Biosynthesis Of Linoleic Acid Research Articles

The Carthamus tinctorius L. genome sequence provides insights into synthesis of unsaturated fatty acids

Domesticated safflower (Carthamus tinctorius L.) is a widely cultivated edible oil crop. However, despite its economic importance, the genetic basis underlying key traits such as oil content, resistance to biotic and abiotic stresses, and flowering time remains poorly understood. Here, we present the genome assembly for C. tinctorius variety Jihong01, which was obtained by integrating Oxford Nanopore Technologies (ONT) and BGI-SEQ500 sequencing results. The assembled genome was 1,061.1 Mb, and consisted of 32,379 protein-coding genes, 97.71% of which were functionally annotated. Safflower had a recent whole genome duplication (WGD) event in evolution history and diverged from sunflower approximately 37.3 million years ago. Through comparative genomic analysis at five seed development stages, we unveiled the pivotal roles of fatty acid desaturase 2 (FAD2) and fatty acid desaturase 6 (FAD6) in linoleic acid (LA) biosynthesis. Similarly, the differential gene expression analysis further reinforced the significance of these genes in regulating LA accumulation. Moreover, our investigation of seed fatty acid composition at different seed developmental stages unveiled the crucial roles of FAD2 and FAD6 in LA biosynthesis. These findings offer important insights into enhancing breeding programs for the improvement of quality traits and provide reference resource for further research on the natural properties of safflower.

FgFAD12 Regulates Vegetative Growth, Pathogenicity and Linoleic Acid Biosynthesis in Fusarium graminearum.

Polyunsaturated fatty acids (PUFAs), as important components of lipids, play indispensable roles in the development of all organisms. ∆12 fatty acid desaturase (FAD12) is a speed-determining step in the biosynthesis of PUFAs. Here, we report the characterization of FAD12 in Fusarium graminearum, which is the prevalent agent of Fusarium head blight, a destructive plant disease worldwide. The results demonstrated that deletion of the FgFAD12 gene resulted in defects in vegetative growth, conidial germination and plant pathogenesis but not sexual reproduction. A fatty acid analysis further proved that the deletion of FgFAD12 restrained the reaction of oleic acid to linoleic acid, and a large amount of oleic acid was detected in the cells. Moreover, the ∆Fgfad12 mutant showed increased resistance to osmotic stress and reduced tolerance to oxidative stress. The expression of FgFAD12 did show a temperature-dependent manner, which was not affected at a low temperature of 10 °C when compared to 25 °C. RNA-seq analysis further demonstrated that most genes enriched in fatty acid metabolism, the biosynthesis of unsaturated fatty acids, fatty acid biosynthesis, fatty acid degradation, steroid biosynthesis and fatty acid elongation pathways were significantly up-regulated in the ∆Fgfad12 mutants. Overall, our results indicate that FgFAD12 is essential for linoleic acid biosynthesis and plays an important role in the infection process of F. graminearum.

Biological Characteristics and Functional Analysis of the Linoleic Acid Synthase Gene ZjFAD2 in Jujube.

Jujube fruit is rich in linoleic acid and other bioactive components and has great potential to be used for the development of functional foods. However, the roles of FAD2 genes in linoleic acid biosynthesis in jujube fruit remain unclear. Here, we identified 15 major components in jujube and found that linoleic acid was the main unsaturated fatty acid; major differences in the content and distribution of linoleic acid in the pulp and seeds were observed, and levels of linoleic acid decreased during fruit maturation. Analysis of the fatty acid metabolome, genome, and gene expression patterns of cultivated and wild-type jujube revealed five ZjFAD2 family members highly related to linoleic acid biosynthesis. The heterologous expression of these five ZjFAD2 family members in tobacco revealed that all five of these genes increased the content of linoleic acid. Additionally, transient expression of these genes in jujube fruit and the virus-induced gene silencing (VIGS) test further confirmed the key roles of ZjFAD2-11 and ZjFAD2-1 in the biosynthesis of linoleic acid. The results of this research provide valuable insights into the molecular mechanism underlying linoleic acid synthesis in jujube and will aid the development of quality-oriented breeding strategies.

