Liquid chromatography high‐resolution mass spectrometry for fatty acid profiling

Quantification of fatty acids has been crucial to elucidate lipid biosynthesis pathways in plants. To date, fatty acid identification and quantification has relied mainly on gas chromatography (GC) coupled to flame ionization detection (FID) or mass spectrometry (MS), which requires the derivatization of samples and the use of chemical standards for annotation. Here we present an alternative method based on a simple procedure for the hydrolysis of lipids, so that fatty acids can be quantified by liquid chromatography mass spectrometry (LC‐MS) analysis. Proper peak annotation of the fatty acids in the LC‐MS‐based methods has been achieved by LC‐MS measurements of authentic standard compounds and elemental formula annotation supported by ¹³C isotope‐labeled Arabidopsis. As a proof of concept, we have compared the analysis by LC‐MS and GC‐FID of two previously characterized Arabidopsis thaliana knock‐out mutants for FAD6 and FAD7 desaturase genes. These results are discussed in light of lipidomic profiles obtained from the same samples. In addition, we performed untargeted LC‐MS analysis to determine the fatty acid content of two diatom species. Our results indicate that both LC‐MS and GC‐FID analyses are comparable, but that because of higher sensitivity and selectivity the LC‐MS‐based method allows for a broader coverage and determination of novel fatty acids.     

The green microalga Lobosphaera incisa harbours an arachidonate 15S‐lipoxygenase

• The green microalga Lobosphaera incisa is an oleaginous eukaryotic alga that is rich in arachidonic acid (20:4). Being rich in this polyunsaturated fatty acid (PUFA), however, makes it sensitive to oxidation. In plants, lipoxygenases (LOXs) are the major enzymes that oxidise these molecules.
• Here, we describe, to our best knowledge, the first characterisation of a cDNA encoding a LOX (LiLOX) from a green alga. To obtain first insights into its function, we expressed it in E. coli, purified the recombinant enzyme and analysed its enzyme activity.
• The protein sequence suggests that LiLOX and plastidic LOXs from bryophytes and flowering plants may share a common ancestor. The fact that LiLOX oxidises all PUFAs tested with a consistent oxidation on the carbon n‐6, suggests that PUFAs enter the substrate channel through their methyl group first (tail first). Additionally, LiLOX form the fatty acid hydroperoxide in strict S configuration.
• LiLOX may represent a good model to study plastid LOX, because it is stable after heterologous expression in E. coli and highly active in vitro. Moreover, as the first characterised LOX from green microalgae, it opens the possibility to study endogenous LOX pathways in these organisms.

     

Intrapopulation variability in a functional trait: Susceptibility of Daphnia to limitation by dietary fatty acids

1. Functional traits are measurable characteristics of an organism that have an impact on its fitness. Variation in functional traits between and among species has been suggested to represent the basis for competition and selection, thus allowing for evolution in natural populations.
2. In freshwater ecosystems, the availability of essential polyunsaturated fatty acids (PUFAs), in particular ω3‐ and ω6‐PUFAs, determines the food quality of phytoplankton for the herbivorous zooplankton Daphnia, an unselective filter feeder. The content of such essential PUFAs in the phytoplankton is thus a functional phytoplankton trait affecting the trophic transfer efficiency and dynamics at the pelagic plant–herbivore interface.
3. In turn, the susceptibility of consumers to become limited by the availability of essential PUFAs is a fitness‐determining trait of Daphnia genotypes, and variability of this herbivore trait may thus affect the daphnids’ intrapopulation competition. To estimate the intrapopulation variation in susceptibility, we isolated clonal lines of Daphnia longispina from a natural population and compared the strength of their limitation by dietary PUFA availability via standardised laboratory growth assays. We used a liposome supplementation technique to enrich a PUFA‐poor green alga with essential ω3‐ and ω6‐PUFAs and determined juvenile somatic growth rate of different D. longispina genotypes as a fitness proxy.
4. As expected, D. longispina genotypes that coexisted in a natural population differed markedly in their specific patterns of susceptibility to dietary PUFA availability. On average, the D. longispina population was more strongly susceptible to limitations in the availability of the ω6‐PUFA arachidonic acid (20:4ω6) than to limitations in the availability of ω3‐PUFAs α‐linolenic acid (18:3ω3) and eicosapentaenoic acid (20:5ω3).
5. The ability to cope with PUFA limitation is thus a crucial trait that can probably affect intraspecific competition and Daphnia population structure. Therefore, we suggest that such intrapopulation variation in susceptibility to absence of dietary PUFAs might be one of the driving forces of natural selection and local adaptation among freshwater zooplankton.

