component of foods, but are also used in a range of high value oleochemical applications. Despite being biodegradable, nontoxic and renewable current plant oils suffer from the presence of residual polyunsaturated fatty acids that are prone to free radical formation that limit their oxidative stability, and consequently shelf life and functionality. Many decades of plant breeding have been successful in raising the oleic content to ~90%, but have come at the expense of overall field performance, including poor yields. Here, we engineer superhigh oleic (SHO) safflower producing a seed oil with 93% oleic generated from seed produced in multisite field trials spanning five generations. SHO safflower oil is the result of seed-specific hairpin-based RNA interference of two safflower lipid biosynthetic genes, FAD2.2 and FATB, producing seed oil containing less than 1.5% polyunsaturates and only 4% saturates but with no impact on lipid profiles of leaves and roots. Transgenic SHO events were compared to non-GM safflower in multisite trial plots with a wide range of growing season conditions, which showed no evidence of impact on seed yield. The oxidative stability of the field-grown SHO oil produced from various sites was 50 h at 110°C compared to 13 h for conventional ~80% oleic safflower oils. SHO safflower produces a uniquely stable vegetable oil across different field conditions that can provide the scale of production that is required for meeting the global demands for high stability oils in food and the oleochemical industry.

Genetic enhancement of palmitic acid accumulation in cotton seed oil through RNAi down-regulation of ghKAS2 encoding ß-ketoacyl-ACP synthase II (KASII).

Palmitic acid (C16:0) already makes up approximately 25% of the total fatty acids in the conventional cotton seed oil. However, further enhancements in palmitic acid content at the expense of the predominant unsaturated fatty acids would provide increased oxidative stability of cotton seed oil and also impart the high melting point required for making margarine, shortening and confectionary products free of trans fatty acids. Seed-specific RNAi-mediated down-regulation of ß-ketoacyl-ACP synthase II (KASII) catalysing the elongation of palmitoyl-ACP to stearoyl-ACP has succeeded in dramatically increasing the C16 fatty acid content of cotton seed oil to well beyond its natural limits, reaching up to 65% of total fatty acids. The elevated C16 levels were comprised of predominantly palmitic acid (C16:0, 51%) and to a lesser extent palmitoleic acid (C16:1, 11%) and hexadecadienoic acid (C16:2, 3%), and were stably inherited. Despite of the dramatic alteration of fatty acid composition and a slight yet significant reduction in oil content in these high-palmitic (HP) lines, seed germination remained unaffected. Regiochemical analysis of triacylglycerols (TAG) showed that the increased levels of palmitic acid mainly occurred at the outer positions, while C16:1 and C16:2 were predominantly found in the sn-2 position in both TAG and phosphatidylcholine. Crossing the HP line with previously created high-oleic (HO) and high-stearic (HS) genotypes demonstrated that HP and HO traits could be achieved simultaneously; however, elevation of stearic acid was hindered in the presence of high level of palmitic acid.

Dedicated Industrial Oilseed Crops as Metabolic Engineering Platforms for Sustainable Industrial Feedstock Production.

Feedstocks for industrial applications ranging from polymers to lubricants are largely derived from petroleum, a non-renewable resource. Vegetable oils with fatty acid structures and storage forms tailored for specific industrial uses offer renewable and potentially sustainable sources of petrochemical-type functionalities. A wide array of industrial vegetable oils can be generated through biotechnology, but will likely require non-commodity oilseed platforms dedicated to specialty oil production for commercial acceptance. Here we show the feasibility of three Brassicaceae oilseeds crambe, camelina, and carinata, none of which are widely cultivated for food use, as hosts for complex metabolic engineering of wax esters for lubricant applications. Lines producing wax esters >20% of total seed oil were generated for each crop and further improved for high temperature oxidative stability by down-regulation of fatty acid polyunsaturation. Field cultivation of optimized wax ester-producing crambe demonstrated commercial utility of these engineered crops and a path for sustainable production of other industrial oils in dedicated specialty oilseeds.

Comparative transcriptomic analysis of developing cotton cotyledons and embryo axis.

