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Moth insects rely on sex pheromones for long distance attraction and searching for sex partners. The biosynthesis of moth sex pheromones involves the catalytic action of multiple enzymes, with desaturases playing a crucial role in the process of carbon chain desaturation. However, the specific desaturases involved in sex pheromone biosynthesis in fall armyworm (FAW), Spodoptera frugiperda, have not been clarified. In this study, a Δ11 desaturase (SfruDES1) gene in FAW was knocked out using the CRISPR/Cas9 genome editing system. A homozygous mutant of SfruDES1 was obtained through genetic crosses. The gas chromatography-mass spectrometry (GC-MS) analysis results showed that the three main sex pheromone components (Z7-12:Ac, Z9-14:Ac, and Z11-16:Ac) and the three minor components (Z9-14:Ald, E11-14:Ac and Z11-14:Ac) of FAW were not detected in homozygous mutant females compared to the wild type. Furthermore, behavioral assay demonstrated that the loss of SfruDES1 resulted in a significant reduction in the attractiveness of females to males, along with disruptions in mating behavior and oviposition. Additionally, in a heterologous expression system, recombinant SfruDES1 could introduce a cis double bond at the Δ11 position in palmitic acid, which resulted in the changes in components of the synthesized products. These findings suggest desaturase plays a key role in the biosynthesis of sex pheromones, and knockout of the SfruDES1 disrupts sex pheromone biosynthesis and mating behavior in FAW. The SfruDES1 could serve as tool to develop a control method for S. frugiperda., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Various bacterial species are capable of producing highly modified fatty acid derivatives with conjugated triple bonds, which play important ecological roles as antifungals and toxins in mutualistic and pathogenic interactions. Furthermore, the terminal polyyne moiety is of interest as pharmacophore and as tag in bioorthogonal chemistry and live imaging. To gain insight into the assembly of these highly reactive natural products, we investigated tetrayne (caryoynencin and protegencin) biosynthesis genes (cay and pgn) from Trinickia caryophylli and Pseudomonas protegens. Pathway dissection and reconstitution in the heterologous host Burkholderia graminis revealed the genes minimally required for polyyne formation. Mutational analyses and biochemical assays demonstrated that polyyne biosynthesis is thiotemplated, involving a fatty acyl-AMP ligase, a designated acyl carrier protein, and a thioesterase. Heterologous expression of point-mutated desaturase genes showed that three desaturases work synergistically to introduce four triple bonds. These findings point to an intricate desaturase complex and provide important information for future bioengineering experiments., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

This review highlights the key role of fatty acid desaturases in the synthesis of naturally occurring, more common and not unsaturated fatty acids. The three major classes of fatty acid desaturases, such as acyl-lipid, acyl-acyl carrier protein and acyl-coenzyme A, are described in detail, with particular attention to the cellular localisation, the structure, the substrate and product specificity and the expression and regulation of desaturase genes. The review also gives an insight into the biocatalytic reaction of fatty acid desaturation by covering the general and more class-specific mechanistic studies around the synthesis of unsaturated fatty acids Finally, we conclude the review by looking at the numerous novel applications for desaturases in order to meet the very high demand for polyunsaturated fatty acids, taking into account the opportunity for the development of new, more efficient, easily reproducible, sustainable bioengineering advances in the field., (© 2022 The Authors. IUBMB Life published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Fatty acid desaturase (FADS) generates double bond at a certain position of the corresponding polyunsaturated fatty acids (PUFAs) with high selectivity, the enzyme activity and PUFAs products of which are essential to biological systems and are associated with a variety of physiological diseases. Little is known about the