     

Membrane lipid polyunsaturation mediated by FATTY ACID DESATURASE 2 (FAD2) is involved in endoplasmic reticulum stress tolerance in Arabidopsis thaliana

Unsaturation of membrane glycerolipid classes at their hydrophobic fatty acid tails critically affects the physical nature of the lipid molecule. In Arabidopsis thaliana, 7 fatty acid desaturases (FADs) differently desaturate each glycerolipid class in plastids and the endoplasmic reticulum (ER). Here, we showed that polyunsaturation of ER glycerolipids is required for the ER stress response. Through systematic screening of FAD mutants, we found that a mutant of FAD2 resulted in a hypersensitive response to tunicamycin, a chemical inducer of ER stress. FAD2 converts oleic acid to linoleic acid of the fatty acyl groups of ER‐synthesized phospholipids. Our functional in vivo reporter assay revealed the ER localization and distinct tissue‐specific expression patterns of FAD2. Moreover, glycerolipid profiling of both mutants and overexpressors of FAD2 under tunicamycin‐induced ER stress conditions, along with phenotypic screening of the mutants of the FAD family, suggested that the ratio of monounsaturated fatty acids to polyunsaturated fatty acids, particularly 18:1 to 18:2 species, may be an important factor in allowing the ER membrane to cope with ER stress. Therefore, our results suggest that membrane lipid polyunsaturation mediated by FAD2 is involved in ER stress tolerance in Arabidopsis.     

Successful high‐level accumulation of fish oil omega‐3 long‐chain polyunsaturated fatty acids in a transgenic oilseed crop

Omega‐3 (also called n‐3) long‐chain polyunsaturated fatty acids (≥C20; LC‐PUFAs) are of considerable interest, based on clear evidence of dietary health benefits and the concurrent decline of global sources (fish oils). Generating alternative transgenic plant sources of omega‐3 LC‐PUFAs, i.e. eicosapentaenoic acid (20:5 n‐3, EPA) and docosahexaenoic acid (22:6 n‐3, DHA) has previously proved problematic. Here we describe a set of heterologous genes capable of efficiently directing synthesis of these fatty acids in the seed oil of the crop Camelina sativa, while simultaneously avoiding accumulation of undesirable intermediate fatty acids. We describe two iterations: RRes_EPA in which seeds contain EPA levels of up to 31% (mean 24%), and RRes_DHA, in which seeds accumulate up to 12% EPA and 14% DHA (mean 11% EPA and 8% DHA). These omega‐3 LC‐PUFA levels are equivalent to those in fish oils, and represent a sustainable, terrestrial source of these fatty acids. We also describe the distribution of these non‐native fatty acids within C. sativa seed lipids, and consider these data in the context of our current understanding of acyl exchange during seed oil synthesis.     

Glucose‐6‐Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose‐6‐phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19‐fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09‐fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose‐6‐phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.     

Temperature affects the limitation of Daphnia magna by eicosapentaenoic acid,and the fatty acid composition of body tissue and eggs

1. Poikilothermic animals incorporate more polyunsaturated fatty acids (PUFAs) into their cellular membranes as temperature declines, suggesting an increased sensitivity to PUFA limitation in cool conditions. To test this we raised Daphnia magna at different temperatures and investigated the effect of varying dietary PUFA on life history parameters (i.e. growth, reproduction) and the PUFA composition of body tissue and eggs. 2. Upon a PUFA‐rich diet (Cryptomonas sp.) females showed higher concentrations of several ω3 PUFAs in their body tissue at 15 °C than at 20 °C and 25 °C, indicating a greater structural requirement for ω3 PUFAs at low temperature. Their eggs had an equal but higher concentration of ω3 PUFAs than their body tissue. 3. In a life history experiment at 15 and 20 °C we supplemented a diet of a PUFA‐free cyanobacterium with the ω3 PUFA eicosapentaenoic acid (EPA). The growth of D. magna was more strongly EPA limited at low temperature. A greater requirement for structural EPA at 15 °C was indicated by a steeper increase in somatic EPA content with dietary EPA compared to 20 °C. 4. At 20 °C the development of eggs to successful hatching was high when EPA was supplied to the mothers. At 15 °C the hatching success was generally poor, despite of a higher maternal provision of EPA to eggs, compared to that at 20 °C, suggesting that EPA alone was insufficient for proper neonatal development at the low temperature. The growth of offspring from mothers raised at 20 °C without EPA supplementation was very low, indicating that the negative effects of EPA deficiency can be carried on to the next generation. 5. The fatty acid composition of Daphnia sp. in published field studies shows increasing proportions of saturated fatty acids with increasing environmental temperature, whereas ω3 PUFAs and EPA show no clear pattern, suggesting that variations in dietary PUFA may mask temperature‐dependent adjustments in ω3 PUFA concentrations of cladocerans in nature.     