BACKGROUND: As a by product of higher value cotton fibre, cotton seed has been increasingly recognised to have excellent potential as a source of additional food, feed, biofuel stock and even a renewable platform for the production of many diverse biological molecules for agriculture and industrial enterprises. The large size difference between cotyledon and embryo axis that make up a cotton seed results in the under-representation of embryo axis gene transcript levels in whole seed embryo samples. Therefore, the determination of gene transcript levels in the cotyledons and embryo axes separately should lead to a better understanding of metabolism in these two developmentally diverse tissues. RESULTS: A comparative study of transcriptome changes between cotton developing cotyledon and embryo axis has been carried out. 17,384 unigenes (20.74% of all the unigenes) were differentially expressed in the two adjacent embryo tissues, and among them, 7,727 unigenes (44.45%) were down-regulated and 9,657 unigenes (55.55%) were up-regulated in cotyledon. CONCLUSIONS: Our study has provided a comprehensive dataset that documents the dynamics of the transcriptome at the mid-maturity of cotton seed development and in discrete seed tissues, including embryo axis and cotyledon tissues. The results showed that cotton seed is subject to many transcriptome variations in these two tissue types and the differential gene expression between cotton embryo axis and cotyledon uncovered in our study should provide an important starting point for understanding how gene activity is coordinated during seed development to make a seed. Further, the identification of genes involved in rapid metabolite accumulation stage of seed development will extend our understanding of the complex molecular and cellular events in these developmental processes and provide a foundation for future studies on the metabolism, embryo differentiation of cotton and other dicot oilseed crops.

Characterisation of the FAD2 gene family from Hiptage benghalensis: a ricinoleic acid accumulating plant.

We have characterised the FAD2 gene family from Hiptage benghalensis, a tropical plant that accumulates high levels of ricinoleic acid in its seeds. Functional characterisation of six FAD2 gene family members showed that two of them were capable of functioning as Δ12-hydroxylases while the other FAD2 members were confirmed to be Δ12-desaturases. The Δ12-hydroxylation function of these two genes was confirmed in yeast cells, using C16:1(Δ9) and C18:1(Δ9) monounsaturated fatty acids as substrates. These Δ12-hydroxylases, like the other Δ12-hydroxylases previously cloned from plants Ricinus communis (castor), Physaria fendleri and fungus Claviceps purpurea, also showed some Δ12-desaturase activity. The hydroxylation activity of the two Hiptage hydroxylases was further confirmed by their expression in the Arabidopsis fad2/fae1 double mutant where they were able to produce equivalent or higher levels hydroxylated fatty acids in the seed oil when compared with the other known hydroxylases.

A large and functionally diverse family of Fad2 genes in safflower (Carthamus tinctorius L.).

BACKGROUND: The application and nutritional value of vegetable oil is highly dependent on its fatty acid composition, especially the relative proportion of its two major fatty acids, i.e oleic acid and linoleic acid. Microsomal oleoyl phosphatidylcholine desaturase encoded by FAD2 gene is known to introduce a double bond at the Δ12 position of an oleic acid on phosphatidylcholine and convert it to linoleic acid. The known plant FAD2 enzymes are encoded by small gene families consisting of 1-4 members. In addition to the classic oleate Δ12-desaturation activity, functional variants of FAD2 that are capable of undertaking additional or alternative acyl modifications have also been reported in a limited number of plant species. In this study, our objective was to identify FAD2 genes from safflower and analyse their differential expression profile and potentially diversified functionality. RESULTS: We report here the characterization and functional expression of an exceptionally large FAD2 gene family from safflower, and the temporal and spatial expression profiles of these genes as revealed through Real-Time quantitative PCR. The diversified functionalities of some of the safflower FAD2 gene family members were demonstrated by ectopic expression in yeast and transient expression in Nicotiana benthamiana leaves. CtFAD2-1 and CtFAD2-10 were demonstrated to be oleate desaturases specifically expressed in developing seeds and flower head, respectively, while CtFAD2-2 appears to have relatively low oleate desaturation activity throughout the plant. CtFAD2-5 and CtFAD2-8 are specifically expressed in root tissues, while CtFAD2-3, 4, 6, 7 are mostly expressed in the cotyledons and hypocotyls in young safflower seedlings. CtFAD2-9 was found to encode a novel desaturase operating on C16:1 substrate. CtFAD2-11 is a tri-functional enzyme able to introduce a carbon double bond in either cis or trans configuration, or a carbon triple (acetylenic) bond at the Δ12 position. CONCLUSIONS: In this study, we isolated an unusually large FAD2 gene family with 11 members from safflower. The seed expressed FAD2 oleate Δ12 desaturase genes identified in this study will provide candidate targets to manipulate the oleic acid level in safflower seed oil. Further, the divergent FAD2 enzymes with novel functionality could be used to produce rare fatty acids, such as crepenynic acid, in genetically engineered crop plants that are precursors for economically important phytoalexins and oleochemical products.

