Lipidomic and transcriptional analysis of the linoleoyl-omega-hydroxyceramide biosynthetic pathway in human psoriatic lesions.

A complex assembly of lipids including fatty acids, cholesterol, and ceramides is vital to the integrity of the mammalian epidermal barrier. The formation of this barrier requires oxidation of the substrate fatty acid, linoleic acid (LA), which is initiated by the enzyme 12R-lipoxygenase (LOX). In the epidermis, unoxidized LA is primarily found in long-chain acylceramides termed esterified omega-hydroxy sphingosine (EOS)/phytosphingosine/hydroxysphingosine (collectively EOx). The precise structure and localization of LOX-oxidized EOx in the human epidermis is unknown, as is their regulation in diseases such as psoriasis, one of the most common inflammatory diseases affecting the skin. Here, using precursor LC/MS/MS, we characterized multiple intermediates of EOx, including 9-HODE, 9,10-epoxy-13-HOME, and 9,10,13-TriHOME, in healthy human epidermis likely to be formed via the epidermal LOX pathways. The top layers of the skin contained more LA, 9-HODE, and 9,10,13-TriHOME EOSs, whereas 9,10-epoxy-13-HOME EOS was more prevalent deeper in the stratum corneum. In psoriatic lesions, levels of native EOx and free HODEs and HOMEs were significantly elevated, whereas oxidized species were generally reduced. A transcriptional network analysis of human psoriatic lesions identified significantly elevated expression of the entire biosynthetic/metabolic pathway for oxygenated ceramides, suggesting a regulatory function for EOx lipids in reconstituting epidermal integrity. The role of these new lipids in progression or resolution of psoriasis is currently unknown. We also discovered the central coordinated role of the zinc finger protein transcription factor, ZIC1, in driving the phenotype of this disease. In summary, long-chain oxygenated ceramide metabolism is dysregulated at the lipidomic level in psoriasis, likely driven by the transcriptional differences also observed, and we identified ZIC1 as a potential regulatory target for future therapeutic interventions.

De novo biosynthesis of linoleic acid is widespread in parasitic wasps.

Linoleic acid (C18:2∆9,12 , LA) is an important metabolite with numerous essential functions for growth, health, and reproduction of organisms. It has long been assumed that animals lack ∆12-desaturases, the enzymes needed to produce LA from oleic acid (C18:1∆9 , OA). There is, however, increasing evidence that this is not generally true for invertebrates. In the insect order Hymenoptera, LA biosynthesis has been shown for only two parasitic wasp species of the so-called "Nasonia group," but it is unknown whether members of other taxa are also capable of synthesizing LA. Here, we demonstrate LA biosynthesis in 13 out of 14 species from six families of parasitic wasps by gas chromatography-mass spectrometry analysis using two different stable isotope labeling techniques. Females of the studied species converted topically applied fully 13 C-labeled OA into LA and/or produced labeled LA after feeding on fully 13 C-labeled α- d-glucose. These results indicate that ∆12-desaturases are widespread in parasitic Hymenoptera and confirm previous studies demonstrating that these insects are capable of synthesizing fatty acids de novo.

Functional characterization and overexpression of Δ12-desaturase in the oleaginous yeast Rhodotorula toruloides for production of linoleic acid-rich lipids.

Linoleic acid (LA) has garnered much attention due to its potential applications in the oleochemical and nutraceutical industries. The oleaginous yeast Rhodotorula toruloides has outstanding lipogenecity, and is considered a potential alternative to the current plant-based platforms for LA production. Δ12-fatty acid desaturases (Δ12-Fads) are involved in LA synthesis in various fungi and yeasts, but their functions in R. toruloides remain poorly understood. To achieve the production of LA-rich lipids in R. toruloides, we investigated the function of the native Δ12-FAD (RtFAD2). First, the overexpression of RtFAD2 and its co-overexpression with RtFAD1 (encoding R. toruloides Δ9-Fad) and their effects on LA production in R. toruloides were investigated. The function of RtFad2 was confirmed by heterologous expression in Saccharomyces cerevisiae. Overexpression of RtFAD2 significantly elevated the LA contents and titers in the wild-type strain R. toruloides DMKU3-TK16 (TK16) and in a thermotolerant derivative of TK16 (L1-1). Additionally, overexpression of RtFAD2 in R. toruloides strains also increased the lipid titer and content. Overexpression of RtFAD1 was down-regulated in the RtFAD1 and RtFAD2 co-overexpressing strains, suggesting that the elevated LA content may function as a key regulator of RtFAD1 expression to control C18 fatty-acid synthesis in R. toruloides. We characterized the function of RtFAD2 and showed that its overexpression in R. toruloides increased the lipid and LA production. These findings may assist in the rational design of metabolic engineering related to LA or polyunsaturated fatty acid production in R. toruloides.

