Multifunctional Acyltransferases from <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Multifunctional acyltransferases are able to catalyze the esterification of various acyl-acceptors with activated fatty acids. Here we describe the identification of four proteins from Tetrahymena thermophila that share certain properties with mammalian acyltransferases regarding their predicted transmembrane structure, their molecular mass and the typical acyltransferase motif. Expression of the Tetrahymena sequences results in production of triacylglycerols and wax esters in recombinant yeast when appropriate substrates are provided. The in vitro characterization shows, that these enzymes are capable of esterifying different acyl-acceptors including fatty alcohols, diols, diacylglycerols and isoprenols with acyl-CoA thioesters. Based on these catalytic activities and the sequence similarities of the Tetrahymena proteins with acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) family members, we conclude that we identified a new group of DGAT2-related multifunctional acyltransferases from protozoan organisms.     

Properties and Biotechnological Applications of Acyl‐CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae

Triacylglycerol (TAG) is the major storage lipid in most terrestrial plants and microalgae, and has great nutritional and industrial value. Since the demand for vegetable oil is consistently increasing, numerous studies have been focused on improving the TAG content and modifying the fatty‐acid compositions of plant seed oils. In addition, there is a strong research interest in establishing plant vegetative tissues and microalgae as platforms for lipid production. In higher plants and microalgae, TAG biosynthesis occurs via acyl‐CoA‐dependent or acyl‐CoA‐independent pathways. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step in the acyl‐CoA‐dependent biosynthesis of TAG, which appears to represent a bottleneck in oil accumulation in some oilseed species. Membrane‐bound and soluble forms of DGAT have been identified with very different amino‐acid sequences and biochemical properties. Alternatively, TAG can be formed through acyl‐CoA‐independent pathways via the catalytic action of membrane‐bound phospholipid:diacylglycerol acyltransferase (PDAT). As the enzymes catalyzing the terminal steps of TAG formation, DGAT and PDAT play crucial roles in determining the flux of carbon into seed TAG and thus have been considered as the key targets for engineering oil production. Here, we summarize the most recent knowledge on DGAT and PDAT in higher plants and microalgae, with the emphasis on their physiological roles, structural features, and regulation. The development of various metabolic engineering strategies to enhance the TAG content and alter the fatty‐acid composition of TAG is also discussed.     

The Utilization of the Acyl-CoA and the Involvement PDAT and DGAT in the Biosynthesis of Erucic Acid-Rich Triacylglycerols in Crambe Seed Oil

The triacylglycerol of Crambe abyssinica seeds consist of 95 % very long chain (>18 carbon) fatty acids (86 % erucic acid; 22:1^?13) in the sn‐1 and sn‐3 positions. This would suggest that C. abyssinica triacylglycerols are not formed by the action of the phospholipid:diacylglycerol acyltransferase (PDAT), but are rather the results of acyl‐CoA:diacylglycerol acyltransferase (DGAT) activity. However, measurements of PDAT and DGAT activities in microsomal membranes showed that C. abyssinica has significant PDAT activity, corresponding to about 10 % of the DGAT activity during periods of rapid seed oil accumulation. The specific activity of DGAT for erucoyl‐CoA had doubled at 19 days after flowering compared to earlier developmental stages, and was, at that stage, the preferred acyl donor, whereas the activities for 16:0‐CoA and 18:1‐CoA remained constant. This indicates that an expression of an isoform of DGAT with high specificity for erucoyl‐CoA is induced at the onset of rapid erucic acid and oil accumulation in the C. abyssinica seeds. Analysis of the composition of the acyl‐CoA pool during different stages of seed development showed that the percentage of erucoyl groups in acyl‐CoA was much higher than in complex lipids at all stages of seed development except in the desiccation phase. These results are in accordance with published results showing that the rate limiting step in erucic acid accumulation in C. abyssinica oil is the utilization of erucoyl‐CoA by the acyltransferases in the glycerol‐3‐phosphate pathway.     