Combined Transcriptomic and Metabolomic Approach Revealed a Relationship between Light Control, Photoprotective Pigments, and Lipid Biosynthesis in Olives.

Olive possesses excellent nutritional and economic values for its main healthy products. Among them, a high content of antioxidant compounds, balanced during the ripening process, are produced under genetic and environmental control, resulting in high variability among cultivars. The genes involved in these complex pathways are mainly known, but despite many studies which indicated the key role of light quality and quantity for the synthesis of many metabolites in plants, limited information on these topics is available in olive. We carried out a targeted gene expression profiling in three olive cultivars, Cellina di Nardò, Ruveia, and Salella, which were selected for their contrasting oleic acid and phenolic content. The -omics combined approach revealed a direct correlation between a higher expression of the main flavonoid genes and the high content of these metabolites in 'Cellina di Nardò'. Furthermore, it confirmed the key role of FAD2-2 in the linoleic acid biosynthesis. More interestingly, in all the comparisons, a co-regulation of genes involved in photoperception and circadian clock machinery suggests a key role of light in orchestrating the regulation of these pathways in olive. Therefore, the identified genes in our analyses might represent a useful tool to support olive breeding, although further investigations are needed.

Evolution of Linoleic Acid Biosynthesis Paved the Way for Ecological Success of Termites.

Termites are dominant animals of tropical terrestrial ecosystems. Their success is due to their eusocial organization as well as their ability to digest dead plant tissues. While being extremely abundant, the termite diet is poor in crucial nutrients, such as fatty acids. Linoleic acid (LA) is a precursor for many vital biomolecules, and most animals depend on its dietary supply. Termites count among the exceptions known to produce LA de novo, presumably via the action of an unknown Δ12 fatty acyl desaturase (FAD) introducing the second double bond into monounsaturated oleic acid. Here, we search for the evolutionary origin of LA biosynthesis in termites. To this end, we compile the repertoire of FAD homologs from 57 species of termites and their closest relatives, the cockroaches, analyze FAD phylogeny, and identify a potential Δ12 FAD branch, which arose through duplication of a likely Δ9 FAD. We functionally characterize both paralogs and identify the Δ9 activity in the ancestral FAD-A1a and the Δ12 activity responsible for LA biosynthesis in FAD-A1b. Through the combination of homology modeling and site-directed mutagenesis, we pinpoint structural features possibly contributing to the distinct functions, regiospecificities, and substrate preferences of the two enzymes. We confirm the presence of both paralogs in all 36 studied species of the Blattoidea lineage (Blattidae, Lamproblattidae, Cryptocercidae, and termites) and conclude that we identified an evolutionary event important for the ecological success of termites, which took place in their cockroach ancestors roughly 160 My and remained conserved throughout termite diversification into 3,000 extant species.

Identification of lncRNAs and Their Regulatory Network Involved in Oil Biosynthesis in Developing Seeds of Yellowhorn (Xanthoceras sorbifolium)

Yellowhorn (Xanthoceras sorbifolium) seeds can have as high as 67% oil content and are especially rich in oleic acid, linoleic acid, and nervonic acid. Exploration of the lipid biosynthesis regulatory network is essential for increasing the yellowhorn oil content. Long non-coding RNAs (lncRNAs) play important roles in various plant biological processes; however, there is no report on the identification of lncRNAs involved in yellowhorn seed development and lipid biosynthesis affecting oil production. We performed whole transcriptome sequencing of yellowhorn seeds at four developmental stages and identified 16,920 putative lncRNAs. Among them, 325 lncRNAs were revealed to trans-regulate 58 key genes in fatty acid (FA) and triacylglycerol (TAG) biosynthesis pathways. Of these, ECR-2–LNC_009778 was found to be involved in nervonic acid biosynthesis and DGAT-1–LNC_009778 was beneficial to TAG accumulation. sRNA-seq was performed, and 55 microRNAs (miRNAs) were found to target 26 genes involved in FA and TAG biosynthesis; miR396a-4 targets FAD2, affecting linoleic acid biosynthesis, and miR156f-5p targets PDAT-2, contributing to TAG accumulation. Interestingly, 30 lncRNA–miRNA–gene modules involved in FA and TAG biosynthesis were identified, in which the KCS11-1–miR156g-2–LNC_000849 module was found to participate in nervonic acid synthesis, and the DGAT-2–miR172j–LNC_005874 module was assumed to contribute to the accumulation of TAG. Our results constitute the first comprehensive identification of lncRNAs in developing seeds of yellowhorn and serve as a new theoretical reference for improving oil content in the future.

