Fatty acid desaturase enzymes perform dehydrogenation reactions leading to the insertion of double bonds in fatty acids, and are divided into soluble and integral membrane classes. Crystal structures of soluble desaturases are available; however, membrane desaturases have defied decades of efforts due largely to the difficulty of generating recombinant desaturase proteins for crystallographic analysis. Mortierella alpina is an oleaginous fungus which possesses eight membrane desaturases involved in the synthesis of saturated, monounsaturated and polyunsaturated fatty acids. Here, we describe the successful expression, purification and enzymatic assay of three M. alpina desaturases (FADS15, FADS12, and FADS9-I). Estimated yields of desaturases with purity >95% are approximately 3.5% (Ca. 4.6 mg/L of culture) for FADS15, 2.3% (Ca. 2.5 mg/L of culture) for FADS12 and 10.7% (Ca. 37.5 mg/L of culture) for FADS9-I. Successful expression of high amounts of recombinant proteins represents a critical step towards the structural elucidation of membrane fatty acid desaturases.

Fatty acid desaturases are enzymes that introduce double bonds into the hydrocarbon chains of fatty acids. The fatty acid desaturases from 37 cyanobacterial genomes were identified and classified based upon their conserved histidine-rich motifs and phylogenetic analysis, which help to determine the amounts and distributions of desaturases in cyanobacterial species. The filamentous or N(2)-fixing cyanobacteria usually possess more types of fatty acid desaturases than that of unicellular species. The pathway of acyl-lipid desaturation for unicellular marine cyanobacteria Synechococcus and Prochlorococcus differs from that of other cyanobacteria, indicating different phylogenetic histories of the two genera from other cyanobacteria isolated from freshwater, soil, or symbiont. Strain Gloeobacter violaceus PCC 7421 was isolated from calcareous rock and lacks thylakoid membranes. The types and amounts of desaturases of this strain are distinct to those of other cyanobacteria, reflecting the earliest divergence of it from the cyanobacterial line. Three thermophilic unicellular strains, Thermosynechococcus elongatus BP-1 and two Synechococcus Yellowstone species, lack highly unsaturated fatty acids in lipids and contain only one Delta9 desaturase in contrast with mesophilic strains, which is probably due to their thermic habitats. Thus, the amounts and types of fatty acid desaturases are various among different cyanobacterial species, which may result from the adaption to environments in evolution.

A detailed analysis of the trypanosomatids' genome projects revealed the presence of genes predicted to encode fatty-acid desaturases of the methyl-end type (MED). After cloning and functional characterization of all identified genes, it can be concluded that Trypanosoma cruzi contains two MEDs with oleate desaturase (OD) activities whereas Leishmania major contains one OD and two active linoleate desaturases (LD). All characterized ODs are highly specific for oleate (18:1Δ9) as substrate, presenting a ν+3 regioselectivity, although palmitoleate (16:1Δ9) can be desaturated as well, but to a lesser extent. L. major LD appears to use exclusively linoleate (18:2n-6), converting it into α-linolenate (18:3n-3). This strong specificity assures no further conversion of polyunsaturated fatty acids (PUFAs) of the n-6 series into the n-3 series, downstream in the PUFA biosynthesis pathway. This characterization completes the identification of all enzymes involved in PUFA biosynthesis in a parasitic protist. Differently from their Trypanosoma brucei orthologue, T. cruzi and L. major ODs were more active when expressed either, in the presence of trienoic fatty acids or at higher temperatures. This could be evidence for a differential post-translational regulation of these enzymes as a result of direct sensing of environmentally dependent parameters such as membrane fluidity.