Substrate Specificity and Regioselectivity of Δ12 and ω3 Fatty Acid Desaturases from Saccharomyces kluyveri

Δ12 and ω3 fatty acid desaturases are key enzymes in the synthesis of polyunsaturated fatty acids (PUFAs), which are important constituents of membrane glycerolipids and also precursors to signaling molecules in many organisms. In this study, we determined the substrate specificity and regioselectivity of the Δ12 and ω3 fatty acid desaturases from Saccharomyces kluyveri (Sk-FAD2 and Sk-FAD3). Based on heterologous expression in Saccharomyces cerevisiae, it was found that Sk-FAD2 converted C16–20 monounsaturated fatty acids to diunsaturated fatty acids by the introduction of a second double bond at the ν+3 position, while Sk-FAD3 recognized the ω3 position of C18 and C20. Furthermore, fatty acid analysis of major phospholipids suggested that Sk-FAD2 and Sk-FAD3 have no strong substrate specificity toward the lipid polar head group or the sn-positions of fatty acyl groups in phospholipids.     

NUCLEAR MONOPLOIDY AND ASEXUAL PROPAGATION OF NANNOCHLOROPSIS OCEANICA (EUSTIGMATOPHYCEAE) AS REVEALED BY ITS GENOME SEQUENCE1

Species in genus Nannochloropsis are promising candidates for both biofuel and biomass production due to their ability to accumulate rich fatty acids and grow fast; however, their sexual reproduction has not been studied. It is clear that the construction of their metabolic pathways, such as that of polyunsaturated fatty acid (PUFA) biosynthesis, and understanding of their biological characteristics, such as nuclear ploidy and reproductive strategy, will certainly facilitate their genetic improvement through gene engineering and mutation and clonal expansion. In this study, the genome of N. oceanica S. Suda et Miyashita was sequenced with the next‐generation Illumina GA sequencing technologies. The genome was ～30 Mb in size, which contained 11,129 protein‐encoding genes. Of them, 59.65% were annotated by aligning with those in diverse protein databases, and 29.68% were assigned at least one function described in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Less frequent polymorphic nucleotides (one in 22.06 kb) and the obvious deviation from 1:1 (major:minor, minor ≥10) expectation indicated the nuclear monoploidy of N. oceanica. The lack of the majority of meiosis‐specific proteins implied the asexual reproduction of this alga. In combination, the nuclear monoploidy and asexual propagation led us to favor the hypothesis that N. oceanica was a premeiotic or ameiotic alga. In addition, sequence similarity‐based searching identified the elongase‐ and desaturase‐encoding genes involved in the biosynthesis of long‐chain PUFAs, which provided the genetic basis of its rich content of eicosapentaenoic acid (EPA). The functional genes and their metabolic pathways profiled against its genome sequence will facilitate its integrative investigations.     

Improved production of various polyunsaturated fatty acids through filamentous fungus Mortierella alpina breeding

Studies on the application of functional lipids such as polyunsaturated fatty acids (PUFAs) have proceeded in various fields regarding health and dietary requirements in a search for novel and rich sources. Filamentous fungus Mortierella alpina 1S-4 produces triacylglycerols rich in arachidonic acid, ones reaching 20 g/L and containing 30–70% arachidonic acid as to the total fatty acids. Mutants derived from M. alpina 1S-4, defective in Δ5 and Δ6 desaturases, accumulate triacylglycerols rich in unique PUFAs, i.e., dihomo-γ-linolenic acid and Mead acid, respectively. Furthermore, various mutants derived from M. alpina 1S-4 have led to the production of oils containing n−1, n−3, n−4, n−6, n−7, and n−9 PUFAs. A variety of genes encoding fatty acid desaturases and elongases involved in PUFA biosynthesis in M. alpina 1S-4 has been isolated and characterized. Molecular breeding of M. alpina strains by means of manipulation of these genes facilitates improvement of PUFA productivity and elucidation of the functions of enzymes involved in PUFA biosynthesis.     

