Search

The degumming step in seed oil refining is essential to avoid deleterious oxidative reactions caused by the presence of gums, which may impact on the refined oil stability and shelf life. However, conventional degumming techniques produce gums with significant amounts of entrained oil that is not recovered. The present work evaluated the application of megasonic waves during acid degumming of canola, soybean, and sunflower oils to recover entrained oil in the gums and increase the gum yield. Oil-acid mixing options were explored at selected temperatures and times, with and without a megasonic treatment (2 MHz, 340 W, 81.6 kJ/kg, 20 min). Megasonics best enhanced the acid degumming of canola oil after a one-pass ultra-turrax shearing for 3 min at 40 °C. Such megasonic-assisted acid degumming conditions were effective in reducing residual phosphorus content in both canola and soybean oil by 54.3% and 67.0%, respectively, but less effective in sunflower oil (39%, relative to the non-megasonic degumming). The megasonic treatment also reduced the loss of gum-entrained canola, soybean, and sunflower oil by 50.0%, 35.0%, and 28.4%, respectively. Furthermore, the gum yield (% w/w oil, dry basis) obtained from canola, soybean, and sunflower oil degumming was also enhanced by 22.7%, 18.7%, and 16.0%, respectively, compared with the control. The three megasonic-assisted degummed oils met industry standard specifications for refined seed oil, including free fatty acids, chlorophyll, peroxide, and phosphorus content. Income from additional oil and gum recovery, resulting from megasonics, was initially indicative of the economic viability of the process. Further cost benefit analysis is required, from pilot to industrial scale process data, to validate economic viability.

Residual oil in post-expeller canola cake, is industrially recovered by solvent extraction using hexane with greater than 90% recovery. However, due to health and environmental risks, industry is seeking safer and air polluting alternatives to hexane. The present work explores the application of ultrasound in the megasonic range for aqueous-based extraction of oil from canola cake. A factorial experiment was designed to examine various ultrasound and canola cake extraction parameters including frequency (0.4, 1, and 2 MHz; 33.6–122.4 kJ/kg), sonication time (10, 20, and 30 min), cake: water ratio (1:9, 2:8 and 3:7 w/w), and temperature (60, 70, and 80 °C). A rapid and simple aqueous-based megasonic extraction method was established for preliminary screening of studying the effect of processing parameters for maximising oil extractability. We also assessed the oil quality tests for each condition by analysis of free fatty acids (FFA), peroxide value (PV), and chlorophyll for the best conditions. Control (non-megasonic) trials were also carried out in parallel to determine the net ultrasound extractability (NUE). The highest NUE obtained was 49.5% (g oil/100 g hexane extractable oil in meal) at treatment conditions of 2 MHz, 30 min, meal: water 1:9 (w/w), and 80 °C. An aqueous-based centrifugation method was then established and tested under the same conditions without and with ultrasound, giving a net ultrasound yield improvement (NUY) of 31.8% (g oil/100 g meal) and a NUE of 30.6%. The canola oil quality after ultrasound aqueous-based extraction met the industry standard specifications for free fatty acids, chlorophyll, peroxide, and phosphorus content.

The large phagotrophic dinoflagellate Noctiluca has become a prominent red tide organism in southeast Australian waters since the 2000s, raising concerns for beach tourism, grazing impacts as well as ichthyotoxicity for finfish aquaculture. Satisfactory culture growth rates (0.23–0.56 per day) were obtained by feeding with small Thalassiosira diatom and Tetraselmis flagellate diets, while optimal growth rates sustained for up to 8 months (0.69 per day) were achieved by feeding in a plankton wheel with the large chain-forming dinoflagellate Gymnodinium catenatum. Noctiluca was highly tolerant towards salinities from 20 to 35 and growth was stimulated by temperatures increasing from 10 to 23°C, which in combination with the key factor of prey abundance explains the incidence in southeast Australia of predominantly summer and spring but occasionally also winter blooms. Fatty acid biomarkers suggest that Tasmanian field populations indiscriminately feed on available diatom and dinoflagellate mixtures. Noctiluca exhibited very limited ichthyotoxicity, and only at the highest cell concentrations of 2 000 000/L (50% reduction in RTgill W1 cell viability). Only the densest red tide surface slicks contained acutely toxic levels of unionized ammonia of 242 to 510 μg/L while inshore slicks generated oxygen concentrations as low as 0–1.5 ppm. Lipid phycotoxins (eicosapentaenoic acid, docosahexaenoic acid) did not appear to contribute to Noctiluca ichthyotoxicity. The fatty acid 20:0 eicosanoic acid may serve as a potential Noctiluca biomarker in marine food webs and sediments.

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.