ω-3 fatty acid desaturase is a key enzyme for the biosynthesis of ω-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three ω-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new ω-3 desaturases share 55 % identity at the amino acid level with the known Δ-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional Δ-12/Δ-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the ω-6 arachidonic acid to the ω-3 eicosapentanoic acid, with a substrate conversion efficiency of 54-65 %. These enzymes have a broad ω-6 fatty acid substrate spectrum, including both C18 and C20 ω-6 fatty acids although they prefer the C20 substrates, and have strong Δ-17 desaturase activity but weaker Δ-15 desaturase activity. Thus, they belong to the Δ-17 desaturase class. Unlike the previously identified bifunctional Δ-12/Δ-15 desaturase from F. monoliforme, they lack Δ-12 desaturase activity. The newly identified Δ-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these Δ-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce ω-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.

The diatom Phaeodactylum is rich in very long chain polyunsaturated fatty acids (PUFAs). Fatty acid (FA) synthesis, elongation, and desaturation have been studied in depth in plants including Arabidopsis, but for secondary endosymbionts the full picture remains unclear. FAs are synthesized up to a chain length of 18 carbons inside chloroplasts, where they can be incorporated into glycerolipids. They are also exported to the ER for phospho- and betaine lipid syntheses. Elongation of FAs up to 22 carbons occurs in the ER. PUFAs can be reimported into plastids to serve as precursors for glycerolipids. In both organelles, FA desaturases are present, introducing double bonds between carbon atoms and giving rise to a variety of molecular species. In addition to the four desaturases characterized in Phaeodactylum (FAD2, FAD6, PtD5, PtD6), we identified eight putative desaturase genes. Combining subcellular localization predictions and comparisons with desaturases from other organisms like Arabidopsis, we propose a scheme at the whole cell level, including features that are likely specific to secondary endosymbionts.

Membrane fatty acid desaturases are a diverse superfamily of enzymes that catalyze the introduction of double bonds into fatty acids. They are essential in a range of metabolic processes, such as the production of omega-3 fatty acids. However, our structure-function understanding of this superfamily is still developing and their range of activities and substrate specificities are broad, and often overlapping, which has made their systematic characterization challenging. A central issue with characterizing these proteins has been the lack of a structural model, which has been overcome with the recent publication of the crystal structures of two mammalian fatty acid desaturases. In this work, we have used sequence similarity networks to investigate the similarity among over 5000 related membrane fatty acid desaturase sequences, leading to a detailed classification of the superfamily, families and subfamilies with regard to their function and substrate head-group specificity. This work will facilitate rapid prediction of the function and specificity of new and existing sequences, as well as forming a basis for future efforts to manipulate the substrate specificity of these proteins for biotechnology applications.

FADS genes encode fatty acid desaturases that are important for the conversion of short chain polyunsaturated fatty acids (PUFAs) to long chain fatty acids. Prior studies indicate that the FADS genes have been subjected to strong positive selection in Africa, South Asia, Greenland, and Europe. By comparing FADS sequencing data from present-day and Bronze Age (5-3k years ago) Europeans, we identify possible targets of selection in the European population, which suggest that selection has targeted different alleles in the FADS genes in Europe than it has in South Asia or Greenland. The alleles showing the strongest changes in allele frequency since the Bronze Age show associations with expression changes and multiple lipid-related phenotypes. Furthermore, the selected alleles are associated with a decrease in linoleic acid and an increase in arachidonic and eicosapentaenoic acids among Europeans; this is an opposite effect of that observed for selected alleles in Inuit from Greenland. We show that multiple SNPs in the region affect expression levels and PUFA synthesis. Additionally, we find evidence for a gene-environment interaction influencing low-density lipoprotein (LDL) levels between alleles affecting PUFA synthesis and PUFA dietary intake: carriers of the derived allele display lower LDL cholesterol levels with a higher intake of PUFAs. We hypothesize that the selective patterns observed in Europeans were driven by a change in dietary composition of fatty acids following the transition to agriculture, resulting in a lower intake of arachidonic acid and eicosapentaenoic acid, but a higher intake of linoleic acid and α-linolenic acid.