A high-throughput lipidomics and transcriptomic approach reveals novel compounds from sugarcane linked with promising therapeutic potential against COVID-19.

Sugarcane (Saccharum ssp., Poaceae) provides enormous metabolites such as sugars, lipid, and other dietary metabolites to humans. Among them, lipids are important metabolites that perform various functions and have promising pharmacological value. However, in sugarcane, few studies are focusing on lipidomics and few lipid compounds were reported, and their pharmacological values are not explored yet. The transcriptomic and widely targeted lipidomics approach quantified 134 lipid compounds from the rind of six sugarcane genotypes. These lipid compounds include 57 fatty acids, 30 lysophosphatidylcholines, 23 glycerol esters, 21 lysophosphatidylethanolamines, 2 phosphatidylcholines, and 1 sphingolipid. Among them, 119 compounds were first time reported in sugarcane rind. Seventeen lipids compounds including 12 fatty acids, 2 glycerol lipids, LysoPC 16:0, LysoPE 16:0, and choline alfoscerate were abundantly found in the rind of sugarcane genotypes. From metabolic and transcriptomic results, we have developed a comprehensive lipid metabolic pathway and highlighted key genes that are differentially expressed in sugarcane. Several genes associated with α-linolenic acid and linoleic acid biosynthesis pathways were highly expressed in the rind of the ROC22 genotype. ROC22 has a high level of α-linolenic acid (an essential fatty acid) followed by ROC16. Moreover, we have explored pharmacological values of lipid compounds and found that the 2-linoleoylglycerol and gingerglycolipid C have strong binding interactions with 3CLpro of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and these compounds can be utilized against SARS-CoV-2 as therapeutic agents. The transcriptome, metabolome, and bioinformatics analysis suggests that the sugarcane cultivars have a diversity of lipid compounds having promising therapeutic potential, and exploring the lipid metabolism will help to know more compounds that have promising cosmetic and pharmacological value.

Proteomics Insights into the Gene Network of cis9, trans11-Conjugated Linoleic Acid Biosynthesis in Bovine Mammary Gland Epithelial Cells.

Simple SummaryCis9, trans11-conjugated linoleic acid (c9, t11-CLA) is a bioactive compound that is mainly found in ruminant products and has a variety of beneficial biological functions in human health. However, the gene network of c9, t11-CLA biosynthesis in the mammary gland is still not well documented. Therefore, this study aimed to elucidate the stearoyl-coenzyme A desaturase (SCD1)-dependent gene network of c9, t11-CLA biosynthesis in MAC-T cells from the perspective of energy metabolism. The results showed that the deficiency of stearoyl-coenzyme A desaturase (SCD1), led by CAY10566, blocked the biosynthesis of c9, t11-CLA. In total, 60 SCD1-related proteins that were mainly involved in energy metabolism pathways were primary screened by Tandem mass tag-based quantitative proteomics analysis. Moreover, 17 proteins were validated under the parallel reaction monitoring analysis. Then, 11 genes involved in energy metabolism pathways were verified to have negative relationships with SCD1 after small RNA interference analysis. Based on the above results, we concluded that the 11 genes involved in energy metabolism pathways had an impact on the SCD1-dependent molecular mechanism of c9, t11-CLA biosynthesis. This study provided a fresh understanding of the gene network of c9, t11-CLA biosynthesis in the mammary gland from a metabolism perspective.The objective of the study was to elucidate the stearoyl-coenzyme A desaturase (SCD1)-dependent gene network of c9, t11-CLA biosynthesis in MAC-T cells from an energy metabolism perspective. The cells were divided into the CAY group (firstly incubated with CAY10566, a chemical inhibitor of SCD1, then incubated with trans-11-octadecenoic acid, (TVA)), the TVA group (only TVA), and the control group (without CAY, TVA). The c9, t11-CLA, and TVA contents were determined by gas chromatography. The mRNA levels of SCD1 and candidate genes were analyzed via real-time PCR. Tandem mass tag (TMT)-based quantitative proteomics, bioinformatic analysis, parallel reaction monitoring (PRM), and small RNA interference were used to explore genes involved in the SCD1-dependent c9, t11-CLA biosynthesis. The results showed that the SCD1 deficiency led by CAY10566 blocked the biosynthesis of c9, t11-CLA. In total, 60 SCD1-related proteins mainly involved in energy metabolism pathways were primarily screened by TMT-based quantitative proteomics analysis. Moreover, 17 proteins were validated using PRM analysis. Then, 11 genes were verified to have negative relationships with SCD1 after the small RNA interference analysis. Based on the above results, we concluded that genes involved in energy metabolism pathways have an impact on the SCD1-dependent molecular mechanism of c9, t11-CLA biosynthesis.