Comparative Analysis of Different Isolated Oleaginous Mucoromycota Fungi for Their γ-Linolenic Acid and Carotenoid Production.

γ-Linolenic acid (GLA) and carotenoids have attracted much interest due to their nutraceutical and pharmaceutical importance. Mucoromycota, typical oleaginous filamentous fungi, are known for their production of valuable essential fatty acids and carotenoids. In the present study, 81 fungal strains were isolated from different Egyptian localities, out of which 11 Mucoromycota were selected for further GLA and carotenoid investigation. Comparative analysis of total lipids by GC of selected isolates showed that GLA content was the highest in Rhizomucor pusillus AUMC 11616.A, Mucor circinelloides AUMC 6696.A, and M. hiemalis AUMC 6031 that represented 0.213, 0.211, and 0.20% of CDW, respectively. Carotenoid analysis of selected isolates by spectrophotometer demonstrated that the highest yield of total carotenoids (640 µg/g) was exhibited by M. hiemalis AUMC 6031 and M. hiemalis AUMC 6695, and these isolates were found to have a similar carotenoid profile with, ß-carotene (65%), zeaxanthin (34%), astaxanthin, and canthaxanthin (5%) of total carotenoids. The total fatty acids of all tested isolates showed moderate antimicrobial activity against Staphylococcus aureus and Salmonella Typhi, and Penicillium chrysogenum. To the best of our knowledge, this is the first report on the highest yield of total lipid accumulation (51.74% CDW) by a new oleaginous fungal isolate R. pusillus AUMC 11616.A. A new scope for a further study on this strain will be established to optimize and improve its total lipids with high GLA production. So, R. pusillus AUMC 11616.A might be a potential candidate for industrial application.

Role of silica coated magnetic nanoparticle on cell flocculation, lipid extraction and linoleic acid production from Chlorella pyrenoidosa.

In the present work, it has been observed that magnetic (Fe3O4) - silica core- shell nanoparticles helps in flocculation of Chlorella pyrenoidosa cells with simultaneous production of linoleic acid. The mean particle size in Dynamic light scattering (DLS) of the silica coated magnetic nanoparticle was estimated 444.7 nm. The characterization of nanoparticles was also performed by X-ray diffraction technique (XRD). Apart from flocculation, it has been observed that in presence of magnetic silica core- shell nanoparticles the amount of lipid obtained was four times than that of control. On the contrary, in presence of these nanoparticles, linoleic acid (18:2) has been produced in Chlorella pyrenoidosa cells almost by 80% whereas, it has been noticed only 8.73% in control. This is the first report where the linoleic acid has been obtained as major component of microalgal fatty acid methyl esters (FAME) having important application in nutraceuticals and pharmaceutical sectors.

Molecular cloning and function analysis of FAD2 gene in Idesia polycarpa.