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most severe and devastating diseases in viticulture. Unravelling the grapevine defence mechanisms is crucial to develop sustainable disease control measures. Here we provide new insights concerning fatty acid's (FA) desaturation, a fundamental process in lipid remodelling and signalling. Previously, we have provided evidence that lipid signalling is essential in the establishment of the incompatible interaction between grapevine and Plasmopara viticola. In the first hours after pathogen challenge, jasmonic acid (JA) accumulation, activation of its biosynthetic pathway and an accumulation of its precursor, the polyunsaturated α-linolenic acid (C18:3), were observed in the leaves of the tolerant genotype, Regent. This work was aimed at a better comprehension of the desaturation processes occurring after inoculation. We characterised, for the first time in Vitis vinifera, the gene family of the FA desaturases and evaluated their involvement in Regent response to Plasmopara viticola. Upon pathogen challenge, an up-regulation of the expression of plastidial FA desaturases genes was observed, resulting in a higher content of polyunsaturated fatty acids (PUFAs) of chloroplast lipids. This study highlights FA desaturases as key players in membrane remodelling and signalling in grapevine defence towards biotrophic pathogens.

Omega-3 long chain polyunsaturated fatty acids (ω3 LC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5ω3) and docosahexaenoic acid (DHA; 22:6ω3) are important fatty acids for human health. These ω3 LC-PUFAs are produced from their ω3 precursors by a set of desaturases and elongases involved in the biosynthesis pathway and are also converted from ω6 LC-PUFA by omega-3 desaturases (ω3Ds). Here, we have investigated eight ω3-desaturases obtained from a cyanobacterium, plants, fungi and a lower animal species for their activities and compared their specificities for various C18, C20 and C22 ω6 PUFA substrates by transiently expressing them in Nicotiana benthamiana leaves. Our results showed hitherto unreported activity of many of the ω3Ds on ω6 LC-PUFA substrates leading to their conversion to ω3 LC-PUFAs. This discovery could be important in the engineering of EPA and DHA in heterologous hosts.

Very long-chain polyunsaturated fatty acids (VLC-PUFAs) are important dietary requirements for maintaining human health. Many marine microalgae are naturally high in ω-3 VLC-PUFAs, however, the molecular mechanisms underpinning fatty acid (FA) desaturation and elongation in algae are poorly understood. An advanced molecular understanding would facilitate improvements of this nascent industry. We aimed to investigate expression responses of four front-end fatty acid desaturase genes and downstream effects on FA profiles to nitrogen limitation and cultivation growth stage in Isochrysis aff. galbana (TISO). Cultures were grown in nitrogen-replete and -deplete medium; samples were harvested during logarithmic, late logarithmic and stationary growth phases to analyse FA content/composition and gene expression of ∆(6)-, ∆(8)-, ∆(5)- and ∆(4)-desaturases (d6FAD (putative), d8FAD, d5FAD and d4FAD, respectively). d6FAD (putative) exhibited no differential expression, while d8FAD, d5FAD and d4FAD were significantly upregulated during logarithmic growth of nutrient-replete cultures, coinciding with rapid cell division. In conclusion, it is demonstrated that expression of some FADs in I. aff. galbana varies with culture age and nitrogen status which has downstream consequences on FA desaturation levels. This has implications for the commercial production of VLC-PUFAs where a trade-off between total lipid yield and VLC-PUFAs has to be made.

Frequent gene duplications in the genome incessantly supply new genetic materials for functional innovation presumably driven by positive Darwinian selection. This mechanism in the desaturase gene family has been proposed to be important in triggering the pheromonal diversification in insects. With the recent completion of a dozen Drosophila genomes, a genome-wide perspective is possible. In this study, we first identified homologs of desaturase genes in 12 Drosophila species and noted that while gene duplication events are relatively frequent, gene losses are not scarce, especially in the desat1-desat2-desatF clade. By reconciling the gene tree with species phylogeny and the chromosomal synteny of the sequenced Drosophila genomes, at least one gene loss in desat2 and a minimum of six gene gains (resulting in seven desatF homologs, alpha-eta), three gene losses and one relocation in desatF were inferred. Upon branching off the ancestral desat1 lineage, both desat2 and desatF gained novel functions through accelerating protein evolution. The amino acid residues under positive selection located near the catalytic sites and the C-terminal region might be responsible for altered substrate selectivity between closely related species. The association between the expression pattern of desatF-alpha and the chemical composition of cuticular hydrocarbons implies that the ancestral function of desatF-alpha is the second desaturation at the four carbons after the first double bond in diene synthesis, and the shift from bisexual to female-specific expression in desatF-alpha occurred in the ancestral lineage of Drosophila melanogaster subgroup. A relationship between the number of expressed desatF homologs and the diene diversification has also been observed. These results suggest that the molecular diversification of fatty acid desaturases after recurrent gene duplication plays an important role in pheromonal diversity in Drosophila.