The Arginine Repressor ArgR2 Controls Conjugated Linoleic Acid Biosynthesis by Activating the cla Operon in Lactiplantibacillus plantarum.

ABSTRACTCLA (conjugated linoleic acid) has attracted substantial attention due to its physiological functions, including regulating immunity, reducing obesity, and contributing to cancer suppression. In Lactiplantibacillus plantarum, CLA oleate hydratase (CLA-HY), CLA short-chain dehydrogenase (CLA-DH), and CLA acetoacetate decarboxylase (CLA-DC) catalyze the biotransformation of linoleic acid (LA) to CLA. However, the underlying transcriptional regulation mechanism of this pathway remains largely unknown. In this study, the potential transcriptional regulators that might bind to the cla promoter of L. plantarum AR195 were investigated by DNA pulldown. Interestingly, ArgR2, the transcriptional regulator of arginine metabolism, was identified as a potential regulator involved in the regulation of CLA biotransformation. Electrophoretic mobility shift assay (EMSA) and molecular interaction results demonstrated the specific binding of ArgR2 to the regulatory region of the cla operon. The knockout of argR2 led to the downregulation of cla-dh and cla-dc by 91% and 34%, respectively, resulting in a decline in the CLA yield by 14%. A segmental EMSA revealed that ArgR2 bound to three distinct sites in the cla regulatory region, and these binding sites were highly conserved and rich in AT. The regulatory mechanism of ArgR2 on CLA biosynthesis further expanded our knowledge of the regulatory mechanism of CLA biosynthesis in L. plantarum and laid the theoretical foundation for the production and application of CLA.IMPORTANCE CLA (conjugated linoleic acid) has received extensive attention owing to its important physiological functions. CLA from natural sources is far from meeting people’s demands. Lactic acid bacteria can efficiently synthesize cis-9,trans-11-CLA and trans-10,cis-12-CLA, which possess physiological activities. However, little is known about the regulatory mechanism. In this study, we identified that the biosynthesis of CLA in L. plantarum AR195 was transcriptionally regulated by the arginine biosynthesis regulatory protein ArgR2. The regulation mechanism of ArgR2 on CLA biosynthesis lays a theoretical foundation for the regulation of CLA synthesis and industrial production.

Linum usitatissimum FAD2A and FAD3A enhance seed polyunsaturated fatty acid accumulation and seedling cold tolerance in Arabidopsis thaliana

Flax (Linum usitatissimum) seed oil is rich in polyunsaturated fatty acids (PUFAs), particularly linolenic acid, which is converted from linoleic acid. Studies have indicated that the biosynthesis of linoleic acid and linolenic acid is controlled by FAD2 and FAD3, respectively. However, the functional distinctions of different LuFAD2 and LuFAD3 copies from L. usitatissimum in governing the biosynthesis of linoleic acid or linolenic acid, respectively, remain unclear. In this study, five LuFAD2 and three LuFAD3 cDNAs were cloned from the L. usitatissimum cultivar ‘Longya 10’, and GC–MS results demonstrated that LuFAD2A and LuFAD3A play predominant roles in the accumulation of linoleic acid and linolenic acid, respectively. Their simultaneous overexpression in Arabidopsis thaliana seeds led to a significant increase in fatty acid contents, especially PUFAs. Additionally, LuFAD2A and LuFAD3A promoted the biosynthesis of jasmonic acid by increasing the levels of linolenic acid, which, in turn, enhanced plant cold tolerance. When the amount of linolenic acid is not sufficient, plants adapt to low temperature via the accumulation of anthocyanins. These findings provide insights into the higher accumulation of PUFAs in L. usitatissimum seeds, and provide potential targets for improving oil quality of other oil-producing crops through molecular manipulation.