Two-stage solvent extraction of seeds of <Emphasis Type="Italic">Hibiscus abelmoschus</Emphasis> linn: Lipid and FA compositions

The lipid and FA compositions of TAG seed oil of ambrette (Hibiscus abelmoschus), contrary to earlier reports, were found to contain only a small amount of epoxy FA. Higher HBr absorption values were shown to be due to the presence of fragrance components of the seed coat in the oil derived from intact whole seeds. Cyclopropene/cyclopropane acids in the FAME were determined to be about 1.5% by GLC, while epoxy acids were less than 1% by two different methods. Based on the physicochemical characteristics and lipid and FA compositions of the fresh and methanol-extracted seeds, ambrette TAG oil may be a candidate for edible use. The process of selective recovery of the expensive fragrant oily concentrate in higher yield in a first step and of an edible fatty oil in a second step makes it an attractive economic proposal.     

Changes in Oil Content of Transgenic Soybeans Expressing the Yeast <Emphasis Type="Italic">SLC1</Emphasis> Gene

The wild type (Wt) and mutant form of yeast (sphingolipid compensation) genes, SLC1 and SLC1-1, have been shown to have lysophosphatidic acid acyltransferase (LPAT) activities (Nageic et al. in J Biol Chem 269:22156–22163, 1993). Expression of these LPAT genes was reported to increase oil content in transgenic Arabidopsis and Brassica napus. It is of interest to determine if the TAG content increase would also be seen in soybeans. Therefore, the wild type SLC1 was expressed in soybean somatic embryos under the control of seed specific phaseolin promoter. Some transgenic somatic embryos and in both T2 and T3 transgenic seeds showed higher oil contents. Compared to controls, the average increase in triglyceride values went up by 1.5% in transgenic somatic embryos. A maximum of 3.2% increase in seed oil content was observed in a T3 line. Expression of the yeast Wt LPAT gene did not alter the fatty acid composition of the seed oil.     

Chemical Composition and Nutritional Characteristics of the Seed Oil of Wild <Emphasis Type="Italic">Lathyrus</Emphasis>, <Emphasis Type="Italic">Lens</Emphasis> and <Emphasis Type="Italic">Pisum</Emphasis> Species from Southern Spain

The fatty acid composition of the seed oil of 19 wild legume species from southern Spain was analyzed by gas chromatography. The main seed oil fatty acids ranged from C_14:0 to C_20:0. Among unsaturated fatty acids, the most abundant were linoleic, oleic and linolenic acids, except for Lathyrus angulatus, L. aphaca, L. clymenum, L. sphaericus and L. nigricans where C_18:3 contents were higher than C_18:1 contents. Palmitic acid was the most abundant saturated acid in studied species, ranging from 11.6% in Lathyrus sativus to 19.3% in Lens nigricans. All studied species showed higher amounts of total unsaturated fatty acids than saturated ones. Among studied species, the ω6/ω3 ratio was variable, ranging from 2.0% in L. nigricans to 13.8% in L. sativus, there being eight species in which the ω6/ω3 ratio was below 5. The fatty acids observed in these plants supports the use of these plants as a source of important dietary lipids.     

Cyanolipid-rich seed oils from <Emphasis Type="Italic">Allophylus natalensis</Emphasis> and <Emphasis Type="Italic">A. dregeanus</Emphasis>

As a continuation of our study on plants of the Sapindaceae, the chemical composition of the oil extracted from seeds of Allophylus natalensis (Sonder) De Winter and of A. dregeanus (Sonder) De Winter has been investigated. The oil from both species contained approximately equal amounts of TAG and type I cyanolipids (CL), 1-cyano-2-hydroxymethylprop-2-en-1-oldiesters, with minor amounts of type III CL, 1-cyano-2-hydroxymethylprop-1-en-3-ol-diesters. Structural investigation of the oil components was accomplished by chemical, chromatographic (TLC, CC, GC, and GC-MS), and spectroscopic (IR, NMR) means. GC and GC-MS analysis showed that C₂₀ FA were dominant in the CL components of the oil from the two species (44–80% vs. 21–26% in TAG), with cis-11-eicosenoic acid (36–46%) and cis 13-eicosenoic acid (paullinic acid, 23–37%) as the major esterified fatty acyl chains in A. natalensis and A. dregeanus, respectively. cis-Vaccenic acid was particularly abundant (11–31%) in the CL from A. dregeanus, whereas eicosanoic acid (10–22%) was also a major component of CL in both species.     