Microwave (MW) pre-treatment of canola seeds or flaked seeds was found to be a superior alternative to the conventional thermal pre-treatment (steam). Flaked seeds were “cooked” (heat-treated) with steam or using microwave treatments in the temperature range of 62–130 °C prior to expeller pressing. Microwave cooking at 100 °C resulted in the highest increase in the pressed oil yield, which is an increase of 3.7% (w/w) on a pressed oil basis or 9.0% (oil in seed basis) compared with steam cooking. Whole canola seeds conditioning was conducted with microwaves or steam, in the temperature range of 40–75 °C, followed by microwave or steam cooking at 100 °C to evaluate the effect of MW treatment during conditioning on the expeller oil yield. The use of a continuous microwave process for combined conditioning of whole seeds at 55 °C and subsequent cooking of flaked seeds at 100 °C resulted in a 4.0% increase in expeller oil yield, compared with steam conditioning and cooking. The influence of dry basis (db %) moisture contents of 5%, 11.5%, and 16.5% on oil yield after steam or MW treatments of seeds and flaked seeds was also studied. The moisture content of 11.5% (db %) yielded the highest net oil yield for both MW and steam at best conditioning and cooking temperatures of 55 °C and 100 °C, respectively. No significant impact of MW cooking was seen on oil quality compared with conventional steam cooking.

Canola seeds are one of the most important sources of edible vegetable oils globally. Crude canola oil is industrially extracted from canola seeds by expeller-pressing of heat-preconditioned flaked seeds. The residual oil in the pressed “canola cake” is recovered by solvent extraction using hexane. However, hexane extraction may pose adverse safety and environmental impacts compared to safer and environmentally friendly alternatives that have been studied recently. This manuscript provides a review of the conventional canola oil extraction process and the state of the art on novel alternatives currently explored to improve oil yield and quality. The latter includes supercritical extraction, aqueous enzyme extraction, microwave, and ultrasound processing. The last two technologies can be implemented either as novel processes or as processing interventions into traditional processes. The levels of technology readiness and the challenges for industrial uptake of each technology are also discussed.

The Labyrinthulomycetes or Labyrinthulea are a class of protists that produce a network of filaments that enable the cells to glide along and absorb nutrients. One of the main two Labyrinthulea groups is the thraustochytrids, which are becoming an increasingly recognised and commercially used alternate source of long-chain (LC, ≥C20) omega-3 containing oils. This study demonstrates, to our knowledge for the first time, the regiospecificity of the triacylglycerol (TAG) fraction derived from Australian thraustochytrid Aurantiochytrium sp. strain TC 20 obtained using 13C nuclear magnetic resonance spectroscopy (13C NMR) analysis. The DHA present in the TC 20 TAG fraction was determined to be concentrated in the sn-2 position, with TAG (16:0/22:6/16:0) identified as the main species present. The sn-2 preference is similar to that found in salmon and tuna oil, and differs to seal oil containing largely sn-1,3 LC-PUFA. A higher concentration of sn-2 DHA occurred in the thraustochytrid TC 20 oil compared to that of tuna oil.

A series of novel C18–C22trans ω3 polyunsaturated fatty acids (PUFA) with a single trans double bond in the ω3 position was found in Northern and Southern Hemisphere strains of the marine haptophyte Imantonia rotunda. The novel ω3 PUFA were identified as 18:3(9c,12c,15t) (0.2–1.8 % of total fatty acids), 18:4(6c,9c,12c,15t) (1.9–4.1 %), 18:5 (3c,6c,9c,12c,15t) (0.7–8.8 %), 20:5(5c,8c,11c,14c,17t) (1.2–4.1 %) and 22:6(4c,7c,10c,13c,16c,19t) (0.3–4.3 %), and were accompanied by larger proportions of the all cis isomers: 18:3ω3(9,12,15) (2.7–3.5 %), 18:4ω3(6,9,12,15) (9.3–14.3 %), 18:5ω3(3,6,9,12,15) (7.8–18.5 %), 20:5ω3(5,8,11,14,17) (3.2–3.9 %), 22:5ω3(7,10,13,16,19) (0.1–0.3 %) and 22:6ω3(4,7,10,13,16,19) (2.3–5.2 %). GC analysis of FAME using a non-polar column did not reveal the trans isomers as they coeluted with the all cis PUFA. However, GC using a polar column resolved the trans PUFA from the all cis PUFA, with the trans isomers eluting before the all cis isomers. GC-MS of FAME fractionated by argentation solid-phase chromatography confirmed the molecular ions of all components. FAME were derivatized to form 4,4-dimethyloxazoline (DMOX) derivatives, and GC-MS revealed the same double bond positions for each trans and cis FAME. The results suggest that the ω3 trans double bond originated from the Δ15/ω3 desaturation of 18:2(9c,12c), suggesting that this desaturase has dual cis/trans activity in these species. These results indicate that 18:3(9c,12c,15 t) was the precursor trans isomer produced for the trans series and further desaturation by the common Δ6 desaturase to produce the trans tetraene and successive elongations and desaturations led to the subsequent series of trans ω3 PUFA isomers. To our knowledge, this is the first report of these trans ω3 isomers occurring in strains of I. rotunda. These trans ω3 PUFA may be used as biomarkers in marine food webs for this species and with their unique structure may be biologically active.