Transcriptomic Analysis Reveals Key Genes Involved in Oil and Linoleic Acid Biosynthesis during Artemisia sphaerocephala Seed Development.

Artemisia sphaerocephala seeds are rich in polysaccharides and linoleic acid (C18:2), which have been widely used as traditional medicine and to improve food quality. The accumulation patterns and molecular regulatory mechanisms of polysaccharides during A. sphaerocephala seed development have been studied. However, the related research on seed oil and C18:2 remain unclear. For this study, A. sphaerocephala seeds at seven different development stages at 10, 20, 30, 40, 50, 60, and 70 days after flowering (designated as S1~S7), respectively, were employed as experimental samples, the accumulation patterns of oil and fatty acids (FA) and the underlying molecular regulatory mechanisms were analyzed. The results revealed that oil content increased from 10.1% to 20.0% in the early stages of seed development (S1~S2), and up to 32.0% in mature seeds, of which C18:2 accounted for 80.6% of the total FA. FA and triacylglycerol biosynthesis-related genes jointly involved in the rapid accumulation of oil in S1~S2. Weighted gene co-expression network analysis showed that transcription factors FUS3 and bHLH played a critical role in the seed oil biosynthesis. The perfect harmonization of the high expression of FAD2 with the extremely low expression of FAD3 regulated the accumulation of C18:2. This study uncovered the gene involved in oil biosynthesis and molecular regulatory mechanisms of high C18:2 accumulation in A. sphaerocephala seeds; thus, advancing research into unsaturated fatty acid metabolism in plants while generating valuable genetic resources for optimal C18:2 breeding.

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.

The Oleic/Linoleic Acid Ratio in Olive (Olea europaea L.) Fruit Mesocarp Is Mainly Controlled by OeFAD2-2 and OeFAD2-5 Genes Together With the Different Specificity of Extraplastidial Acyltransferase Enzymes.

Fatty acid composition of olive oil has an important effect on the oil quality to such an extent that oils with a high oleic and low linoleic acid contents are preferable from a nutritional and technological point of view. In the present work, we have first studied the diversity of the fatty acid composition in a set of eighty-nine olive cultivars from the Worldwide Olive Germplasm Bank of IFAPA Cordoba (WOGBC-IFAPA), and in a core collection (Core-36), which includes 28 olive cultivars from the previously mentioned set. Our results indicate that oleic and linoleic acid contents displayed the highest degree of variability of the different fatty acids present in the olive oil of the 89 cultivars under study. In addition, the independent study of the Core-36 revealed two olive cultivars, Klon-14 and Abou Kanani, with extremely low and high linoleic acid contents, respectively. Subsequently, these two cultivars were used to investigate the specific contribution of different fatty acid desaturases to the linoleic acid content of mesocarp tissue during olive fruit development and ripening. Fatty acid desaturase gene expression levels, together with lipid analysis, suggest that not only OeFAD2-2 and OeFAD2-5 but also the different specificities of extraplastidial acyltransferase enzymes are responsible for the variability of the oleic/linoleic acid ratio in olive cultivars. All this information allows for an advancement in the knowledge of the linoleic acid biosynthesis in different olive cultivars, which can impact olive breeding programs to improve olive oil quality.