structure of FADSs and their amino acid residues related to catalytic activities. Identifying key residues of Micromonas pusilla delta 6 desaturase (MpFADS6) provides a point of departure for a better understanding of desaturation. In this study, conserved amino acids were anchored through gene consensus analysis, thereby generating corresponding variants by site-directed mutagenesis. To achieve stable and high-efficiency expression of MpFADS6 and its variants in Saccharomyces cerevisiae, the key points of induced expression were optimized. The contribution of conserved residues to the function of enzyme was determined by analyzing enzyme activity of the variants. Molecular modeling indicated that these residues are essential to catalytic activities, or substrate binding. Mutants MpFADS6 [Q409R] and MpFADS6 [M242P] abolished desaturation, while MpFADS6 [F419V] and MpFADS6 [A374Q] significantly reduced catalytic activities. Given that certain residues have been identified to have a significant impact on MpFADS6 activities, it is put forward that histidine-conserved region III of FADS6 is related to electronic transfer during desaturation, while histidine-conserved regions I and II are related to desaturation. These findings provide new insights and methods to determine the structure, mechanism and directed transformation of membrane-bound desaturases., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Living systems provide a promising approach to chemical synthesis, having been optimized by evolution to convert renewable carbon sources, such as glucose, into an enormous range of small molecules. However, a large number of synthetic structures can still be difficult to obtain solely from cells, such as unsubstituted hydrocarbons. In this work, we demonstrate the use of a dual cellular-heterogeneous catalytic strategy to produce olefins from glucose using a selective hydrolase to generate an activated intermediate that is readily deoxygenated. Using a new family of iterative thiolase enzymes, we genetically engineered a microbial strain that produces 4.3 ± 0.4 g l -1 of fatty acid from glucose with 86% captured as 3-hydroxyoctanoic and 3-hydroxydecanoic acids. This 3-hydroxy substituent serves as a leaving group that enables heterogeneous tandem decarboxylation-dehydration routes to olefinic products on Lewis acidic catalysts without the additional redox input required for enzymatic or chemical deoxygenation of simple fatty acids., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Fatty acid desaturases add a second bond into a single bond of carbon atoms in fatty acid chains, resulting in an unsaturated bond between the two carbons. They are classified into soluble and membrane-bound desaturases, according to their structure, subcellular location, and function. The orthologous genes in Camelina sativa were identified and analyzed, and a total of 62 desaturase genes were identified. It was revealed that they had the common fatty acid desaturase domain, which has evolved separately, and the proteins of the same family also originated from the same ancestry. A mix of conserved, gained, or lost intron structure was obvious. Besides, conserved histidine motifs were found in each family, and transmembrane domains were exclusively revealed in the membrane-bound desaturases. The expression profile analysis of C. sativa desaturases revealed an increase in young leaves, seeds, and flowers. C. sativa ω3-fatty acid desaturases CsaFAD7 and CsaDAF8 were cloned and the subcellular localization analysis showed their location in the chloroplast. They were transferred into Arabidopsis thaliana to obtain transgenic lines. It was revealed that the ω3-fatty acid desaturase could increase the C18:3 level at the expense of C18:2, but decreases in oil content and seed weight, and wrinkled phenotypes were observed in transgenic CsaFAD7 lines, while no significant change was observed in transgenic CsaFAD8 lines in comparison to the wild-type. These findings gave insights into the characteristics of desaturase genes, which could provide an excellent basis for further investigation for C. sativa improvement, and overexpression of ω3-fatty acid desaturases in seeds could be useful in genetic engineering strategies, which are aimed at modifying the fatty acid composition of seed oil.