Idesia polycarpa is a valuable oil-producing tree and can potentially be used for edible oil and biofuel production. The fruits of I. polycarpa are unique in that they contain both saturated and unsaturated lipids. Fatty acid desaturase 2 (FAD2), also as known as omega-6 fatty acid desaturase in endoplasmic, is a key enzyme for linoleic acid and α-linolenic acid biosynthesis. However, bioinformatics and expression of FAD2 in I. polycarpa are still absent. Here, to gain insight into the lipid and linoleic synthesis of I. polycarpa, we compared the fruits from different growth stages. Lipid accumulation rates, final lipid content, linoleic accumulation rates and final linoleic content were significantly different among the different stages. In a further step, the FAD2 gene from fruits of I. polycarpa, named IpFAD2, was cloned and characterized. A partial fragment of 169 bp of IpFAD2 was amplified by degenerate PCR. Full cDNA of IpFAD2 was obtained by the RACE technique. The open-reading frame of IpFAD2 was 1149 bp in length, encoding 382 amino acids. A comparison of the deduced amino acids sequence of IpFAD2 with FAD2 from other species showed high similarities, ranging from 78.8 to 92.6%. The IpFAD2-predicted protein has a theoretical molecular mass of 44.03 kDa and an isoelectric point (pI) of 8.04. It has five transmembrane helices located on the endoplasmic reticulum. The IpFAD2-predicted protein was classified as belonging to the Membrane-FADS-like superfamily based on its conserved domain analysis. Expression analysis based on qRT-PCR indicated that IpFAD2 was expressed in different fruit growth stages, with the highest expression level at 80 DAP and the lowest at 130 DAP. The expression of IpFAD2 was positively correlated with the linoleic accumulation rates in I. polycarpa fruits. Prokaryotic expression in Escherichia. Coli BL21(DE3) indicated that IpFAD2 gene could encode a bio-functional omega-6 fatty acid desaturase. Heterologous expression in Arabidopsis thaliana confirmed that the isolated IpFAD2 proteins could catalyse linoleic synthesis. This is the first cloning and expression analysis of FAD2 from I. polycarpa, significantly contributing to our understanding of the role of IpFAD2 in linoleic synthesis, esp. in terms of genetic engineering breeding for linoleic production.

FAD2 Gene Radiation and Positive Selection Contributed to Polyacetylene Metabolism Evolution in Campanulids.

Polyacetylenes (PAs) are bioactive, specialized plant defense compounds produced by some species in the eudicot clade campanulids. Early steps of PA biosynthesis are catalyzed by Fatty Acid Desaturase 2 (FAD2). Canonical FAD2s catalyze desaturation, but divergent forms can catalyze hydroxylation, conjugation, acetylenation, and epoxygenation. These alternate reactions give rise to valuable unusual fatty acids, including the precursors to PAs. The extreme functional diversity of FAD2 enzymes and the origin of PA biosynthesis are poorly understood from an evolutionary perspective. We focus here on the evolution of the FAD2 gene family. We uncovered a core eudicot-wide gene duplication event giving rise to two lineages: FAD2-α and FAD2-ß. Independent neofunctionalizations in both lineages have resulted in functionally diverse FAD2-LIKEs involved in unusual fatty acid biosynthesis. We found significantly accelerated rates of molecular evolution in FAD2-LIKEs and use this metric to provide a list of uncharacterized candidates for further exploration of FAD2 functional diversity. FAD2-α has expanded extensively in Asterales and Apiales, two main clades of campanulids, by ancient gene duplications. Here, we detected positive selection in both Asterales and Apiales lineages, which may have enabled the evolution of PA metabolism in campanulids. Together, these findings also imply that yet uncharacterized FAD2-α copies are involved in later steps of PA biosynthesis. This work establishes a robust phylogenetic framework in which to interpret functional data and to direct future research into the origin and evolution of PA metabolism.

Methyl-end desaturases with ∆12 and ω3 regioselectivities enable the de novo PUFA biosynthesis in the cephalopod Octopus vulgaris.