Metabolic engineering of plant oils and waxes for use as industrial feedstocks.

Society has come to rely heavily on mineral oil for both energy and petrochemical needs. Plant lipids are uniquely suited to serve as a renewable source of high-value fatty acids for use as chemical feedstocks and as a substitute for current petrochemicals. Despite the broad variety of acyl structures encountered in nature and the cloning of many genes involved in their biosynthesis, attempts at engineering economic levels of specialty industrial fatty acids in major oilseed crops have so far met with only limited success. Much of the progress has been hampered by an incomplete knowledge of the fatty acid biosynthesis and accumulation pathways. This review covers new insights based on metabolic flux and reverse engineering studies that have changed our view of plant oil synthesis from a mostly linear process to instead an intricate network with acyl fluxes differing between plant species. These insights are leading to new strategies for high-level production of industrial fatty acids and waxes. Furthermore, progress in increasing the levels of oil and wax structures in storage and vegetative tissues has the potential to yield novel lipid production platforms. The challenge and opportunity for the next decade will be to marry these technologies when engineering current and new crops for the sustainable production of oil and wax feedstocks.

Caenorhabditis elegans Delta12-desaturase FAT-2 is a bifunctional desaturase able to desaturate a diverse range of fatty acid substrates at the Delta12 and Delta15 positions.

Caenorhabditis elegans FAT-2 has been characterized as fatty acid Δ12-desaturase able to desaturate C16 and C18 fatty acids. However, in this report we show that when expressed in yeast cells this enzyme can also catalyze Δ15 desaturation. This results in the production of both linoleic acid (ω6 C18:2Δ9,12) and linolenic acid (ω3 C18:3Δ9,12,15) from oleic acid (C18:1Δ9) substrate, and hexadecadienoic acid (ω4 C16:2Δ9,12) and hexadecatrienoic acid (ω1 C16:3Δ9,12,15) from palmitoleic acid (C16:1Δ9) substrate. In addition, this enzyme can also produce C14:2Δ9,12, C15:2Δ9,12, C17:2Δ9,12, and C18:4Δ6,9,12,15 when C14:1Δ9, C15:1Δ9, C17:1Δ9, and C18:3Δ6,9,12 substrates are available in yeast cells. Mass spectrometry analysis of 2,4-dimethyloxazoline modification of fatty acid methyl esters confirms the positions of all newly formed double bonds. These results indicate that when expressed in yeast the C. elegans Δ12-desaturase CeFAT-2 shows a characteristic of a bifunctional Δ12/Δ15-desaturase and has a great deal of elasticity with respect to fatty acid chain length in being able to accept fatty acids ranging from C14 to C18. Interestingly, despite possessing a bifunctional Δ12/Δ15 desaturation activity, phylogenetic analysis suggests that C. elegans Δ12-desaturase CeFAT-2 might have arisen independently from other reported dual Δ12/Δ15-desaturases from fungi and protozoa.

Replacing fossil oil with fresh oil - with what and for what?

Industrial chemicals and materials are currently derived mainly from fossil-based raw materials, which are declining in availability, increasing in price and are a major source of undesirable greenhouse gas emissions. Plant oils have the potential to provide functionally equivalent, renewable and environmentally friendly replacements for these finite fossil-based raw materials, provided that their composition can be matched to end-use requirements, and that they can be produced on sufficient scale to meet current and growing industrial demands. Replacement of 40% of the fossil oil used in the chemical industry with renewable plant oils, whilst ensuring that growing demand for food oils is also met, will require a trebling of global plant oil production from current levels of around 139 MT to over 400 MT annually. Realisation of this potential will rely on application of plant biotechnology to (i) tailor plant oils to have high purity (preferably >90%) of single desirable fatty acids, (ii) introduce unusual fatty acids that have specialty end-use functionalities and (iii) increase plant oil production capacity by increased oil content in current oil crops, and conversion of other high biomass crops into oil accumulating crops. This review outlines recent progress and future challenges in each of these areas.Practical applications: The research reviewed in this paper aims to develop metabolic engineering technologies to radically increase the yield and alter the fatty acid composition of plant oils and enable the development of new and more productive oil crops that can serve as renewable sources of industrial feedstocks currently provided by non-renewable and polluting fossil-based resources. As a result of recent and anticipated research developments we can expect to see significant enhancements in quality and productivity of oil crops over the coming decades. This should generate the technologies needed to support increasing plant oil production into the future, hopefully of sufficient magnitude to provide a major supply of renewable plant oils for the industrial economy without encroaching on the higher priority demand for food oils. Achievement of this goal will make a significant contribution to moving to a sustainable carbon-neutral industrial society with lower emissions of carbon dioxide to the atmosphere and reduced environmental impact as a result.

Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting.

BACKGROUND: Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T1 progeny. RESULTS: An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T1 seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T1 progeny displayed Mendelian inheritance. CONCLUSIONS: This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications.

Mechanistic and structural insights into the regioselectivity of an acyl-CoA fatty acid desaturase via directed molecular evolution.

Membrane-bound fatty acid desaturases and related enzymes play a pivotal role in the biosynthesis of unsaturated and various unusual fatty acids. Structural insights into the remarkable catalytic diversity and wide range of substrate specificities of this class of enzymes remain limited due to the lack of a crystal structure. To investigate the structural basis of the double bond positioning (regioselectivity) of the desaturation reaction in more detail, we relied on a combination of directed evolution in vitro and a powerful yeast complementation assay to screen for Δx regioselectivity. After two selection rounds, variants of the bifunctional Δ12/Δ9-desaturase from the house cricket (Acheta domesticus) exhibited increased Δ9-desaturation activity on shorter chain fatty acids. This change in specificity was the result of as few as three mutations, some of them near the putative active site. Subsequent analysis of individual substitutions revealed an important role of residue Phe-52 in facilitating Δ9-desaturation of shorter chain acyl substrates and allowed for the redesign of the cricket Δ12/Δ9-desaturase into a 16:0-specific Δ9-desaturase. Our results demonstrate that a minimal number of mutations can have a profound impact on the regioselectivity of acyl-CoA fatty acid desaturases and include the first biochemical data supporting the acyl-CoA acyl carrier specificity of a desaturase able to carry out Δ12-desaturation.

Rapid expression of transgenes driven by seed-specific constructs in leaf tissue: DHA production.

BACKGROUND: Metabolic engineering of seed biosynthetic pathways to diversify and improve crop product quality is a highly active research area. The validation of genes driven by seed-specific promoters is time-consuming since the transformed plants must be grown to maturity before the gene function can be analysed. RESULTS: In this study we demonstrate that genes driven by seed-specific promoters contained within complex constructs can be transiently-expressed in the Nicotiana benthamiana leaf-assay system by co-infiltrating the Arabidopsis thaliana LEAFY COTYLEDON2 (LEC2) gene. A real-world case study is described in which we first assembled an efficient transgenic DHA synthesis pathway using a traditional N. benthamiana Cauliflower Mosaic Virus (CaMV) 35S-driven leaf assay before using the LEC2-extended assay to rapidly validate a complex seed-specific construct containing the same genes before stable transformation in Arabidopsis. CONCLUSIONS: The LEC2-extended N. benthamiana assay allows the transient activation of seed-specific promoters in leaf tissue. In this study we have used the assay as a rapid preliminary screen of a complex seed-specific transgenic construct prior to stable transformation, a feature that will become increasingly useful as genetic engineering moves from the manipulation of single genes to the engineering of complex pathways. We propose that the assay will prove useful for other applications wherein rapid expression of transgenes driven by seed-specific constructs in leaf tissue are sought.

A leaf-based assay using interchangeable design principles to rapidly assemble multistep recombinant pathways.