PEROXIREDOXIN Q stimulates the activity of the chloroplast 16:1Δ3trans FATTY ACID DESATURASE4

Thylakoid membrane lipids, comprised of glycolipids and the phospholipid phosphatidylglycerol (PG), are essential for normal plant growth and development. Unlike other lipid classes, chloroplast PG in nearly all plants contains a substantial fraction of the unusual trans fatty acid 16:1^Δ3trans or 16:1t. We determined that, in Arabidopsis thaliana, 16:1t biosynthesis requires both FATTY ACID DESATURASE4 (FAD4) and a thylakoid‐associated redox protein, PEROXIREDOXIN Q (PRXQ), to produce wild‐type levels of 16:1t. The FAD4–PRXQ biochemical relationship appears to be very specific in planta, as other fatty acids (FA) desaturases do not require peroxiredoxins for their activity, nor does FAD4 require other chloroplast peroxiredoxins under standard growth conditions. Although most of chloroplast PG assembly occurs at the inner envelope membrane, FAD4 was primarily associated with the thylakoid membranes facing the stroma. Furthermore, co‐production of PRXQ with FAD4 was required to produce Δ3‐desaturated FAs in yeast. Alteration of the redox state of FAD4 or PRXQ through site‐directed mutagenesis of conserved cysteine residues impaired Δ3 FA production. However, these mutations did not appear to directly alter disulfide status of FAD4. These results collectively demonstrate that the production of 16:1t is linked to the redox status of the chloroplast through PRXQ associated with the thylakoids.     

Using lipidomics to reveal details of lipid accumulation in developing Siberian apricot (Prunus sibirica L.) seed kernels

Siberian apricot seed kernel (SASK) contains a high of 50% oil with suitable fuel properties conformed to biodiesel standard. To date, Prunus sibirica is a novel non‐crop feedstock for biodiesel production in China. Here, oil contents and fatty acid (FA) compositions were identified in developing SASK from AS‐80 and AS‐84, at intervals of 1 week from 3 weeks after anthesis (WAA) to 9 weeks. The major differences in oil content between C18:1 and C18:2 levels were greater among the AS‐80 (32.69/15.48 g/100 g) than among the AS‐84 (25.78/13.15 g/100 g). Subsequently, the SASKs from 4, 6, and 8 WAA, respectively, representing early, middle, and late phases of oil accumulation, were selected as optimal samples for lipidomics analysis. It was notable that 18:1/18:1/18:2, 18:1/18:1/18:3, and 18:2/18:2/18:2 were the prominent compositions in triacylglycerol (TAG), and their higher content found among the AS‐80 was consistent with FA results. Although phosphatidic acid (PA) is directly connected with diacylglycerol (DAG) in Kennedy pathway, we found significant difference between PA and DAG compositions. The resulting molecular species differ in acyl composition depending on whether they were generated via phosphatidylcholine (PC) or Kennedy pathway. By qRT‐PCR analysis, the expression levels of FAD3, PDCT, and DAG‐CPT related to the biosynthesis of polyunsaturated fatty acids (PUFAs) showed a gradual decrease with SASK mature, explaining the drastic change of DAG‐18:3/18:3 content. Additionally, the lipidomics data coupled with qRT‐PCR analysis suggested that phospholipid:DAG acyltransferase may play a critical role in incorporation of PUFAs into sn‐3 of TAG. Our data contribute significantly to understand the underlying mechanisms of lipid accumulation in P. sibirica, and may also present strategies for engineering oil accumulation in oilseed plants.     