Crude canola oil from expeller pressing contains undesirable gums mainly composed of phospholipids, which must be removed to increase the oil’s shelf life. Degumming is carried out by high shear mixing of the oil with chemical reagents. This often requires multiple energy-consuming steps and is dependent on the phospholipid content of the oil being degummed. A set of process parameters at laboratory scale were selected to reproduce common industrial degumming processes (water, acid, and total degumming) and to compare with megasonic intervention before centrifugation. A factorial experimental design was applied to examine the effect of frequency (0, 0.4, 1, and 2 MHz; 33.6-122.4 kJ/kg), sonication treatment time (10, 20, and 30 min), and temperature (40, 60, and 80 °C) when applying megasonics during phosphoric acid degumming. Process parameters of 2 MHz at 40 °C for 30 min were the most efficient to reduce the residual phosphorus content from 27 mg/kg, on the non-megasonic water degummed oil, to 11 mg/kg, on the megasonic water degummed oil. Furthermore, the phosphoric acid and total degumming processes showed significant phosphorus reduction in the degummed oil when applying similar megasonic conditions (2 MHz at 40 °C for 20 min). The residual phosphorus with acid megasonic-assisted degumming was below the values achieved with a total degumming process without megasonics. This demonstrated that a megasonic intervention may eliminate the industrial alkalisation (neutralisation) while reducing the degumming processing time. A fatty acid composition analysis showed a noticeable reduction in the FAA and polar lipids with megasonic treatment after a total degumming process. However, there were no significant changes on both triacylglycerols (TAG), and diacylglycerols.

New and sustainable sources of long-chain (LC, ≥C20) omega-3 oils containing DHA (docosahexaenoic acid, 22:6ω3) are required to meet increasing demands. The lipid content of the oilseed of a novel transgenic, DHA-producing land plant, Camelina sativa, containing microalgal genes able to produce LC omega-3 oils, contained 36% lipid by weight with triacylglycerols (TAG) as the major lipid class in hexane extracts (96% of total lipid). Subsequent chloroform-methanol (CM) extraction recovered further lipid (~50% polar lipid, comprising glycolipids and phospholipids) and residual TAG. The main phospholipid species were phosphatidyl choline and phosphatidyl ethanolamine. The % DHA was: 6.8% (of total fatty acids) in the TAG-rich hexane extract and 4.2% in the polar lipid-rich CM extract. The relative level of ALA (α-linolenic acid, 18:3ω3) in DHA-camelina seed was higher than the control. Major sterols in both DHA- and control camelina seeds were: sitosterol, campesterol, cholesterol, brassicasterol and isofucosterol. C16–C22 fatty alcohols, including iso-branched and odd-chain alcohols were present, including high levels of iso-17:0, 17:0 and 19:0. Other alcohols present were: 16:0, iso-18:0, 18:0 and 18:1 and the proportions varied between the hexane and CM extracts. These iso-branched odd-chain fatty alcohols, to our knowledge, have not been previously reported. These components may be derived from wax esters, or free fatty alcohols.

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications. © 2013 CSIRO., We would like to thank Nathalie Niesner, Anne Mackenzie, Dawar Hussain, Anna Mechanicos, Cheryl Blundell, Luch Hac, Alex Miller, Lina Ma, Lijun Tian and Jeni Pritchard for technical assistance. We also thank Dr Damien Callahan at Metabolomics Australia, University of Melbourne and Dr. Matt Taylor at CSIRO Ecosystem Sciences for assistance with LC-MS, Dr. Rosemary White for help with microscopy and Dr. Allan Green for helpful discussions and advice. This work was funded by the CSIRO Food Futures National Research Flagship, CSIRO Plant Industry and the CSIRO Office of the Chief Executive Postdoctoral Fellowship Scheme. LC-MS analysis was supported by Bioplatforms Australia Services. Research in the UNT Center for Plant Research on oil accumulation in leaves is supported by a grant from the U.S. Department of Energy, BER Division, DE-FG02-09ER64812. Acquisition of the imaging instruments at UNT was made possible, in part, by grants from the Hoblitzelle Foundation and the US National Science Foundation (MRI#112605).

We describe a transgenic microalgal Δ9-elongase pathway transformed in both Brassica napus and Arabidopsis thaliana seed resulting in the production of arachidonic acid (ARA). This pathway is noteworthy for both the production of ARA in seed tissue and the low levels of intermediate C20 fatty acids that accumulate. We also demonstrate that the arachidonic acid is naturally enriched at the sn2 position in triacylglycerol. This is the first report of ARA production by the Δ9-elongase pathway in an oilseed.

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.