The interest in understanding the capacity of aquatic invertebrates to biosynthesise omega-3 (ω3) long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) has increased in recent years. Using the common octopus Octopus vulgaris as a model species, we previously characterised a ∆5 desaturase and two elongases (i.e. Elovl2/5 and Elovl4) involved in the biosynthesis of LC-PUFA in molluscs. The aim of this study was to characterise both molecularly and functionally, two methyl-end (or ωx) desaturases that have been long regarded to be absent in most animals. O. vulgaris possess two ωx desaturase genes encoding enzymes with ∆12 and ω3 regioselectivities enabling the de novo biosynthesis of the C18 PUFA 18:2ω6 (LA, linoleic acid) and 18:3ω3 (ALA, α-linolenic acid), generally regarded as dietary essential for animals. The O. vulgaris ∆12 desaturase ("ωx2") mediates the conversion of 18:1ω9 (oleic acid) into LA, and subsequently, the ω3 desaturase ("ωx1") catalyses the ∆15 desaturation from LA to ALA. Additionally, the O. vulgaris ω3 desaturase has ∆17 capacity towards a variety of C20 ω6 PUFA that are converted to their ω3 PUFA products. Particularly relevant was the affinity of the ω3 desaturase towards 20:4ω6 (ARA, arachidonic acid) to produce 20:5ω3 (EPA, eicosapentaenoic acid), as supported by yeast heterologous expression, and enzymatic activity exhibited in vivo when paralarvae were incubated in the presence of [1-14C]20:4ω6. These results confirmed that several routes enabling EPA biosynthesis are operative in O. vulgaris whereas ARA and docosahexaenoic acid (DHA, 22:6ω3) should be considered essential fatty acids since endogenous production appears to be limited.

Production of high oleic/low linoleic rice by genome editing.

Rice bran oil (RBO) contains many valuable healthy constituents, including oleic acid. Improvement of the fatty acid composition in RBO, including an increase in the content of oleic acid, which helps suppress lifestyle disease, would increase health benefits. The enzyme fatty acid desaturase 2 (FAD2) catalyzes the conversion of oleic acid to linoleic acid in plants, and FAD2 mutants exhibit altered oleic and linoleic acid content in many crops. There are three functional FAD2 genes in the genome of rice (Oryza sativa L.), and, of these, expression of the OsFAD2-1 gene is highest in rice seeds. In order to produce high oleic/low linoleic RBO, we attempted to disrupt the OsFAD2-1 gene by CRISPR/Cas9-mediated targeted mutagenesis. We succeeded in the production of homozygous OsFAD2-1 knockout rice plants. The content of oleic acid increased to more than twice that of wild type, and, surprisingly, linoleic acid, a catabolite of oleic acid by FAD2, decreased dramatically to undetectable levels in fad2-1 mutant brown rice seeds. In this study, by genome editing based on genome information, we succeeded in the production of rice whose fatty acid composition is greatly improved. We suggest that CRISPR/Cas9-mediated mutagenesis of a major gene that shows dominant expression in the target tissue could be a powerful tool to improve target traits in a tissue-specific manner.

Effects of nitrogen and phosphorous stress on the formation of high value LC-PUFAs in Porphyridium cruentum.

This study systematically examined the effect of nitrogen and phosphorous stress on the formation of linoleic acid (LA), arachidonic acid (ARA), and eicosapentaenoic acid (EPA) in Porphyridium cruentum gy-h56. P. cruentum was cultivated in six different media conferring different conditions of nitrogen (N) sufficiency/deprivation and phosphorous (P) sufficiency/limitation/deprivation. Over a 16-day cultivation process, the dry-weight content, proportion of total fatty acids (TFAs), and the concentration in the medium of linoleic acid (LA) were greatly improved by a maximum of 2.5-, 1.6-, and 1.1-fold, respectively, under conditions of N or P deprivation compared with N and P sufficiency. In contrast, levels of EPA or ARA were not enhanced under N or P stress conditions. Additionally, the results showed that N deprivation weakened the impact of P deficiency on the content and proportions of LA and EPA, while P deprivation enhanced the impact of N starvation on the content and proportions of LA and EPA. The conditions of N sufficiency and P deprivation (N+P-) were the optimal conditions for the production of LA, while the optimal conditions for EPA, ARA, and TFAs production were N sufficiency and P limitation (N+P-lim). This study suggests the potential application of combining N removal from saline wastewater with the production of LA, ARA, EPA, and biodiesel.

Potential of Laurencia obtusa as a substrate for the development of a probiotic Saccharomyces cerevisiae.