The role of Δ6-desaturase acyl-carrier specificity in the efficient synthesis of long-chain polyunsaturated fatty acids in transgenic plants

The role of acyl‐CoA‐dependent Δ6‐desaturation in the heterologous synthesis of omega‐3 long‐chain polyunsaturated fatty acids was systematically evaluated in transgenic yeast and Arabidopsis thaliana. The acyl‐CoA Δ6‐desaturase from the picoalga Ostreococcus tauri and orthologous activities from mouse (Mus musculus) and salmon (Salmo salar) were shown to generate substantial levels of Δ6‐desaturated acyl‐CoAs, in contrast to the phospholipid‐dependent Δ6‐desaturases from higher plants that failed to modify this metabolic pool. Transgenic plants expressing the acyl‐CoA Δ6‐desaturases from either O. tauri or salmon, in conjunction with the two additional activities required for the synthesis of C20 polyunsaturated fatty acids, contained higher levels of eicosapentaenoic acid compared with plants expressing the borage phospholipid‐dependent Δ6‐desaturase. The use of acyl‐CoA‐dependent Δ6‐desaturases almost completely abolished the accumulation of unwanted biosynthetic intermediates such as γ‐linolenic acid in total seed lipids. Expression of acyl‐CoA Δ6‐desaturases resulted in increased distribution of long‐chain polyunsaturated fatty acids in the polar lipids of transgenic plants, reflecting the larger substrate pool available for acylation by enzymes of the Kennedy pathway. Expression of the O. tauriΔ6‐desaturase in transgenic Camelina sativa plants also resulted in the accumulation of high levels of Δ6‐desaturated fatty acids. This study provides evidence for the efficacy of using acyl‐CoA‐dependent Δ6‐desaturases in the efficient metabolic engineering of transgenic plants with high value traits such as the synthesis of omega‐3 LC‐PUFAs.     

Biomass production and fatty acid accumulation in Chlorella sp. (strain DEC1B) isolated from a petrol refinery in Huelva (Spain)

A microalgal strain was established from Cepsa's refinery wastewater treatment plant in Huelva (southwest of Spain). Genetic analysis of the chloroplastic rbcL gene encoding for the large subunit of the ribulose bisphosphate carboxylase enzyme (Rubisco) showed the strain had high homology with other known rbcL sequences of the genus Chlorella. The strain grows well autotrophically in minimum mineral medium, with a growth rate of 0.28 ± 0.012 day^?1 and a biomass productivity of 138.9 ± 6.7 mg L^?1 day^?1. N‐starvation and/or over illumination with 650 µmol photons m^?2 s^?1 of PAR light on the cultures induced a significant increase in the intracellular content of lipids in this microalga. Total lipids were extracted from the strain biomass with 2:1 chloroform‐methanol, and they accounted for approximately 50% of the dry biomass. Polyunsaturated fatty acids (PUFAs) represented 60.4% of the total fatty acids found in the strain, thus making this biomass attractive as a high added‐value product source. The strain was able to grow efficiently in the refinery treated wastewater from which it was isolated, providing an attractive advantage for further development of more sustainable algal biomass production processes at reduced costs close to a petrol refinery area.     

The impact of reducing fatty acid desaturation on the composition and thermal stability of rapeseed oil

Oilseed rape (Brassica napus) is the third largest source of vegetable oil globally. In addition to food uses, there are industrial applications that exploit the ability of the species to accumulate the very‐long‐chain fatty acid (VLCFA) erucic acid in its seed oil, controlled by orthologues of FATTY ACID ELONGASE 1 (Bna.FAE1.A8 and Bna.FAE1.C3). The proportion of polyunsaturated fatty acids (PUFAs) in rapeseed oil is predicted to affect its thermal stability and is controlled by orthologues of FATTY ACID DESATURASE 2, particularly Bna.FAD2.C5. Our aim was to develop rapeseed lines combining high erucic and low PUFA characters and to assess the impact on thermal stability of the oil they produce. The new type of rapeseed oil (high erucic low polyunsaturate; HELP) contained a substantially greater proportion of erucic acid (54%) compared with high erucic rapeseed oil (46%). Although the total VLCFA content was greater in oil from HELP lines (64%) than from high erucic rapeseed (57%), analysis of triacylglycerol composition showed negligible incorporation of VLCFAs into the sn‐2 position. Rancimat analysis showed that the thermal stability of rapeseed oil was improved greatly as a consequence of reduction of PUFA content, from 3.8 and 4.2 h in conventional low erucic and high erucic rapeseed oils, respectively, to 11.3 and 16.4 h in high oleic low PUFA (HOLP) and HELP oils, respectively. Our results demonstrate that engineering of the lipid biosynthetic pathway of rapeseed, using traditional approaches, enables the production of renewable industrial oils with novel composition and properties.