The production of long-chain polyunsaturated fatty acids from precursor molecules linoleic acid (LA; 18:2omega6) and alpha-linolenic acid (ALA; 18:3omega3) is catalysed by sequential desaturase and elongase reactions. We report the isolation of a front-end Delta6-desaturase gene from the microalgae Ostreococcus lucimarinus and two elongase genes, a Delta6-elongase and a Delta5-elongase, from the microalga Pyramimonas cordata. These enzymes efficiently convert their respective substrates when transformed in yeast (39-75% conversion for omega3 substrate fatty acids), and the Delta5-elongase in particular displays higher elongation efficiency (75% for conversion of eicosapentaenoic acid (20:5omega3) to docosapentaenoic acid (22:5omega3)) than previously reported genes. In addition, the Delta6-desaturase is homologous with acyl-CoA desaturases and shows a strong preference for the omega3 substrate ALA.

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 Δ4, Δ5 and Δ8 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.

The presence of long-chain polyunsaturated fatty acids (PUFA; ca. 9% of total fatty acids) in marine sediments near Dover, southern Tasmania, Australia prompted a search for their likely source. Analysis of a number of different species of benthic fauna isolated from these sediments revealed that the brittle star Amphiura elandiformis contained abundant PUFA including high contents of the uncommon long-chain fatty acid 24:6(n-3), but much smaller amounts of the more common animal PUFA 22:6(n-3). This is the first report of the lipid composition of this animal. Identifications of the unsaturated fatty acids were confirmed by formation of DMOX derivatives which gave characteristic and easily interpreted mass spectra. The 24:6(n-3) PUFA has been identified in some genera of brittle stars, but not others. It is rarely found in significant amounts in other marine animals. DMDS adducts were used to identify the positions of double bonds in the monounsaturated fatty acids. The major 20:1 isomer was identified as the rarely reported 20:1(n-13) fatty acid. The two fatty acids 20:1(n-13) and 24:6(n-3) may be useful biomarkers in food-web studies for identifying a brittle star diet and for recognising contributions of organic detritus from this benthic animal to marine sediments.

Nine microalgal species from the classes Bacillariophyceae, Cryptophyceae, Prymnesiophyceae and Dinophyceae were isolated from Australian waters, cultured to stationary phase and analyzed for their lipid and fatty acid composition and yield. Five species (Pavlova pinguis, Heterocapsa niei, Proteomonas sulcata, Navicula jeffreyi and Thalassiosira pseudonana) produced high proportions of triacylglycerol (TAG: 22–57% total lipid). An unidentified Navicula-like diatom (CS-786), despite having a low TAG content, had the highest EPA yield (5.8 mg L−1), due to high biomass and a high relative proportion of EPA. Heterocapsa niei had the highest DHA yield (2.9 mg L−1), due to a high cellular lipid and DHA content (171 pg cell−1 and 13.7 pg cell−1, respectively) despite its relatively low biomass. The desirable PUFA composition and yield of both diatom CS-786 and H. niei make them potential candidates for optimization of biomass and PUFA production for use as live-feeds in aquaculture. In addition, H. niei may have potential as a source of DHA for other uses. Low proportions (< 1.2%) of 24:6(n−3) accompanied by trace proportions of 24:5(n−6) were detected in most strains, while 28:8(n−3) was found in dinoflagellates and also in the prymnesiophyte P. pinguis. All non-diatomaceous species contained 26:7(n−3) in minor quantities. This is the first time these unusual C24 and C26 PUFA have been reported in microalgae and the first report of C28 PUFA in a microalga other than dinoflagellates. Possible biosynthetic reasons why these might occur in stationary phase cultures are considered and the likely dietary transfer of these PUFA to higher aquatic life is discussed.

We have studied the effects of growth phase on the lipid composition in batch cultures of Gymnodinium sp. CS-380/3 over 43 days of culturing. The lipid content increased two fold, from late logarithmic (day 6) to linear growth phase (day 22) then decreased at stationary phase (day 43) while the lipid yield (mg l −1 ) increased 30-fold from day 6 to 30 mg l −1 at day 43. Changes in fatty acid content mirrored those observed for the total lipid, while the sterol content continued to increase with culture age through to stationary phase. The largest changes occurred in the lipid classes, especially the polar lipids and triacylglycerols (oil). The proportion of triacylglycerols increased from 8% (of total lipids) at day 6 to 30% at day 43, with a concomitant decrease in the polar lipid fraction. The proportions of 16:0 and DHA [22:6(n-3)] increased while those of 18:5(n-3) and EPA [20:5(n-3)] decreased with increasing culture age. The proportion of the major sterol, dinosterol, decreased from 41% (day 6) to 29% (day 43), while the major dinostanol epimer (23 R ,24 R ) increased from 33% (day 6) to 38% (day 22). Despite small changes in the proportion of the main sterols, the same sterols were present at all stages of growth, indicating their value as a chemotaxonomic tool for distinguishing between strains within the same genus. Growth phase could be a useful variable for optimising the oil and DHA content with potential for aquaculture feeds and a source of DHA-rich oils for nutraceuticals.