Laurencia obtusa (Ceramiales, Rhodophyta) has tremendous nutritional value, being high in proteins, oligosaccharides, vitamins, essential minerals, and fatty acids, and it is a rich source of amino acids and trace elements. In this study, L. obtusa was extracted and subjected to phenolic, sugar and flavonoid analyses.The fatty acid, vitamin and phytosterol contents in Saccharomyces cerevisiae were evaluated when it was incubated with L. obtusa dry biomass. The fatty acids in the lipid extract were analysed after converting them into methyl esters using gas chromatography, and vitamin concentrations were measured using high-performance liquid chromatography (HPLC). According to the achieved results, the total fatty acid levels and vitamin contents of the S. cerevisiae prepared with algal extract increased at different rates. Our results showed that α-tocopherol decreased in the group in which the S. cerevisiae was added the algal extract. When compared to the control group, ergesterol increased in the group in which L. obtusa extract was added. Additionally, when compared to the control group in which L. obtusa extract was added, stearic acid (18:0), oleic acid (18:1) and linoleic acid (18:2) increased in the other groups. Palmitoleic acid (16:1) increased in the L. obtusa culture medium, but palmitic acid decreased in the L. obtusa culture medium. In conclusion, it was determined that the L. obtusa extract added to the development medium of S. cerevisiae caused differences in the synthesis of some vitamins and fatty acids.

Biosynthetic pathway of aliphatic formates via a Baeyer-Villiger oxidation in mechanism present in astigmatid mites.

Astigmatid mites depend on bioactive glandular secretions, pheromones, and defensive agents to mediate intra- and interspecies interactions. Aliphatic formates, such as (Z,Z)-8,11-heptadecadienyl formate (8,11-F17) and (Z)-8-heptadecenyl formate (8-F17), are rarely encountered natural products that are abundant in Sancassania sp. Sasagawa (Acari: Acaridae) mite secretions. Linoleic acid and oleic acid are predicted as key intermediates in the synthesis of the closely related aliphatic formates. To gain insight in this biosynthetic pathway, acarid mite feeding experiments were conducted using 13C-labeled precursors to precisely track incorporation. Analyses using 13C NMR spectroscopy demonstrated that the 13C-labeling pattern of the precursors was detectable on formates in exocrine secretions and likewise on fatty acids in total lipid pools. Curiously, the results demonstrated that the formates were biosynthesized without the dehomologation of corresponding fatty acids. Careful examination of the mass spectra from labeling experiments revealed that the carbonyl carbon of the formates is originally derived from the C-1 position of the fatty acids. Consistent with a Baeyer-Villiger oxidation reaction, labeling studies support the insertion of an oxygen atom between the carbonyl group and carbon chain. Empirical data support the existence of a Baeyer-Villiger monooxygenase responsible for the catalyzation of the Baeyer-Villiger oxidation. The predicted existence of a Baeyer-Villiger monooxygenase capable of converting aliphatic aldehydes to formates represents an exciting opportunity to expand the enzymatic toolbox available for controlled biochemical synthesis.

Overexpression of Δ12-Fatty Acid Desaturase in the Oleaginous Yeast Rhodosporidium toruloides for Production of Linoleic Acid-Rich Lipids.

Under nutrient-limited conditions, the red yeast Rhodosporidium toruloides can accumulate neutral lipids, of which the compositional fatty acids are mainly saturated and mono-unsaturated ones with 16 or 18 carbon atoms. To improve the linoleic acid content in the lipids, we enabled galactose-inducible expression of the gene encoding Δ12-fatty acid desaturase (FADS) from Mortierella alpina or Fusarium verticillioides by integration of the corresponding expression cassettes into the genome of R. toruloides haploid and diploid strains. The relative linoleic acid content increased up to fivefold and the final linoleic acid titer reached 1.3 g/L under flask culture conditions. Our results suggested that R. toruloides may be further explored as cell factory for production of high-valued lipids and other fatty acid derivatives as bio-based chemicals and fuels.

Identification of a Δ12 fatty acid desaturase from oil palm (Elaeis guineensis Jacq.) involved in the biosynthesis of linoleic acid by heterologous expression in Saccharomyces cerevisiae.