Fatty acid desaturase catalyzes the desaturation reactions by insertion of double bonds into the fatty acyl chain, producing unsaturated fatty acids. Though soluble fatty acid desaturases have been studied widely in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to the difficulty of generating recombinant desaturase. Brassica napus is a rapeseed, which possesses a range of different membrane-bound desaturases capable of producing fatty acids including Δ3, Δ4, Δ8, Δ9, Δ12, and Δ15 fatty acids. The 1155 bp open reading frame of Δ12 fatty acid desaturase (FAD12) from Brassica napus codes for 383 amino acid residues with a molecular weight of 44 kDa. It was expressed in Escherichia coli at 37 °C in soluble and insoluble forms when induced with 0.5 mM IPTG. Soluble FAD12 has been purified using Ni 2+ -Sepharose affinity chromatography with a total protein yield of 0.728 mg/mL. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that desaturase activity of FAD12 could produce linoleic acid from oleic acid at a retention time of 17.6 with a conversion rate of 47%. Characterization of purified FAD12 revealed the optimal temperature of FAD12 was 50 °C with 2 mM preferred substrate concentration of oleic acid. Analysis of circular dichroism (CD) showed FAD12 was made up of 47.3% and 0.9% of alpha-helix and β-sheet secondary structures. The predicted Tm value was 50.2 °C., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Fatty acid desaturases (FADs) represent a class of oxygen-dependent enzymes that dehydrogenate C-C bonds in the fatty acids (FAs) producing unsaturated CC double bonds that markedly change the properties of biological membranes. FADs are highly specific towards their acyl substrates, the position and configuration of the introduced double bonds. The double bond positioning of soluble acyl-carrier-protein Δ9-FADs was determined relative to the carboxyl end of a FA. Similar mode was suggested for the acyl-lipid Δ12-FADs (also known as ω6-FADs), however, their exact counting order remain unknown. Here we used monounsaturated odd- (17:1Δ 10 ) and even-chain (18:1Δ 11 ) FAs to show that acyl-lipid Δ12-FADs of, at least, two cyanobacterial species, Gloeobacter violaceus and Synechocystis sp. strain PCC 6803, use neither end of the fatty acid (Δ or ω) as a counting reference point; but count three carbons toward the methyl end from an existing double bond in the monoene precursors irrespective of a FA chain length., Competing Interests: Declaration of competing interest Authors declare no conflict of interests., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Fatty acid desaturases are key enzymes involved in unsaturated fatty acid biosynthesis, which insert double bonds at specific positions of fatty acids, playing a pivotal role in unsaturated fatty acid synthesis required for membrane lipid fluidity. The ∆5 and ∆6 desaturases are responsible for producing long chain-polyunsaturated fatty acids (LC-PUFA) through their precursors α-linolenic acid and linoleic acid in organisms lacking or with very low ability to synthesize LC-PUFA by themselves. Extensive studies of fatty acid desaturases are available in model organisms, such as humans and mouse; however, the diversity of these genes in the marine biodiversity is less known. This study performed an exhaustive analysis to identify the ∆5 and ∆6 desaturases in the available marine genomes in databases, as well as transcriptomes and EST databases, and their coding sequences were compared to the well-characterized ∆5 and ∆6 desaturases from humans. The FADS1 and FADS2 genetic structures are well conserved among all the organisms analyzed. A common amino acid pattern was identified to discriminate between ∆5 and ∆6 desaturases. The analysis of the conserved motif involved in catalysis showed that 20% of the desaturases, ∆5 and ∆6, have lost motifs required for catalysis. Additionally, bifunctional ∆5/∆6 desaturases were able to be identified by amino acid sequence patterns found in previously described enzymes. A revision of the expression profiles and functional activity on sequences in databases and scientific literature provided information regarding the function of these marine organism enzymes., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Δ9 fatty acyl desaturases introduce a cis -double bond between C9 and C10 of saturated fatty acyl chains. From the crystal structure of the mouse stearoyl-CoA desaturase (mSCD1) it was proposed that Tyr-104, a surface residue located at the distal end of the fatty acyl binding pocket plays a key role in specifying 18C selectivity. We created mSCD1-Y104G to test the hypothesis that eliminating this bulky side chain would create an opening and permit the substrate's methyl end to protrude through the enzyme into the lipid bilayer, facilitating the desaturation of very-long-chain (VLC) substrates. Consistent with this hypothesis, Y104G acquired the ability to desaturate 24C and 26C acyl-CoAs while maintaining its Δ9-regioselectivity. We