The compositions of 4-desmethyl sterols and 4-methyl sterols in four species of marine dinoflagellates of the genus Prorocentrum (viz., P. micans Ehrenberg, P. minimum (Pavillard) Schiller, P. balticum (Lev.) Lemm and P. mexicanum Tafall) were identified by capillary gas chromatography–mass spectrometry as part of a study to identify signature lipids for dinoflagellates in marine organic matter. Complex mixtures were found in each species with over 20 sterols identified in all. All species contained the same core group of sterols, but there were significant differences in the proportions of the various sterols. Two distinct groupings could be discerned in the sterol patterns. The 4-methyl sterol 4α,23,24-trimethyl-5α-cholest-22 E -en-3β-ol (dinosterol), which is common in many dinoflagellates, predominated in P. balticum and in P. minimum whereas in the closely related species P. micans and P. mexicanum the major sterol was cholesterol. A novel monounsaturated C 23 sterol having a much shortened side-chain was found in P. balticum and P. minimum and both P. balticum and P. minimum contained peridinosterol (4α,23,24-trimethyl-5α-cholest-17(20)-en-3β-ol). 24-Methylenecholesterol was only found in P. minimum , where it comprised over one-third of the sterols. The steroid ketone dinosterone occurred in P. balticum , but none of the other species contained steroid ketones. Although all the sterol distributions were broadly similar, the presence or absence of specific components might be a useful chemotaxonomic tool for distinguishing between closely related species.

The fatty acid and sterol compositions of five species of marine dinoflagellates (Scrippsiella sp. Symbiodinium microadriaticum Freud, Gymnodinium sp., Gymnodinium sanguineum Hirasaki, and Fragilidium sp.) are reported. All contained the major fatty acids that are considered common in dinoflagellates, but the proportions were quite variable, and some species contained low contents of some polyunsaturated fatty acids. Concentration ranges for the major fatty acids were: 16:0 (9.0%–24.8%), 18:4(n-3) (2.5%–11.5%), 18:5(n-3) (7.0%–43.1%), 20:5(n-3) (EPA) (1.8%–20.9%), and 22:6(n-3) (DHA) (9.9%– 26.3%). Small amounts of novel very-long-chain highly unsaturated C28 fatty acids occurred in all species. Each dinoflagellate contained a complex mixture of 4-methyl sterols and 4-desmethyl sterols. Four species contained cholesterol, although the amounts were highly variable (from 0.2% of total sterols in Scrippsiella sp. to 45.6% in Fragilidium sp.). All but G. sanguineum contained the 4-methyl sterol dinosterol, and all species contained sterols lacking a double bond in the ring system (i.e. stanols); in Scrippsiella sp. cholestanol composed 24.3% of the total sterols. Other common features of the 4-methylsterol profiles were the presence of 23,24-dimethyl alkylation and unsaturation at Δ22 in the side chain. In Scrippsiella sp., four steroidal ketones were identified: cholestanone, dinosterone, 4α,23,24-trimethyl-5α-cholest-8(14)-en-3-one, and dinostanone. The structures of these corresponded to the major sterols in this species, suggesting that the sterols and steroidal ketones are biosynthetically linked. Steroidal ketones were not detected in the other species. Although fatty acid profiles can be used to distinguish among algal classes, they were not useful for differentiating among dinoflagellate species. In contrast, whereas some taxonomic groupings of dinoflagellates display similar sterol patterns, others, such as the gymnodinoids studied here, clearly do not. The combination of fatty acid, sterol, and steroidal ketone profiles may be useful complementary chemotaxonomic tools for distinguishing morphologically similar species. The identification of steroidal ketones supports earlier suggestions that certain dinoflagellates might be a significant source of such components in marine environments.

Very-long-chain (C28) highly unsaturated fatty acids (VLC-HUFA), octacosaheptaenoic acid (28:7(n-6)(4,7,10,13,16,19,22)) and octacosaoctaenoic acid (28:8(n-3)(4,7,10,13,16,19,22,25)) were identified in seven marine dinoflagellate species: Prorocentrum mexicanum, P. micans, Scrippsiella sp., Symbiodinium microadriaticum, Gymnodinium sp., G. sanguineum and Fragilidium sp. The proportion of these fatty acids accounted for less than 2.3% of the total fatty acids in these species. The main VLC-HUFA in S. microadriaticum was 28:7(n-6); in the other species it was 28:8(n-3), with Scrippsiella sp., Gymnodinium sp. and Fragilidium sp. containing only 28:8(n-3) and Gymnodinium sanguineum containing a small proportion of 28:7(n-6) in addition to 28:8(n-3). These findings show that some marine dinoflagellates have the ability to produce VLC-HUFA, but precursors of these VLC-HUFA with chain lengths between 22 and 28 carbon atoms were below the detection level. Both 28:7(n-6) and 28:7(n-3), as well as other VLC-HUFAs, have been detected previously in Baltic herring; our results suggest that they may have originated from microalgae.