Physicochemical Characteristics of Nigella Seed (<Emphasis Type="Italic">Nigella sativa</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) Oil as Affected by Different Extraction Methods

The physicochemical properties of crude Nigella seed (Nigella sativa L.) oil which was extracted using Soxhlet, Modified Bligh–Dyer and Hexane extraction methods were determined. The effect of different extraction methods which includes different parameters, such as temperature, time and solvent on the extraction yield and the physicochemical properties were investigated. The experimental results showed that temperature, different solvents and extraction time had the most significant effect on the yield of the Nigella oil extracts. The fatty acid (FA) compositions of Nigella seed oil were further analyzed by gas chromatography to compare the extraction methods. The C16:0, C18:1 and C18:2 have been identified to be the dominant fatty acids in the Nigella seed oils. However, the main triacylglycerol (TAG) was LLL followed by OLL and PLL. The FA and TAG content showed that the composition of the Nigella seed oil extracted by different methods was mostly similar, whereas relative concentration of the identified compounds were apparently different according to the extraction methods. The melting and crystallization temperatures of the oil extracted by Soxhlet were −2.54 and −55.76 °C, respectively. The general characteristics of the Nigella seed oil obtained by different extraction methods were further compared. Where the Soxhlet extraction method was considered to be the optimum process for extracting Nigella seed oil with a higher quality with respect to the other two processes.     

Identification of Crepenynic Acid in the Seed Oil of <Emphasis Type="Italic">Atractylodes lancea</Emphasis> and <Emphasis Type="Italic">A. macrocephala</Emphasis>

Atractylodes rhizome is widely used in traditional Chinese herbal medicine. Although the chemical composition of the root has been studied in detail, the oil content and fatty acid composition of the seeds of Atractylodes species have not been reported. Fatty acyl composition of seeds from Atractylodes lancea and A. macrocephala was determined by gas chromatography and mass spectrometry of fatty acid methyl esters and 3-pyridylcarbinol esters. The predominant fatty acid in the seeds of both species was linolenic acid, but the unusual acetylenic fatty acid, crepenynic acid (cis-9-octadecen-12-ynoic acid), was also observed at levels of 18% in A. lancea and 13–15% in A. macrocephala. Fatty acid content was 24% for the samples of A. lancea and 16–17% for samples from A. macrocephala. sn-1,3 regioselective lipase digestion of seed lipids revealed that crepenynic acid was absent from the sn-2 position of the seed triacylglycerol. Crepenynic acid was also found in the seed oil of Jurinea mollis at 24% and was not present in the sn-2 position of the TAG. A contrasting distribution of crepenynic acid was found in the oil of Crepis rubra, suggesting differences in crepenynic acid synthesis or TAG assembly between these species.     

Identification of the Unsaturated Heptadecyl Fatty Acids in the Seed Oils of <Emphasis Type="Italic">Thespesia populnea</Emphasis> and <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

The fatty acid composition of the seed oils of Thespesia populnea and cotton variety SG-747 (Gossypium hirsutum) were studied to identity their 17-carbon fatty acids. With a combination of chemical derivatization, gas chromatography, and mass spectrometry, 8-heptadecenoic acid, 9-heptadecenoic acid, and 8,11-heptadecadienoic acids were identified in both oils. Additionally, traces of 10-heptadecenoic acid were identified in the T. populnea oil. Although these odd-carbon number fatty acids are present in only minor amounts in cottonseed oil, they make up about ~2 % of the fatty acids in T. populnea seed oil. The identification of these acids indicates that fatty acid α-oxidation is not restricted to cyclopropene fatty acids in these plants, but also occurs with unsaturated fatty acids. Combined with malvalic acid (generally accepted as being formed by α-oxidation of sterculic acid), ~7 % of the fatty acids in T. populnea seed have under gone α-oxidization. The results should help clarify the composition of T. populnea seed oil, which has been reported inconsistently in the literature.     

Properties of lysophosphatidylcholine acyltransferase from <Emphasis Type="Italic">Brassica napus</Emphasis> cultures