The assembly of multistep recombinant pathways in stably transformed plants is a cornerstone of crops producing new products yet can be a laborious and time-consuming process. Any heterologous expression platform capable of providing a rapid estimation of the functional assembly of an entire pathway would guide the design of such transgenic traits. In this study, we use a Nicotiana benthamiana transient leaf expression system to simultaneously express five genes, from five independent T(DNA) binary vectors, to assemble a complete recombinant pathway in five days. In this study, we demonstrate the production of long-chain polyunsaturated fatty acids (LC-PUFA) requiring five transgene-encoded reactions to convert endogenous fatty acids to LC-PUFA. The addition of a triacylglycerol assembly enzyme, Arabidopsis thaliana diacylglyceride-O-acyltransferase, and fractionation of the total lipid profile demonstrated that leaf oils contained 37% newly synthesised LC-PUFA, including 7% arachidonic acid (AA), 6% eicosopentaenoic acid and 3% docosahexaenoic acid. The calculation of enzymatic conversion efficiencies at each step of LC-PUFA synthesis suggests that this transient assembly of a complicated multistep pathway is highly efficient. Unlike experiments using stably transformed plants our assembly of an intricate pathway maintained full gene-for-gene interchangeability and required a fraction of the time and glasshouse space. Furthermore, an exogenous LC-PUFA fatty acid substrate, AA, was fed and metabolised by a transiently expressed Delta17-desaturase enzyme, and provided results similar to those obtained in yeast feeding experiments. Although the assay was ideal for LC-PUFA pathways, this assay format may become a powerful tool for the characterisation and step-wise improvement of other recombinant pathways and multigenic traits.

Isolation and characterisation of a delta5-fatty acid elongase from the marine microalga Pavlova salina.

The marine microalga Pavlova salina (Haptophyta, Pavlovophyceae) produces lipids containing approximately 50% n-3 long-chain polyunsaturated fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). A full-length cDNA sequence, designated PsElo5, was isolated from P. salina. Sequence alignment showed that the gene was homologous to corresponding ELO-type elongases from other microalgae. Heterologous expression of PsElo5 in yeast and in higher plants confirmed that it encodes a specific Delta5-elongase activity as predicted and, furthermore, within the n-3 pathway, the elongation activity was confined exclusively to EPA.

High-value oils from plants.

The seed oils of domesticated oilseed crops are major agricultural commodities that are used primarily for nutritional applications, but in recent years there has been increasing use of these oils for production of biofuels and chemical feedstocks. This is being driven in part by the rapidly rising costs of petroleum, increased concern about the environmental impact of using fossil oil, and the need to develop renewable domestic sources of fuel and industrial raw materials. There is also a need to develop sustainable sources of nutritionally important fatty acids such as those that are typically derived from fish oil. Plant oils can provide renewable sources of high-value fatty acids for both the chemical and health-related industries. The value and application of an oil are determined largely by its fatty acid composition, and while most vegetable oils contain just five basic fatty acid structures, there is a rich diversity of fatty acids present in nature, many of which have potential usage in industry. In this review, we describe several areas where plant oils can have a significant impact on the emerging bioeconomy and the types of fatty acids that are required in these various applications. We also outline the current understanding of the underlying biochemical and molecular mechanisms of seed oil production, and the challenges and potential in translating this knowledge into the rational design and engineering of crop plants to produce high-value oils in plant seeds.

Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis.

The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Delta4, Delta5 and Delta8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b(5) domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants.

Metabolic engineering of new fatty acids in plants.

Metabolic engineering of plants to express high levels of new fatty acids that are of nutritional and industrial importance has proven to be highly challenging. Significant advances have been made recently, however, particularly in the development of the first plant oils to contain long-chain polyunsaturated fatty acids, such as arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid. Methods of increasing the accumulation of Delta12-modified fatty acids synthesized by transgenically expressed FAD2-like enzymes have also been investigated. Biochemical analyses of plants that express these introduced fatty-acid metabolic pathways have highlighted the central importance of ensuring the removal of novel fatty acids from their site of synthesis on phosphatidylcholine to enable their further modification, exclusion from membrane lipids and accumulation in seed triacylglycerols.

Metabolic engineering of Arabidopsis to produce nutritionally important DHA in seed oil.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are nutritionally important long-chain (≥ C20) omega-3 polyunsaturated fatty acids (ω3 LC-PUFA) currently obtained mainly from marine sources. A set of genes encoding the fatty acid chain elongation and desaturation enzymes required for the synthesis of LC-PUFA from their C18 PUFA precursors was expressed seed-specifically in Arabidopsis thaliana. This resulted in the synthesis of DHA, the most nutritionally important ω3 LC-PUFA, for the first time in seed oils, along with its precursor EPA and the ω6 LC-PUFA arachidonic acid (ARA). The assembled pathway utilised Δ5 and Δ6 desaturases that operate on acyl-CoA substrates and led to higher levels of synthesis of LC-PUFA than previously reported with acyl-PC desaturases. This demonstrates the potential for development of land plants as alternative sources of DHA and other LC-PUFA to meet the growing demand for these nutrients.