Plant fatty acid desaturases (FADs) catalyze the desaturation of fatty acids in various forms and play important roles in regulating fatty acid composition and maintaining membrane fluidity under temperature stress. A total of 30 FADs were identified from a maize genome, including 13 soluble and 17 membrane-bound FADs, which were further classified into two and five sub-groups, respectively, via phylogenetic analysis. Although there is no evolutionary relationship between the soluble and the membrane-bound FADs, they all harbor a highly conserved FA_desaturase domain, and the types and the distributions of conserved motifs are similar within each sub-group. The transcriptome analysis revealed that genes encoding FADs exhibited different expression profiles under cold and heat stresses. The expression of ZmFAD2.1&2.2, ZmFAD7, and ZmSLD1&3 were significantly up-regulated under cold stress; moreover, the expression of ZmFAD2.1&2.3 and ZmSLD1&3 were obviously down-regulated under heat stress. The co-expression analysis demonstrated close correlation among the transcription factors and the significant responsive FAD genes under cold or heat stress. This study helps to understand the roles of plant FADs in temperature stress responses.

For vertebrates, the adequate supply of polyunsaturated fatty acids (PUFA) by the diet, in particular ω3 long-chain PUFA, is considered essential for neural development, growth and reproduction. In contrast to aquatic ecosystems, ω3 long-chain PUFA apparently are not widely available in the terrestrial food chain. Their de novo synthesis requires the presence of Δ12 and ω3 fatty acid desaturase enzymes, which are absent in vertebrates but present, for example, in the nematode Caenorhabditis elegans (FAT-2 and FAT-1). This raises the question if soil-dwelling nematodes offer substantial supply of these valuable nutritional compounds in terrestrial food webs. BLAST searches in available nematode genomes revealed the existence of fat-2 like genes in almost all clade III-V species, but failed to identify orthologs in clade I-II nematodes. An additional RT-PCR screen across soil-dwelling nematode species identified six novel fat-2 like genes. Hints for the genetic basis of a ω3 (fat-1) desaturase activity was found only in selected clade IV-V species, but not in clades I to III nematodes. Fatty acid pattern analyses following a PUFA-free cultivation and enzymatic characterization of six selected fat-2 or fat-1 like desaturases in yeast confirmed the findings from the genetic approaches. Thus, in similar soil habitats, taxa exist that can synthesize ω3 long-chain PUFA (as Panagrolaimus, Mesorhabditis and Caenorhabditis) whereas others are unable to do so (Acrobeloides, Cephalobus and Oscheius). While these nematodes do not differ in trophic position or major diet, distinction in reproduction mode may have led to the observed variations in desaturase genes.

Demand for Cocoa butter is steadily increasing, but the supply of cocoa beans is naturally limited and under threat from global warming. One route to meeting the future demand for cocoa butter equivalent (CBE) could be to utilize microbial cell factories such as the oleaginous yeast Yarrowia lipolytica.