also investigated two distantly related very-long-chain fatty acyl (VLCFA) desaturases from Arabidopsis, ADS1.2 and ADS1.4, which have Ala and Gly, respectively, in place of the gatekeeping Tyr found in mSCD1. Substitution of Tyr for Ala and Gly in ADS1.2 and ADS1.4, respectively, blocked their ability to desaturate VLCFAs. Further, we identified a pair of fungal desaturase homologs which contained either an Ile or a Gly at this location and showed that only the Gly-containing desaturase was capable of very-long-chain desaturation. The conserved desaturase architecture wherein a surface residue with a single bulky side chain forms the end of the substrate binding cavity predisposes them to single amino acid substitutions that enable a switch between long- and very-long-chain selectivity. The data presented here show that such changes have independently occurred multiple times during evolution., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Cheese lacks essential fatty acids (EFAs). Delta 12 fatty acid desaturase (FADS12) is a critical enzyme required for EFA biosynthesis in fermentation of the predominant strains of cheese. Previously, we identified the FADS12 gene and characterized its function for the first time in Geotrichum candidum, a dominant strain used to manufacture soft cheese with white rind. In this study, we analyzed the molecular mechanism of FADS12 function by swapping domains from Mortierella alpina and G. candidum that had, respectively, high and low oleic acid conversion rates. The results revealed three regions that are essential to this process, including regions from the end of the second transmembrane domain to the beginning of the third transmembrane domain, from the end of the third transmembrane domain to the beginning of the fourth transmembrane domain, and from the 30-amino acid from the end of the sixth transmembrane domain to the C-terminal end region. Based on our domain swapping analyses, nine pairs of amino acids including H112, S118, H156, Q161, K301, R306, E307, A309 and S323 in MaFADS12 (K123, A129, N167, M172, T302, D307, I308, E310 and D324 in GcFADS12) were identified as having a significantly effect on FADS12 catalytic efficiency, and linoleic acid and its analogues (12,13-cyclopropenoid fatty acid) were found to inhibit the catalytic activity of FADS12 and related recombinant enzymes. Furthermore, the molecular mechanism of FADS12 inhibition was analyzed. The results revealed two allosteric domains, including one domain from the N-terminal region to the beginning of the first transmembrane domain and another from the 31 st amino acid from the end of the sixth transmembrane domain to the C terminus. Y4 and F398 amino acid residues from MaFADS12 and eight pairs of amino acids including G56, L60, L344, G10, Q13, S24, K326 and L344 in MaFADS12 (while Y66, F70, F345, F20, Y23, Y34, F327 and F345 in GcFADS12) played a pivotal role in FADS12 inhibition. Finally, we found that both allosteric and active sites were responsible for the catalytic activity of FADS12 at various temperatures, pH, and times. This study offers a solid theoretical basis to develop preconditioning methods to increase the rate at which GcFADS12 converts oleic and linoleic acids to produce higher levels of EFAs in cheese., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA), a process to convert C18 polyunsaturated fatty acids into eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or arachidonic acid (ARA), requires the concerted activities of two enzymes, the fatty acyl desaturase (Fads) and elongase (Elovl). This study highlights the cloning, functional characterisation and tissue expression pattern of a Fads and an Elovl from the Boddart's goggle-eyed goby (Boleophthalmus boddarti), a mudskipper species widely distributed in the Indo-Pacific region. Phylogenetic analysis revealed that the cloned fads and elovl are clustered with other teleost orthologs, respectively. The investigation of the genome of several mudskipper species, namely Boleophthalmus pectinirostris, Periophthalmus schlosseri and Periophthalmus magnuspinnatus, revealed a single Fads2 and two elongases, Elovl5 and Elovl4 for each respective species. A heterologous yeast assay indicated that the B. boddarti Fads2 possessed low desaturation activity on C18 PUFA and no desaturation on C20 and C22 PUFA substrates. In comparison, the Elovl5 showed a wide range of substrate specificity, with a capacity to elongate C18, C20 and C22 PUFA substrates. An amino acid residue that affects the capacity to elongate C22:5n-3 was identified in the B. boddarti Elovl5. Both genes are highly expressed in brain tissue. Among all tissues, DHA is highly concentrated in neuron-rich tissues, whereas EPA is highly deposited in gills. Taken together, the results showed that due to the inability to perform desaturation steps, B. boddarti is unable to biosynthesise LC-PUFA, relying on dietary intake to acquire these nutrients., (© 2020 The Fisheries Society of the British Isles.)