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT; EC 2.3.1.23) catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine (LPC) to produce PC and CoA. LPCAT activity may affect the incorporation of fatty acyl moieties at the sn-2 position of PC where PUFA are formed and may indirectly influence seed TAG composition. LPCAT activity in microsomes prepared from microspore-derived cell suspension cultures of oilseed rape (Brassica napus L. cv Jet Neuf) was assayed using [1-¹⁴C]acyl-CoA as the fatty acyl donor. LPCAT activity was optimal at neutral pH and 35°C, and was inhibited by 50% at a BSA concentration of 3 mg mL⁻¹. At acyl-CoA concentrations above 20 μM, LPCAT activity was more specific for oleoyl (18∶1)-CoA than stearoyl (18∶0)- and palmitoyl (16∶0)-CoA. Lauroyl (12∶0)-CoA, however, was not an effective acyl donor. LPC species containing 12∶0, 16∶0, 18∶0, or 18∶1 as the fatty acyl moiety all served as effective acyl acceptors for LPCAT, although 12∶0-LPC was somewhat less effective as a substrate at lower concentrations. The failure of LPCAT to catalyze the incorporation of a 12∶0 moiety from acyl-CoA into PC is consistent with the tendency of acyltransferases to discriminate against incorporation of this fatty acyl moiety at the sn-2 position of TAG from the seed oil of transgenic B. napus expressing a medium-chain thioesterase.     

Identification of Diacylglycerol Acyltransferase Inhibitors from <Emphasis Type="Italic">Rosa centifolia</Emphasis> Petals

Diacylglycerol acyltransferase (DGAT) catalyzes the final step of triacylglycerol (TAG) synthesis, and is considered as a potential target to control hypertriglyceridemia or other metabolic disorders. In this study, we found that the extract of rose petals suppressed TAG synthesis in cultured cells, and that the extract showed DGAT inhibitory action in a dose-dependent manner. Fractionation of the rose extract revealed that the DGAT inhibitory substances in the extract were ellagitannins; among them rugosin B, and D, and eusupinin A inhibited DGAT activity by 96, 82, and 84% respectively, at 10 μM. These substances did not inhibit the activities of other hepatic microsomal enzymes, glucose-6-phosphatase and HMG-CoA reductase, or pancreatic lipase, suggesting that ellagitannins inhibit DGAT preferentially. In an oral fat load test using mice, postprandial plasma TAG increase was suppressed by rose extract; TAG levels 2 h after the fat load were significantly lower in mice administered a fat emulsion containing rose extract than in control mice (446.3 ± 33.1 vs 345.3 ± 25.0 mg/dL, control vs rose extract group; P < 0.05). These results suggest that rose ellagitannins or rose extract could be beneficial in controlling lipid metabolism and used to improve metabolic disorders.     

<Emphasis Type="Italic">Annona squamosa</Emphasis> and <Emphasis Type="Italic">Catunaregam nilotica</Emphasis> Seeds,the Effect of the Extraction Method on the Oil Composition

Annona squamosa and Catunaregam nilotica seeds and oils were characterized for their approximate analysis and physico-chemical properties. The oil and protein contents were 26.8, 17.5 and 40.0, 22.2%, in A. squamosa and C. nilotica seeds, respectively. The oils were extracted using cold extraction (CE) and Soxhlet extraction (SE) methods. Fatty acids and tocopherols were determined by GC–MS and HPLC, respectively. Generally the physico-chemical properties and fatty acids were not significantly (P ≤ 0.05) affected by the extraction methods. The major fatty acids of A. squamosa oil extracted by CE and SE were oleic 49.2 and 50.5%, linoleic 22.3 and 22.7%, palmitic 15.6 and 15.2%, and stearic 10.6 and 9.3%, respectively. While the major fatty acids in C. nilotica oil extracted by CE and SE were oleic 10.5, and 10.4%, linoleic 63.1 and 63.4%, palmitic 9.7 and 9.8% and stearic 5.1 and 5.4%, respectively. The tocopherol content of CE and SE extracted oils from A. squamosa amounted to 16.6 and 15.5 and from C. nilotica amounted to 110.5 and 107.7 mg/100 g oil, respectively, with delta-tocopherol as the predominant tocopherol in A. squamosa oil, and beta-tocopherol in C. nilotica oil. The total amount of amino acids was found to be 7.266 and 14.202 g/100 g protein, in seeds of A. squamosa and C. nilotica, respectively.     

Proximate Composition and Fatty Acid Profile of <Emphasis Type="Italic">Pongamia pinnata</Emphasis>, a Potential Biodiesel Crop

The chemical characteristics of Pongamia pinnata seeds, focussing on proximate composition and the fatty acid profile of its oil, are presented. The proximate composition of P. pinnata seeds was: 3.8% ash, 9.7% sugar, 7.07% protein, 24% oil, 10.7% free amino acids, and 0.24% free fatty acids. The oil was extracted from seeds by use of different solvents and the highest yield (29%) was obtained by use of n-hexane. Monounsaturated and polyunsaturated fatty acids accounted for 63.3 and 22.9%, respectively, of the seed oil. Oleic acid was the major fatty acid but a substantial amount of erucic acid was also detected; this was not reported in previous studies. The level of erucic acid and the presence of toxic flavonoids, for example karanjin, pongapin, and pongaglabrin, render the oil inedible according to WHO recommendations. However, low levels of saturated and polyunsaturated fatty acids with desirable cetane number and iodine value suggest potential for application as a biodiesel fuel.     