Systematic assessment of oat β-glucan catabolism during in vitro digestion and fermentation

β-Glucan as a component of grain cell walls is consumed daily. However, little is known about whether β-glucan is influenced by the gastrointestinal environment. In this study, we aim to investigate the integrated metabolic process of cereal β-glucan. In vitro simulated digestion and fermentation combined with microbiome and metabolome analysis were used to profile the metabolism of β-glucan. Intriguingly, we found that β-glucan was not hydrolyzed by digestive enzymes but partially degraded by gastric acid environment during in vitro digestion. Moreover, β-glucan was utilized by gut microbiota to produce acetate, propionate and butyrate, concurrently, the relative abundance of Lactobacillus significantly increased and Escherichia-Shigella significantly decreased. The correlation analysis between metabolomics datasets and microorganisms revealed that β-glucan catabolism was also accompanied by amino acid catabolism and linoleic acid biosynthesis. Our study offered a forceful basis for the further exploration of the role of β-glucan and gut microbiota in host health.

Association Study of the 5'UTR Intron of the FAD2-2 Gene With Oleic and Linoleic Acid Content in Olea europaea L.

Cultivated olive (Olea europaea L. subsp. europaea var. europaea) is the most ancient and spread tree crop in the Mediterranean basin. An important quality trait for the extra virgin olive oil is the fatty acid composition. In particular, a high content of oleic acid and low of linoleic, linolenic, and palmitic acid is considered very relevant in the health properties of the olive oil. The oleate desaturase enzyme encoding-gene (FAD2-2) is the main responsible for the linoleic acid content in the olive fruit mesocarp and, therefore, in the olive oil revealing to be the most important candidate gene for the linoleic acid biosynthesis. In this study, an in silico and structural analysis of the 5′UTR intron of the FAD2-2 gene was conducted with the aim to explore the natural sequence variability and its role in the gene expression regulation. In order to identify functional allele variants, the 5′UTR intron was isolated and partially sequenced in 97 olive cultivars. The sequence analysis allowed to find a 117-bp insertion including two long duplications never found before in FAD2-2 genes in olive and the existence of many intron-mediated enhancement (IME) elements. The sequence polymorphism analysis led to detect 39 SNPs. The candidate gene association study conducted for oleic and linoleic acids content revealed seven SNPs and one indel significantly associated able to explain a phenotypic variation ranging from 7% to 16% among the years. Our study highlighted new structural variants within the FAD2-2 gene in olive, putatively involved in the regulation mechanisms of gene expression associated with the variation of the content of oleic and linoleic acid.

Cloning and functional analysis of the FAD2 gene family from desert shrub Artemisia sphaerocephala

BackgroundLinoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes. Microsomal delta-12 (Δ12) oleate desaturase (FAD2) is a key enzyme for linoleic acid biosynthesis. Desert shrub Artemisia sphaerocephala is rich in linoleic acid, it has a large FAD2 gene family with twenty-six members. The aim of this work is to unveil the difference and potentially functionality of AsFAD2 family members.ResultsFull-length cDNAs of twenty-one AsFAD2 genes were obtained from A. sphaerocephala. The putative polypeptides encoded by AsFAD2 family genes showed a high level of sequence similarity and were relatively conserved during evolution. The motif composition was also relatively conservative. Quantitative real-time PCR analysis revealed that the AsFAD2–1 gene was strongly expressed in developing seeds, which may be closely associated with the high accumulating ability of linoleic acid in A. sphaerocephala seeds. Although different AsFAD2 family members showed diverse response to salt stress, the overall mRNA levels of the AsFAD2 family genes was stable. Transient expression of AsFAD2 genes in the Nicotiana benthamiana leaves revealed that the encoded proteins were all located in the endoplasmic reticulum. Heterologous expression in Saccharomyces cerevisiae suggested that only three AsFAD2 enzymes, AsFAD2–1, − 10, and − 23, were Δ12 oleate desaturases, which could convert oleic acid to linoleic acid, whereas AsFAD2–1 and AsFAD2–10 could also produce palmitolinoleic acid.ConclusionsThis research reported the cloning, expression studies, subcellular localization and functional identification of the large AsFAD2 gene family. These results should be helpful in understanding fatty acid biosynthesis in A. sphaerocephala, and has the potential to be applied in the study of plant fatty acids traits.

Oxidative Stress Response of Aspergillus oryzae Induced by Hydrogen Peroxide and Menadione Sodium Bisulfite.