Microalgae are major sources of lipids in lacustrine and marine environments. This paper provides a review of some recent advances in our knowledge of the wide variety of lipid types that have been isolated from microalgae with an emphasis on those likely to be useful biomarkers for identifying sources of organic matter in sediments. Extensive data are now available on the fatty acids in all of the major classes of microalgae and some useful characteristic features have been observed in the abundance of particular polyunsaturated fatty acids. Despite several decades of study, it is now apparent that some of the biosynthetic steps leading to the formation of these unsaturated fatty acids are still not known with certainty as shown by the occurrence of C28 polyunsaturated fatty acids in some dinoflagellates and the likely involvement of chain-shortening reactions. Considerable data have also been obtained on the sterols in microalgae, but some classes of organisms are still not well documented (e.g. cryptomonads, eustigmatophytes, xanthophytes and raphidophytes). Diatoms show a great variety of sterol compositions and no sterol appears to be either unique or representative. However, 24-methylenecholesterol in sediments is probably derived in most cases from diatoms. High contents of C25 highly branched isoprenoid (HBI) alkenes have been identified in the diatom Haslea ostrearia and both C25 and C30 HBI alkenes have been found in diatom strains thought to be Rhizosolenia setigera. Genetic and environmental factors appear to be important controls on the relative abundances of the various homologues identified. Microalgae are also suspected to be a source of long-chain saturated fatty acids having an even carbon number predominance and of long-chain alkanes with no odd over even carbon number predominance, although the available data are not conclusive. An exciting development in recent years is the identification of highly aliphatic biopolymers (algaenans) in some species of marine and freshwater green algae and eustigmatophytes. This material persists in sediments and may be a source of the alkyl chains in ancient kerogens and crude oil constituents. Algaenans do not occur in all algal species and may be absent from some classes, such as diatoms. This implies that the organic matter preserved in sediments is strongly influenced by a subset of the microalgal contributors of organic matter. Although reasonable sources have been identified for many of the lipids in sediments, there are still many gaps in our knowledge and further studies are clearly required.

Background Omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) such as eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) are critical for human health and development. Numerous studies have indicated that deficiencies in these fatty acids can increase the risk or severity of cardiovascular, inflammatory and other diseases or disorders. EPA and DHA are predominantly sourced from marine fish although the primary producers are microalgae. Much work has been done to engineer a sustainable land-based source of EPA and DHA to reduce pressure on fish stocks in meeting future demand, with previous studies describing the production of fish oil-like levels of DHA in the model plant species, Arabidopsis thaliana. Principal Findings In this study we describe the production of fish oil-like levels (>12%) of DHA in the oilseed crop species Camelina sativa achieving a high ω3/ω6 ratio. The construct previously transformed in Arabidopsis as well as two modified construct versions designed to increase DHA production were used. DHA was found to be stable to at least the T5 generation and the EPA and DHA were found to be predominantly at the sn-1,3 positions of triacylglycerols. Transgenic and parental lines did not have different germination or seedling establishment rates. Conclusions DHA can be produced at fish oil-like levels in industrially-relevant oilseed crop species using multi-gene construct designs which are stable over multiple generations. This study has implications for the future of sustainable EPA and DHA production from land-based sources.

BACKGROUND: Omega-3 long-chain (≥C(20)) polyunsaturated fatty acids (ω3 LC-PUFA) have critical roles in human health and development with studies indicating that deficiencies in these fatty acids can increase the risk or severity of cardiovascular and inflammatory diseases in particular. These fatty acids are predominantly sourced from fish and algal oils, but it is widely recognised that there is an urgent need for an alternative and sustainable source of EPA and DHA. Since the earliest demonstrations of ω3 LC-PUFA engineering there has been good progress in engineering the C(20) EPA with seed fatty acid levels similar to that observed in bulk fish oil (∼18%), although undesirable ω6 PUFA levels have also remained high. METHODOLOGY/PRINCIPAL FINDINGS: The transgenic seed production of the particularly important C(22) DHA has been problematic with many attempts resulting in the accumulation of EPA/DPA, but only a few percent of DHA. This study describes the production of up to 15% of the C(22) fatty acid DHA in Arabidopsis thaliana seed oil with a high ω3/ω6 ratio. This was achieved using a transgenic pathway to increase the C(18) ALA which was then converted to DHA by a microalgal Δ6-desaturase pathway. CONCLUSIONS/SIGNIFICANCE: The amount of DHA described in this study exceeds the 12% level at which DHA is generally found in bulk fish oil. This is a breakthrough in the development of sustainable alternative sources of DHA as this technology should be applicable in oilseed crops. One hectare of a Brassica napus crop containing 12% DHA in seed oil would produce as much DHA as approximately 10,000 fish.