Arbuscular mycorrhizal (AM) fungi are oleaginous organisms, and the most abundant fatty acyl moiety usually found in their lipids is palmitvaccenic acid (16:1 Δ11cis ). However, it is not known how this uncommon fatty acid species is made. Here, we have cloned two homologues of lepidopteran fatty acyl-coenzyme A Δ11 desaturases from the AM fungus Rhizophagus irregularis. Both enzymes, DES1 and DES2, are expressed in intraradical mycelium and can complement the unsaturated fatty acid-requiring auxotrophic growth phenotype of the Saccharomyces cerevisiae ole1Δ mutant. DES1 expression leads almost exclusively to oleic acid (18:1 Δ9cis ) production, whereas DES2 expression results in the production of 16:1 Δ11cis and vaccenic acid (18:1 Δ11cis ). DES2 therefore encodes a Δ11 desaturase that is likely to be responsible for the synthesis of 16:1 Δ11cis in R. irregularis., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Oilseed crop oils contain a variety of unsaturated fatty acids that are synthesized and regulated by fatty acid desaturases (FADs). In this study, 14 FAD3 (ω3 desaturase) protein sequences from oilseeds are analyzed and presented through the application of several computational tools. The results indicated a close relationship between Brassica napus and Camelina sativa, as well as between Salvia hispanica and Perilla frutescens FAD3s, due to a high similarity in codon preferences in codon usage clusters and the phylogenetic tree. The cis-acting element results reveal that the seed-specific promoter region of BnFAD3 contains the critical conserved boxes such as HSE and ABRE, which are involved in responsiveness to heat stress and abscisic acid. The presence of the aforementioned conserved boxes may increase cold acclimation as well as tolerance to drought and high salinity. Omega(ω)3 desaturases contain a Skn-1 motif which is a cis-acting regulatory element required involved in endosperm development. In oilseed FAD3s, leucine is the most repeated amino acid in FAD3 proteins. The study conveyed that B. napus, Camelina sativa, Linum usitatissimum, Vernicia fordii, Gossypium hirsutum, S. hispanica, Cannabis sativa, and P. frutescens have retention signal KXKXX/XKXX at their c-terminus sites, which is one of the most important characteristics of FADs. Additionally, it was found that BnFAD3 is a transmembrane protein that can convert ω6 to ω3 fatty acids and may simultaneously act as a potassium ion channel in the ER.

Arbuscular mycorrhiza (AM) fungi deliver mineral nutrients to the plant host in exchange for reduced carbon in the form of sugars and lipids. Colonization with AM fungi upregulates a specific host lipid synthesis pathway resulting in the production of fatty acids. Predominantly palmitic acid (16:0) and the unusual palmitvaccenic acid (16:1 Δ11cis ) accumulate in the fungus Rhizophagus irregularis . Here, we present the isolation and characterization of Ri OLE1-LIKE, the desaturase involved in palmitvaccenic acid synthesis, by heterologous expression in yeast and plants. Results are in line with the scenario in which RiOLE1-LIKE encodes an acyl-CoA desaturase with substrate specificity for C15-C18 acyl groups, in particular C16. Phylogenetic analysis of RiOLE1-LIKE -related sequences revealed that this gene is conserved in AM fungi from the Glomales and Diversisporales but is absent from nonsymbiotic Mortierellaceae and Mucoromycotina fungi, suggesting that 16:1 Δ11cis provides a specific function during AM colonization.

The soybean fatty acid desaturase family is composed of seven genes, but the function of each gene has not been reported. Bioinformatics was used to analyse the structure of genes in this family, as well as the correlation between Δ12-fatty acid desaturase II (FAD2) expression and oleic acid content on different days after flowering of soybean. In the present study, CRISPR/Cas9 technology was used to construct single and double mutant knockout vectors of functional genes in the FAD2 family. Analysis of the molecular biology and expression patterns of genes in the FAD2 family, namely, GmFAD2-1A ( Glyma.10G278000 ) and GmFAD2-2A ( Glyma.19G147300 ), showed that they had little homology with other soybean FAD2 genes, and that their function was slightly changed. Sequencing of the target showed that the editing efficiency of the GmFAD2-1A and GmFAD2-2A genes was 95% and 55.56%, respectively, and that the double mutant editing efficiency was 66.67%. The mutations were divided into two main types, as follows: base deletion and insertion. A near-infrared grain analyser determined the following results: In the T 2 generation, the oleic acid content increased from 17.10% to 73.50%; the linoleic acid content decreased from 62.91% to 12.23%; the protein content increased from 37.69% to 41.16%; in the T 3 generation, the oleic acid content increased from 19.15% to 72.02%; the linoleic acid content decreased from 56.58% to 17.27%. In addition, the protein content increased from 37.52% to 40.58% compared to that of the JN38 control variety., Competing Interests: The authors declare no conflict of interest. Any role of the funders in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript, or in the decision to publish the results must be declared in this section. If there is no role, please state “The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results”.