Seed oil composition of <Emphasis Type="Italic">Paullinia cupana</Emphasis> var. <Emphasis Type="Italic">sorbilis</Emphasis> (Mart.) Ducke

The chemical composition of the oil extracted from the seeds of Paullinia cupana var. sorbilis (Mart.) Ducke (syn. P. sorbilis) was investigated. Cyanolipids constituted 3% of the total oil from guaraná seeds, whereas acylglycerols accounted for 28%. ¹H and ¹³C NMR analyses indicated that type 1 cyanolipids (1-cyano-2-hydroxymethylprop-2-ene-1-ol diesters) are present in the oil from P. cupana. GC and GC-MS analysis showed that cis-11-octadecenoic (cis-vaccenic acid) and cis-11-eicosenoic acids were the main FA (30.4 and 38.7%) esterified to the nitrile group. Paullinic acid (7.0%) was also an abundant component. Oleic acid (37.4%) was the dominant fatty acyl chain in the acylglycerols.     

Overexpression of a FAD3 Desaturase Increases Synthesis of a Polymethylene-Interrupted Dienoic Fatty Acid in Seeds of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> L.

A cDNA encoding the Arabidopsis extraplastidic linoleate desaturase (FAD3) was overexpressed in the seeds of wild-type Arabidopsis and in a mutant line that accumulates high levels of oleic acid. In the transformed wild-type plants, linolenic acid (18:3Δ9,12,15) increased from 19% to nearly 40% of total seed fatty acids, with a corresponding decrease in linoleate content (18:2Δ9,12). In the high oleate mutant, a large increase in the level of a fatty acid identified by gas-chromatography/mass-spectrometry as mangiferic acid (18:2Δ9,15) was observed. The results demonstrate that the polymethylene-interrupted dienoic fatty acid, mangiferic acid, can be produced in seed oil through the overexpression of a fatty acid n-3 desaturase.

Metabolism of <Emphasis Type="Italic">sn</Emphasis>-1(3)-Monoacylglycerol and <Emphasis Type="Italic">sn</Emphasis>-2-Monoacylglycerol in Caecal Enterocytes and Hepatocytes of Brown Trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>)

sn‐2‐Monoacylglycerol (2‐MAG) and sn‐1(3)‐monoacylglycerol [1(3)‐MAG] are important but yet little studied intermediates in lipid metabolism. The current study compared the metabolic fate of 2‐MAG and 1(3)‐MAG in isolated caecal enterocytes and hepatocytes of brown trout (Salmo trutta). 1(3)‐Oleoyl [9,10‐3H(N)]‐glycerol and 2‐Oleoyl [9,10‐3H(N)]‐glycerol were prepared by pancreatic lipase digestion of triolein [9,10‐3H(N)]. The 1(3)‐MAG and 2‐MAG were efficiently absorbed by enterocytes and hepatocytes at similar rates. The 2‐MAG was quickly resynthesized into TAG through the monoacylglycerol acyltransferase (EC: 2.3.1.22, MGAT) pathway in both tissues, whereas 1(3)‐MAG was processed into TAG and phospholipids at a much slower rate, suggesting 2‐MAG was the preferred substrates for MGAT. Further analysis showed that 1(3)‐MAG was synthesized into 1,3‐DAG, but there were no accumulation of 1,3‐DAG in either enterocytes or hepatocytes, which contrasts that of mammalian studies. Some of the 1(3)‐MAG may be acylated to 1,2(2,3)‐DAG and then utilized for TAG synthesis. Alternatively, 1(3)‐MAG can be hydrolyzed to free fatty acid and glycerol, and re‐synthesized into TAG through the glycerol‐3‐phosphate (Gro‐3‐P) pathway. The overall data suggested that the limiting step of the intracellular 1(3)‐MAG metabolism is the conversion of 1(3)‐MAG itself.