A series of unusual odd-chain fatty acids (OC-FA) were identified in two thraustochytrid strains, TC 01 and TC 04, isolated from waters off the south east coast of Tasmania, Australia. FA compositions were determined by capillary GC and GC–MS, with confirmation of polyunsaturated fatty acids (PUFA) structure performed by analysis of 4,4-dimethyloxazoline derivatives. PUFA constituted 68–74% of the total FA, with the essential PUFA; eicosapentaenoic acid (20:5ω3, EPA), arachidonic acid (20:4ω6, AA) and docosahexaenoic acid (22:6ω3, DHA), accounting for 42–44% of the total FA. High proportions of the saturated OC-FA 15:0 (7.1% in TC 01) and 17:0 (6.2% in TC 04) were detected. The OC-FA 17:1ω8 was present at 2.8% in TC 01. Of particular interest, the C21 PUFA 21:5ω5 and 21:4ω7 were detected at 3.5% and 4.1%, respectively, in TC 04. A proposed biosynthesis pathway for these OC-PUFA is presented. It is possible that the unsaturated OC-PUFA found previously in a number of marine animals were derived from dietary thraustochytrids and they could be useful biomarkers in environmental and food web studies.

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.

Long-chain (or = C20) polyunsaturated fatty acids (LC-PUFA) EPA and DHA (20:5(Delta5,8,11,14,17) and 22:6(Delta4,7,10,13,16,19)) have well-documented health benefits against coronary heart disease, rheumatoid arthritis and other disorders. Currently, the predominant sources of these fatty acids are marine fish and algal oils, but research is being conducted to ensure that a sustainable, land-based production system can be developed. We here describe the metabolic engineering of an artificial pathway that produces 26% EPA in leaf triacylglycerol using a newly-identified Delta6-desaturase from the marine microalga Micromonas pusilla. We also demonstrate that this enzyme appears to function as an acyl-CoA desaturase that has preference for omega3 substrates both in planta and in yeast. Phylogenetic analysis indicates that this desaturase shares highly conserved motifs with previously described acyl-CoA Delta6-desaturases.

To evaluate the effects of physiologic doses of levothyroxine replacement on the lipoprotein profile in patients with subclinical hypothyroidism (SCH).In a prospective, double-blind, placebo-controlled study, we enrolled 120 patients--mostly, but not exclusively, premenopausal women--with SCH. Patients were randomly assigned to either a levothyroxine-treated group (n = 60) or a placebo (control) group (n = 60). Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) were measured before and 52 weeks after assignment to either group.In the levothyroxine-treated group, the lipoprotein mean values before and after the 52-week study were as follows: TC, 5.05 +/- 0.98 mmol/L versus 4.74 +/- 0.87 mmol/L (P.0001); LDL-C, 3.30 +/- 0.90 mmol/L versus 2.89 +/- 0.59 mmol/L (P.01); TG, 1.18 +/- 0.71 mmol/L versus 0.95 +/- 0.53 mmol/L (P.002); and HDL-C, 1.20 +/- 0.33 mmol/L versus 1.19 +/- 0.32 mmol/L (P = .29). In the control group, TC, HDL-C, and TG values remained unchanged after 52 weeks in comparison with baseline, but LDL-C mean values increased from 2.79 +/- 0.60 mmol/L to 3.11 +/- 0.77 mmol/L, a change that was statistically significant (P.001). At the end of the study, the lipid profile changes between levothyroxine-treated and control groups were compared. Total cholesterol and LDL-C were significantly lower in the levothyroxine-receiving group (P.029 and P.0001, respectively) in comparison with the control group. The difference did not reach statistical significance for TG and HDL-C values.In premenopausal women, SCH has a negative effect on the lipoprotein profile and may translate into a sizable cardiovascular risk if left untreated.

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.

Long-chain (> or = C(20)) polyunsaturated fatty acids, such as docosahexaenoic acid and eicosapentaenoic acid, are nutritionally important and provide protection against cardiovascular disease, stroke, and cancer. Structural variants of these compounds may have the potential to be used as pharmaceuticals. Marine microalgae are the key producers of long-chain polyunsaturated fatty acids in the global food web. Assuming vast biological and biochemical diversity, we devised a screen to identify microalgae that produce novel fatty acids. The herbicide metolachlor, an inhibitor of long-chain fatty acid biosynthesis, was used in microcosms containing field-collected microalgae to identify naturally resistant strains. We show that one diatom, Melosira cf. moniliformis, is naturally resistant to concentrations of metolachlor, which were cytostatic or lethal to all the other microalgae. Gas chromatography and gas chromatography-mass spectrometry revealed three fatty acids that have not previously been described-18:4 (Delta5,8,11,14), 18:4 (Delta5,9,12,15), and 18:5 (Delta5,8,11,14,17). We propose that this type of screen may be generally applicable to the search of novel compounds produced by marine microorganisms.