Fatty acid desaturase 2 (Fads2) is the key enzyme of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis. Endogenous production of these biomolecules in vertebrates, if present, is insufficient to meet demand. Hence, LC-PUFA are considered as conditionally essential. At present, however, LC-PUFA are globally limited nutrients due to anthropogenic factors. Research attention has therefore been paid to finding ways to maximize endogenous LC-PUFA production, especially in production species, whereby deeper knowledge on molecular mechanisms of enzymatic steps involved is being generated. This review first briefly informs about the milestones in the history of LC-PUFA essentiality exploration before it focuses on the main aim-to highlight the fascinating Fads2 potential to play roles fundamental to adaptation to novel environmental conditions. Investigations are summarized to elucidate on the evolutionary history of fish Fads2, providing an explanation for the remarkable plasticity of this enzyme in fish. Furthermore, structural implications of Fads2 substrate specificity are discussed and some relevant studies performed on organisms other than fish are mentioned in cases when such studies have to date not been conducted on fish models. The importance of Fads2 in the context of growing aquaculture demand and dwindling LC-PUFA supply is depicted and a few remedies in the form of genetic engineering to improve endogenous production of these biomolecules are outlined., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Claviceps purpurea bifunctional Δ12-hydroxylase/desaturase, CpFAH12, and monofunctional desaturase CpFAD2, share 86% of sequence identity. To identify the underlying determinants of the hydroxylation/desaturation specificity, chimeras of these two enzymes were tested for their fatty acid production in an engineered Yarrowia lipolytica strain. It reveals that transmembrane helices are not involved in the hydroxylation/desaturation specificity whereas all cytosolic domains have an impact on it. Especially, replacing the CpFAH12 cytosolic part near the second histidine-box by the corresponding CpFAD2 part annihilates all hydroxylation activity. Further mutagenesis experiments within this domain identified isoleucine 198 as the crucial element for the hydroxylation activity of CpFAH12. Monofunctional variants performing the only desaturation were obtained when this position was exchanged by the threonine of CpFAD2. Saturation mutagenesis at this position showed modulation in the hydroxylation/desaturation specificity in the different variants. The WT enzyme was demonstrated as the most efficient for ricinoleic acid production and some variants showed a better desaturation activity. A model based on the recently discovered membrane desaturase structures indicate that these changes in specificity are more likely due to modifications in the di-iron center geometry rather than changes in the substrate binding mode., (© 2019 The Authors Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.)

FAD synthase, the last enzyme of the pathway converting riboflavin to FAD, exists in humans in different isoforms, with isoforms 1, 2 and 6 being characterized at the functional and molecular levels. Isoform 2, the cytosolic and most abundant FADS, consists of two domains: a PAPS reductase C-terminus domain (here named FADSy) responsible for FAD synthesis, and an N-terminus molybdopterin-binding resembling domain (MPTb - here named FADHy), whose FAD hydrolytic activity is hidden unless both Co 2+ and chemical mercurial reagents are added to the enzyme. To investigate the hFADS2 hydrolytic function under conditions closer to the physiological context, the hydrolytic activity was further characterized. Co 2+ induced FAD hydrolysis was strongly stimulated in the presence of K + , reaching a Vmax higher than that of FAD synthesis. The pH dependence together with the inhibition of the hydrolysis by NaF and KI allow excluding that the reaction occurs via a NUDIX type catalysis. The K 0.5 for K + or Co 2+ was 7.2 or 0.035 mM, respectively. Other monovalent or divalent cations can partially substitute K + or Co 2+ . Reduced glutathione stimulated whereas NADH inhibited the hydrolytic activity. The latter aspects correlate with an interconnection of the homeostasis of NAD and FAD., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The long-chain (≥C 20 ) polyunsaturated fatty acid biosynthesis capacity of fish varies among species, with trophic level hypothesised as a major factor. The biosynthesis capacity is largely dependent upon the presence of functionally diversified fatty acyl desaturase 2 (Fads2) enzymes, since many teleosts have lost the gene encoding a Δ5 desaturase (Fads1). The present study aimed to characterise Fads2 from four teleosts occupying different trophic levels, namely Sarpa salpa, Chelon labrosus, Pegusa lascaris and Atherina presbyter, which were selected based on available data on functions of Fads2 from closely related species. Therefore, we had insight into the variability of Fads2 within the same phylogenetic group. Our results showed that Fads2 from S. salpa and C. labrosus were both Δ6 desaturases with further Δ8 activity while P. lascaris and A. presbyter Fads2 showed Δ4 activity. Fads2 activities of herbivorous S. salpa are consistent with those reported for carnivorous Sparidae species. The results suggested that trophic level might not directly drive diversification of teleost Fads2 as initially hypothesised, and other factors such as the species' phylogeny appeared to be more influential. In agreement, Fads2 activities from P. lascaris and A. presbyter were similar to their corresponding phylogenetic counterparts Solea senegalensis and Chirostoma estor.