Oil palm (Elaeis guineensis Jacq.) is one of the highest oil-yield crops in the world. A Δ12-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from oil palm and their functions identified. The open reading frames (ORFs) of egFAD2 (GenBank accession: KT023602) consisted of 1176bp and code for 391 amino acids. Their deduced polypeptides showed 75-93% identity to microsomal Δ12-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. RT-PCR experiment indicated that the egFAD2 gene exhibited the highest accumulation in the mesocarp of fruits at 120-140 DAP (i.e. the fourth period of fruit development) and, despite having different expression levels, the other four stages were at significantly lower levels compared with the fourth stage. Plasmid pYES2-egFAD2 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Yeast cells transformed with plasmid constructs containing egFAD12 produced an appreciable amount of linoleic acids (18:2(Δ9,)(12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ12-desaturase enzymes.

Rhodotorula glutinis-potential source of lipids, carotenoids, and enzymes for use in industries.

Rhodotorula glutinis is capable of synthesizing numerous valuable compounds with a wide industrial usage. Biomass of this yeast constitutes sources of microbiological oils, and the whole pool of fatty acids is dominated by oleic, linoleic, and palmitic acid. Due to its composition, the lipids may be useful as a source for the production of the so-called third-generation biodiesel. These yeasts are also capable of synthesizing carotenoids such as ß-carotene, torulene, and torularhodin. Due to their health-promoting characteristics, carotenoids are commonly used in the cosmetic, pharmaceutical, and food industries. They are also used as additives in fodders for livestock, fish, and crustaceans. A significant characteristic of R. glutinis is its capability to produce numerous enzymes, in particular, phenylalanine ammonia lyase (PAL). This enzyme is used in the food industry in the production of L-phenylalanine that constitutes the substrate for the synthesis of aspartame-a sweetener commonly used in the food industry.

Temperature effect on triacylglycerol species in seed oil from high stearic sunflower lines with different genetic backgrounds.

BACKGROUND: This study characterized the influence of temperature during grain filling on the saturated fatty acid distribution in triacylglycerol molecules from high stearic sunflower lines with different genetic backgrounds. Two growth chamber experiments were conducted with day/night temperatures of 16/16, 26/16, 26/26 and 32/26 °C. RESULTS: In all genotypes, independently of the genetic background, higher temperatures increased palmitic and oleic acid and reduced linoleic acid concentrations. Increasing night temperature produced an increase in saturated-unsaturated-saturated species, indicating a more symmetrical distribution of saturated fatty acids. The solid fat index was more affected by temperature during grain filling in lines with high linoleic than high oleic background. Higher variations in symmetry among night temperatures were observed in lines with high oleic background, which are more stable in fatty acid composition. CONCLUSION: The effect of temperature on triacylglycerol composition is not completely explained by its effect on fatty acid composition. Thus night temperature affects oil properties via its effects on fatty acid synthesis and on the distribution of fatty acids in the triacylglycerol molecules. © 2016 Society of Chemical Industry.

Simultaneous silencing of five lipoxygenase genes increases the contents of α-linolenic and linoleic acids in tomato (Solanum lycopersicum L.) fruits.

α-Linolenic and linoleic acids are essential fatty acids (EFAs) for humans and required for maintenance of optimal health, but they cannot be synthesized by the human body and must be obtained from dietary sources. Using TomloxC fragment, TomloxD fragment, and partial TomloxA sequence that is highly identical with TomloxB and TomloxE, a RNAi expression vector was constructed. The construct was used to transform tomato cotyledon explants with the Agrobacterium-mediated co-cultivation method. The real-time reverse transcription polymerase chain reaction analysis showed that the expression of TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE in transgenic tomato plants was drastically repressed, which led to a marked decrease in the levels of lipoxygenase activity. Finally, higher accumulations of the endogenous α-linolenic and linoleic acids were detected in the transgenic tomato fruits, which were 1.65-3.99 and 2.91-4.98 times that of the non-transformed tomato fruits, respectively.

Expression, purification and refolding of active durum wheat (Triticum durum Desf.) secretory phospholipase A2 from inclusion bodies of Escherichia coli.