A preparative reversed-phase (RP; C(18)) high-performance liquid chromatography (HPLC) method with gradient elution using acetonitrile (MeCN)-chloroform (CHCl(3)) (or dichloromethane (DCM)) and evaporative light-scattering detection (ELSD) with automatic multiple injection and fraction collection was used to purify milligram quantities of microalgal polyunsaturated fatty acids (PUFA), separated as methyl esters (ME). PUFA-ME purified included methyl esters of docosahexaenoic acid (DHA; 22:6(n-3)), eicosapentaenoic acid (EPA; 20:5(n-3)) and the unusual very long-chain (C(28)) highly unsaturated fatty acid (VLC-HUFA), octacosaoctaenoic acid [28:8(n-3)(4, 7, 10, 13, 16, 19, 22, 25)] from the marine dinoflagellate Scrippsiella sp. CS-295/c. Other PUFA purified from various microalgae using this RP-HPLC method to greater than 95% purity included 16:3(n-4), 16:4(n-3), 16:4(n-1) and 18:5(n-3). The number of injections required was variable and depended on the abundance of the desired PUFA-ME, and resolution from closely eluting PUFA-ME, which determined the maximum loading. The purity of these fatty acids was determined by electron impact (EI) GC-MS and the chain length and location of double bonds was determined by EI GC-MS of 4,4-dimethyl oxazoline (DMOX) derivatives formed using a low temperature method. Advantages over silver-ion HPLC for purifying PUFA-ME is that separation occurs according to chain length as well as degree of unsaturation enabling separation of PUFA-ME with the same degree of unsaturation but different chain length (i.e. between 18:5(n-3) and 20:5(n-3)). In addition, PUFA-ME are not strongly adsorbed, but elute earlier than their more saturated corresponding FAME of the same chain length. This method is robust, simple, and requires only a short re-equilibration time. It is a useful tool for preparing milligram quantities of pure PUFA-ME for bioactive screening (as free fatty acids), although many multiple injections may be required for minor PUFA-ME. It also enabled dose-response and structure-activity studies to be carried out. It can be used for the enrichment of low levels of VLC-HUFA-ME to facilitate elucidation of their chemical structure and so is a useful adjunct to EI GC-MS of DMOX derivatives and other techniques such as NMR, which requires milligram quantities of purified compounds.

Objective: The aim of this study was to examine the distribution of trans fatty acids (TFA) in plasma lipid classes and the relationship with dietary intake of TFA. Design: After a 2 week baseline (habitual) diet, all subjects consumed a moderate fat (MF) diet for 3 weeks with the fat being derived mainly from margarine and the rest from lean beef, and then a very low fat (VLF) diet for 3 weeks with the TFA being derived only from the lean beef. Blood samples were collected 2 days prior to the end and also on the last day of each dietary period. Setting: Deakin Institute of Human Nutrition, Deakin University, Geelong, Australia. Subjects: Ten free-living mildly hypercholesterolaemic subjects aged 22–66 were recruited in Geelong. Outcome measures: TFA intake was calculated from analyses of Australian margarines, butter, lean meat and animal fat. The TFA in plasma lipid fractions were separated by AgNO3 thin-layer chromatography and quantitated by capillary gas–liquid chromatography using internal standards. Results: The phospholipid (PL) fraction contained more than 60% of the trans-18:1 isomers in the plasma lipids in all subjects. On the baseline diet, the predominant positional isomer of trans-18:1 in PL was Δ11, whereas in the other lipid classes it was the Δ9 isomer. The concentration of the Δ9 isomer increased on the MF diet, particularly in the PL fraction, while the concentration of the Δ11 isomer decreased in all fractions. On the VLF diet, the total TFA level decreased by approximately 50%, mainly due to decreases in the TFA isomers in the PL and TG fractions. Changes in plasma total and PL TFA, PL Δ9, Δ10 and Δ11 were strongly correlated with dietary TFA intake (P

BACKGROUND: Monoacylglycerol acyltransferases (MGATs) are predominantly associated with lipid absorption and resynthesis in the animal intestine where they catalyse the first step in the monoacylglycerol (MAG) pathway by acylating MAG to form diacylglycerol (DAG). Typical plant triacylglycerol (TAG) biosynthesis routes such as the Kennedy pathway do not include an MGAT step. Rather, DAG and TAG are synthesised de novo from glycerol-3-phosphate (G-3-P) by a series of three subsequent acylation reactions although a complex interplay with membrane lipids exists. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that heterologous expression of a mouse MGAT acyltransferase in Nicotiana benthamiana significantly increases TAG accumulation in vegetative tissues despite the low levels of endogenous MAG substrate available. In addition, DAG produced by this acyltransferase can serve as a substrate for both native and coexpressed diacylglycerol acyltransferases (DGAT). Finally, we show that the Arabidopsis thaliana GPAT4 acyltransferase can produce MAG in Saccharomyces cerevisiae using oleoyl-CoA as the acyl-donor. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the concept of a new method of increasing oil content in vegetative tissues by using MAG as a substrate for TAG biosynthesis. Based on in vitro yeast assays and expression results in N. benthamiana, we propose that co-expression of a MAG synthesising enzyme such as A. thaliana GPAT4 and a MGAT or bifunctional M/DGAT can result in DAG and TAG synthesis from G-3-P via a route that is independent and complementary to the endogenous Kennedy pathway and other TAG synthesis routes.