Fatty acid desaturases (Fads) are a key enzyme in the process of biosynthesis of highly unsaturated fatty acids (HUFAs). In this study, we cloned the full-length sequence of the SiFad1 gene (SiFad1) and analyzed its expression profiles during different developmental stages and in different tissues of Strongylocentrotus intermedius. The full-length cDNA of SiFad1 is composed of 1086 bp, with a putative open reading frame of 885 bp encoding a polypeptide of 294 amino acid (AA) residues. The predicted molecular mass of SiFad1 is 34.67 kDa and its theoretical pI is 8.41. The presence of conserved motifs including three histidine boxes (HXXXH, HXXHH, XXXHH), a FA_desaturases domain and three transmembrane domains suggests that SiFad1 belongs to the microsomal fatty acid desaturases family. Its tissue distribution showed that the highest expression of SiFad1 is in the intestine and the weakest expression is in Aristotle's lantern of S. intermedius. Time-course expression measurements in different developmental stages showed the highest expression of SiFad1 occurs in the gastrula and the weakest expression in the juvenile sea urchin. Knock-down of SiFad1 by specific siRNA revealed that the significantly depressed expression of Elovl5 had decreased in the coelomocytes, intestines and gonads at 24 h post transfection, indicating that the downstream target gene of SiFad1 is Elovl5 and SiFad1 and Elovl5 have positive regulatory effects. When we examined the changes in fatty acids in the gonads before and after interference, the results showed that after 24 h of interference, the content of C20:4n-6 produced by SiFad1 had decreased. Taken together, these results will enable us to understand the role of SiFad1 in fatty acid anabolism, which will help us to understand the fatty acid synthesis pathways and regulatory mechanisms of Strongylocentrotus intermedius and provide a theoretical experimental basis for improving the ability of sea urchins to synthesize fatty acids and cultivating sea urchins of higher quality and nutritional value., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Carboxylases are biocatalysts that capture and convert carbon dioxide (CO 2 ) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO 2 molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO 2 within the active site of Ecr from Kitasatospora setae Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO 2 -fixing enzyme. Together, these 4 residues anchor and position the CO 2 molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO 2 fixation. Altogether, our study reveals unprecedented molecular details of selective CO 2 binding and C-C-bond formation during the catalytic cycle of nature's most efficient CO 2 -fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO 2 in biology and chemistry., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

In the present paper, we investigated the molecular cloning and functional characterization of elongase of very long chain fatty acid (elovl) and fatty acyl desaturase (fads) genes in a marine teleost, Nibea diacanthus. The elongase cDNA sequence encoded a polypeptide of 294 amino acids exhibiting Elovl5 activity, which effectively elongated both C18 (18:2n-6, 18:3n-3 and 18:3n-6) and C20 (20:4n-6 and 20:5n-3) polyunsaturated fatty acids. The desaturase cDNA sequence specified a polypeptide of 445 amino acids indicating Δ6 desaturation activity, which coul converted C18:2n-6 and C18:3n-3 to C18:3n-6 and C18:4n-3, respectively. Tissue distribution analysis by quantitative real-time PCR revealed that the elovl5 was primarily expressed in intestine and liver, while the fads2 was mainly expressed in liver and brain. These results increase our knowledge of the ability of endogenous highly unsaturated fatty acids (HUFA) biosynthesis in marine carnivorous fish., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Although various ω-3 fatty acid desaturases (ω3Des) have been identified and well-studied regarding substrate preference and regiospecificity, the molecular mechanism of their substrate specificities remains to be investigated. Here we compared two ω3Des, FADS15 from Mortierella alpina and oRiFADS17 from Rhizophagus irregularis , which possessed a substrate preference for linoleic acid and arachidonic acid, respectively. Their sequences were divided into six sections and a domain-swapping strategy was used to test the role of each section in catalytic activity. Heterologous expression and fatty acid experiments of hybrid enzymes in Saccharomyces cerevisiae INVSc1 indicated that the sequences between his-boxes I and II played critical roles in influencing substrate preference. Based on site-directed mutagenesis and molecular docking, the amino acid substitutions W129T and T144W, located in the upper part of the hydrocarbon chain, were found to be involved in substrate specificity, while V137T and V152T were confirmed to interfere with substrate recognition. This study provides significant insight into the structure-function relationship of ω3Des.