To explore the contributions of ω-3 fatty acid desaturases (FADs) to cold stress response in a special cryophyte, Chorispora bungeana, two plastidial ω-3 desaturase genes (CbFAD7, CbFAD8) were cloned and verified in an Arabidopsis fad7fad8 mutant, before being compared with the microsomal ω-3 desaturase gene (CbFAD3). Though these genes were expressed in all tested tissues of C. bungeana, CbFAD7 and CbFAD8 have the highest expression in leaves, while CbFAD3 was mostly expressed in suspension-cultured cells. Low temperatures resulted in significant increases in trienoic fatty acids (TAs), corresponding to the cooperation of CbFAD3 and CbFAD8 in cultured cells, and the coordination of CbFAD7 and CbFAD8 in leaves. Furthermore, the cold induction of CbFAD8 in the two systems were increased with decreasing temperature and independently contributed to TAs accumulation at subfreezing temperature. A series of experiments revealed that jasmonie acid and brassinosteroids participated in the cold-responsive expression of ω-3 CbFAD genes in both C. bungeana cells and leaves, while the phytohormone regulation in leaves was complex with the participation of abscisic acid and gibberellin. These results point to the hormone-regulated non-redundant contributions of ω-3 CbFADs to maintain appropriate level of TAs under low temperatures, which help C. bungeana survive in cold environments.

The green microalga Chromochloris zofingiensis is capable of producing high levels of triacylglycerol rich in C18 unsaturated fatty acids (UFAs). FA desaturation degree is regulated by FA desaturases (FADs). Nevertheless, it remains largely unknown regarding what FADs are involved in FA desaturations and how these FADs collaborate to contribute to the high abundance of C18 UFAs in triacylglycerol in C. zofingiensis.

Two fatty acid desaturase genes have been cloned: HpFAD2 and HpFAD3 encode Hansenula polymorpha Δ12-fatty acid desaturase (HpFad2) and Δ15-fatty acid desaturase (HpFad3), which are responsible for the production of linoleic acid (LA, C18:2, Δ9, Δ12) and α-linolenic acid (ALA, αC18:3, Δ9, Δ12, Δ15), respectively. The open reading frame of the HpFAD2 and HpFAD3 genes is 1215bp and 1239bp, encoding 405 and 413 amino acids, respectively. The putative amino acid sequences of HpFad2 and HpFad3 share more than 60% similarity and three conserved histidine-box motifs with other known yeast Fad homologs. Hpfad2Δ disruptant cannot produce C18:2 and αC18:3, while the deletion of HpFAD3 only causes the absence of αC18:3. Heterologous expression of either the HpFAD2 or the HpFAD3 gene in Saccharomyces cerevisiae resulted in the presence of C18:2 and αC18:3 when the C18:2 precursor was added. Taken together, these observations indicate that HpFAD2 and HpFAD3 indeed encode Δ12- and Δ15-fatty acid desaturases that function as the only ones responsible for desaturation of oleic acid (C18:1) and linoleic acid (C18:2), respectively, in H. polymorpha. Because a Fatty Acid Regulated (FAR) region and a Low Oxygen Response Element (LORE), which are responsible for regulation of a Δ9-fatty acid desaturase gene (ScOLE1) in S. cerevisiae, are present in the upstream regions of both genes, we investigated whether the transcriptional levels of HpFAD2 and HpFAD3 are affected by supplementation with nutrient unsaturated fatty acids or by low oxygen conditions. Whereas both genes were up-regulated under low oxygen conditions, only HpFAD3 transcription was repressed by an excess of C18:1, C18:2 and C18:3, while the HpFAD2 transcript level did not significantly change. These observations indicate that HpFAD2 expression is not controlled at the transcriptional level by fatty acids even though it contains a FAR-like region. This study indicates that HpFAD2 may be regulated by post-transcriptional mechanisms, whereas HpFAD3 may be mainly controlled at a transcriptional level.

Arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the most biologically active polyunsaturated fatty acids, but their biosyntheses in mammals are very limited. The biosynthesis of DHA is the most difficult, because this undergoes the Sprecher pathway--a further elongation step from docosapentaenoic acid (DPA), a Δ6-desaturase acting on a C24 fatty acid substrate followed by a peroxisomal chain shortening step. This paper reports the successful heterologous expression of two non-mammalian genes (with modification of codon usage), coding for Euglena gracilis Δ4-desaturase and Siganus canaliculatus Δ4-desaturase respectively, in mammalian cells (HEK293 cell line). Both of the Δ4-desaturases can efficiently function, directly converting DPA into DHA. Moreover, the cooperation of the E. gracilis Δ4-desaturase with C. elegans Δ15-desaturase (able to convert a number of n-6 PUFAs to their corresponding n-3 PUFAs) in transgenic HEK293 cells made a more desirable fatty acid composition--a drastically reduced n-6/n-3 PUFAs ratio and a high level of DHA as well as EPA and ARA. Our findings provide a basis for potential applications of the gene constructs for expression of Δ15/Δ4-desaturases in transgenic livestock to produce such a fatty acid profile in the related products, which certainly will bring benefit to human health.

Rationale: Malignant ascites in peritoneal metastases is a lipid-enriched microenvironment and is frequently involved in the poor prognosis of epithelial ovarian cancer (EOC). However, the detailed mechanisms underlying ovarian cancer (OvCa) cells dictating their lipid metabolic activities in promoting tumor progression remain elusive. Methods: The omental conditioned medium (OCM) was established to imitate the omental or ascites microenvironment. Mass spectrometry, RT-qPCR, IHC, and western blot assays were applied to evaluate human fatty acid desaturases expressions and activities. Pharmaceutical inhibition and genetic ablation of SCD1/FADS2 were performed to observe the oncogenic capacities. RNA sequencing, lipid peroxidation, cellular iron, ROS, and Mito-Stress assays were applied to examine ferroptosis. OvCa patient-derived organoid and mouse model of peritoneal metastases were used to evaluate the combined effect of SCD1/FADS2 inhibitors with cisplatin. Results: We found that two critical fatty acid desaturases, stearoyl-CoA desaturase-1 (SCD1) and acyl-CoA 6-desaturase (FADS2), were aberrantly upregulated, accelerating lipid metabolic activities and tumor aggressiveness of ascites-derived OvCa cells. Lipidomic analysis revealed that the elevation of unsaturated fatty acids (UFAs) was positively associated with SCD1/FADS2 levels and the oncogenic capacities of OvCa cells. In contrast, pharmaceutical inhibition and genetic ablation of SCD1/FADS2 retarded tumor growth, cancer stem cell (CSC) formation and reduced platinum resistance. Inhibition of SCD1/FADS2 directly downregulated GPX4 and the GSH/GSSG ratio, causing disruption of the cellular/mitochondrial redox balance and subsequently, iron-mediated lipid peroxidation and mitochondrial dysfunction in ascites-derived OvCa cells. Conclusions: Combinational treatment with SCD1/FADS2 inhibitors and cisplatin synergistically repressed tumor cell dissemination, providing a promising chemotherapeutic strategy against EOC peritoneal metastases.

Recent advances in targeting leukemic stem cells (LSCs) using venetoclax with azacitidine (ven + aza) has significantly improved outcomes for de novo acute myeloid leukemia (AML) patients. However, patients who relapse after traditional chemotherapy are often venetoclax-resistant and exhibit poor clinical outcomes. We previously described that fatty acid metabolism drives oxidative phosphorylation (OXPHOS) and acts as a mechanism of LSC survival in relapsed/refractory AML. Here, we report that chemotherapy-relapsed primary AML displays aberrant fatty acid and lipid metabolism, as well as increased fatty acid desaturation through the activity of fatty acid desaturases 1 and 2, and that fatty acid desaturases function as a mechanism of recycling NAD+ to drive relapsed LSC survival. When combined with ven + aza, the genetic and pharmacologic inhibition of fatty acid desaturation results in decreased primary AML viability in relapsed AML. This study includes the largest lipidomic profile of LSC-enriched primary AML patient cells to date and indicates that inhibition of fatty acid desaturation is a promising therapeutic target for relapsed AML.