Oxidative stress response protects organisms from deleterious effects of reactive oxygen species (ROS), which can damage cellular components and cause disturbance of the cellular homeostasis. Although the defensive biochemical mechanisms have been extensively studied in yeast and other filamentous fungi, little information is available about Aspergillus oryzae. We investigated the effect of two oxidant agents (menadione sodium bisulfite, MSB, and hydrogen peroxide, H2O2) on cellular growth and antioxidant enzyme induction in A. oryzae. Results indicated severe inhibition of biomass and conidia production when high concentration of oxidants was used. Transcriptomic analysis showed an up-regulated expression of genes involved in oxidoreduction, such as catalase, glutathione peroxidase, and superoxide dismutase. In addition, it was observed that oxidative stress stimuli enhanced the expression of Yap1 and Skn7 transcription factors. Further, metabolomic analysis showed that glutathione content was increased in the oxidative treatments when compared with the control. Moreover, the content of unsaturated fatty acid decreased with oxidative treatment accompanying with the down-regulated expression of genes involved in linoleic acid biosynthesis. This study provided a global transcriptome characterization of oxidative stress response in A. oryzae, and can offer multiple target genes for oxidative tolerance improvement via genetic engineering.

Functional characterisation of two Δ12-desaturases demonstrates targeted production of linoleic acid as pheromone precursor in Nasonia.

Insect pheromones are often derived from fatty acid metabolism. Fatty acid desaturases, enzymes introducing double bonds into fatty acids, are crucial for the biosynthesis of these chemical signals. Δ12-desaturases catalyse the biosynthesis of linoleic acid by introducing a second double bond into oleic acid, but have been identified in only a few animal species. Here, we report the functional characterisation of two Δ12-desaturases, Nvit_D12a and Nvit_D12b, from the parasitic wasp Nasonia vitripennis. We demonstrate that Nvit_D12a is expressed in the rectal vesicle of males where they produce a linoleic acid-derived sex pheromone to attract virgin females. 13C-labelling experiments with Urolepis rufipes, a closely related species belonging to the 'Nasonia group', revealed that females, but not males, are able to synthesise linoleic acid. U. rufipes males produce an isoprenoid sex pheromone in the same gland and do not depend on linoleic acid for pheromone production. This suggests that Δ12-desaturases are common in the 'Nasonia group', but acquired a specialised function in chemical communication of those species that use linoleic acid as a pheromone precursor. Phylogenetic analysis suggests that insect Δ12-desaturases have evolved repeatedly from Δ9-desaturases in different insect taxa. Hence, insects have developed a way to produce linoleic acid independent of the omega desaturase subfamily which harbours all of the eukaryotic Δ12-desaturases known so far.

Characterization and functional analysis of a plastidial FAD6 gene and its promoter in the mesocarp of oil palm (Elaeis guineensis)

Abstract Oil palm (Elaeis guineensis Jacq.) is one of the most important oil crops in the tropics with special fatty acid compositions. In this paper, a cDNA sequence putatively encoding the plastidial ω6 fatty acid desaturase (FAD) which plays a key role in the linoleic acid biosynthesis, designated EgFAD6, was isolated from the mesocarp of oil palm. Sequence analysis indicated that it had an open reading frame (ORF) of 1320 base pairs (bp) encoding 349 amino acids (aa) of 51 KDa. The real-time fluorescent quantitative PCR results indicated that EgFAD6 mRNA exhibited an extremely high expression level in the mature leaves, and it showed quite flat during the mesocarp ripening with a slight increase at the phase 2. The heterologous expression of EgFAD6 in yeast accumulated a considerable amount of linoleic acid, while no significant difference in other fatty acids when compared with the control pYES2. Furthermore, the promoter of EgFAD6 (proEgFAD6) with 1474 bp in length was isolated and used for functional analysis. The bioinformatics analysis indicated proEgFAD6 contains several potential cis-acting elements, such as light and hormone-responsive elements. Expression analysis in transgenic Arabidopsis revealed that proEgFAD6 could drive β-glucuronidase (GUS) reporter gene express in the leaf, silique coat, stem and flower. These results revealed the function of EgFAD6 and the feature of its promoter, which will be useful for understanding the function and regulation mechanism of plastidial linoleic acid synthesis in other tropical palm plants.

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 (18:2Δ9,12), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ12-desaturase enzymes.