Recently, a durum wheat (Triticum durum Desf.) secretory phospholipase A2 (TdsPLA2III) was identified in leaves as potentially involved in plant responses to conditions of limiting water supply. Therefore, to allow future functional studies on TdsPLA2III and shed further light on the involvement of sPLA2 isoforms in specific plant functions, here we report a protocol for the overexpression of TdsPLA2III in Escherichia coli in the form of inclusion bodies, and for its purification and refolding. The use of the Gateway system (Invitrogen) allows the expression of a large quantity of the mature form (without the signal peptide) of TdsPLA2III with an N-terminal 6×His-tag, for purification using Ni-affinity chromatography. The purified recombinant 6×His-TdsPLA2III fusion protein is then refolded using a step-wise dialysis approach. About 40mg purified and active protein was obtained from 1L of cell culture. This recombinant 6×His-TdsPLA2III protein shows PLA2 activity, as it can hydrolyze linoleate from the sn-2 position of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. Moreover, it has some features that are typical of other known plant sPLA2s: Ca(2+)-dependence, inhibition by the disulfide bond reducing agent dithiothreitol, and resistance to high temperature.

Oleic acid is a precursor of linoleic acid and the male sex pheromone in Nasonia vitripennis.

Linoleic acid (C18:2(Δ9,12), LA) is crucial for many cell functions in organisms. It has long been a paradigm that animals are unable to synthesize LA from oleic acid (C18:1(Δ9), OA) because they were thought to miss Δ(12)-desaturases for inserting a double bound at the Δ(12)-position. Today it is clear that this is not true for all animals because some insects and other invertebrates have been demonstrated to synthesize LA. However, the ability to synthesize LA is known in only five insect orders and no examples have been reported so far in the Hymenoptera. LA plays a particular role in the parasitic wasp Nasonia vitripennis, because it is the precursor of the male sex pheromone consisting of (4R,5R)- and (4R,5S)-5-hydroxy-4-decanolides. Here we demonstrate by stable isotope labeling that N. vitripennis is able to incorporate externally applied fully (13)C-labeled OA into the male sex pheromone suggesting that they convert initially OA into LA. To verify this assumption, we produced fly hosts (Lucilia caesar) which were experimentally enriched in (13)C-labeled OA and reared male parasitoids on these hosts. Chemical analysis of transesterified lipid raw extracts from hosts and parasitoids revealed that N. vitripennis but not L. caesar contained (13)C-labeled LA methyl ester. Furthermore, male wasps from the manipulated hosts produced significant amounts of (13)C-labeled sex pheromone. These results suggest that N. vitripennis possesses a Δ(12)-desaturase. The additional fitness relevant function as pheromone precursor might have favored the evolution of LA biosynthesis in N. vitripennis to make the wasps independent of the formerly essential nutrient.

Bioinformatics study of delta-12 fatty acid desaturase 2 (FAD2) gene in oilseeds.

Fatty acid desaturases constitute a group of enzymes that introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids. In plants, seed-specific delta-12 fatty acid desaturase 2 (FAD2) is responsible for the high content of linoleic acid by inserting a double bond at the delta-12 (omega-6) position of oleic acid. In this study, sixteen FAD2 and FAD2-2 protein sequences from oilseeds were analyzed by computational tools including two databases of the NCBI and EXPASY and data management tools such as SignalP, TMHMM, Psort, ProtParam, TargetP, PLACE and PlantCARE. These services were used to predict the protein properties such as molecular mass, pI, signal peptide, transmembrane and conserved domains, secondary and spatial structures. The polypeptide sequences were aligned and a neighbour-joining tree was constructed using MEGA5.1 to elucidate phylogenetic relationships among FAD2 genes. Based on the phylogenetic analysis species with high similarity in FAD2 sequence grouped together. FAD2 proteins include highly conserved histidine-rich motifs (HECGHH, HRRHH and HV[A/C/T]HH) that are located by three to five transmembrane anchors. For further investigations Sesamum indicum FAD2 was selected and analyzed by bioinformatics tools. Analysis showed no N-terminal signal peptide for probable localization of FAD2 protein in cytoplasmic organelles such as chloroplast, mitochondria and Golgi. Instead the C-terminal signaling motif YNNKL, Y(K/N)NKF or YRNKI allows FAD2 protein to selectively bind to and embed in the endoplasmic reticulum. FAD2 promoter contains different cis-regulatory elements involve in the biotic and abiotic stresses response or control of gene expression specifically in seeds.