Desaturases are essentially required for unsaturated fatty acid (UFA) biosynthesis. We identified 10 genes encoding putative desaturases in the transcriptome database of the brown planthopper (BPH), Nilaparvata lugens . These include eight First Desaturase family genes, one cytochrome b5 fused desaturase gene ( Nlug-Cytb5r ) and one Sphingolipid Desaturase gene ( Nlug-ifc ). Transcript level profiling revealed significant variation in the expression patterns of these genes across tissues and developmental stages, which occur in a gene-specific manner. Interestingly, their expression was also modulated by the insect food source: the mRNA levels of Nlug-desatC and Nlug-Cytb5r were down-regulated, but the expression level of Nlug-desatA1-b and Nlug-desatA1-c were elevated in the BPH fed on the resistant rice variety Babawee as compared to the non-resistant variety Taichun Native 1 (TN1). Silencing Nlug-desatA1-b , Nlug-desatA1-c , or Nlug-Ifc reduced fatty acid composition and abundance in female BPH 1-d-old-adults compared to controls. Whereas, single knockdown of all ten desaturase genes significantly increased mortality of BPH nymphs compared with controls. Of the ten desaturase genes, knockdown of Nlug-desatA1-b and Nlug-desatA2 caused the highest mortality in BPH (91% and 97%, respectively). Our findings offer a base for expression and functional characterization of newly identified desaturase genes in BPH, and may contribute to RNA interference-based pest management strategies.

Δ9-Fatty acid desaturase (Δ9-desaturase) is a rate-limiting enzyme of unsaturated fatty acid biosynthesis in animal cells and specifically introduces a cis-double bond at the Δ9-position of acyl-CoA. Since the chemical structure of fatty acids determines the physicochemical properties of cellular membrane and modulates a broad range of cellular functions, double bond introduction into a fatty acid by Δ9-desaturase should be specifically carried out. Reported crystal structures of stearoyl-CoA desaturase (SCD)1, one of the most studied Δ9-desaturases, have revealed the mechanism underlying the determination of substrate preference, as well as the position (Δ9) and conformation (cis) of double bond introduction. The crystal structures of SCD1 have also provided insights into the function of other Δ9-desaturases, including Drosophila homologs. Moreover, the amino-terminal sequences of Δ9-desaturases are shown to have unique roles in protein degradation. In this review, we introduce recent advances in the understanding of the function and regulation of Δ9-desaturase from the standpoint of protein structure.

In vertebrates, the essential fatty acids (FA) that satisfy the dietary requirements for a given species depend upon its desaturation and elongation capabilities to convert the C 18 polyunsaturated fatty acids (PUFA), namely linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), into the biologically active long-chain (C 20-24 ) polyunsaturated fatty acids (LC-PUFA), including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). Recent studies have established that tambaqui (Colossoma macropomum), an important aquaculture-produced species in Brazil, is a herbivorous fish that can fulfil its essential FA requirements with dietary provision C 18 PUFA LA and ALA, although the molecular mechanisms underpinning such ability remained unclear. The present study aimed at cloning and functionally characterizing genes encoding key desaturase and elongase enzymes, namely fads2, elovl5 and elovl2, involved in the LC-PUFA biosynthetic pathways in tambaqui. First, a fads2-like desaturase was isolated from tambaqui. When expressed in yeast, the tambaqui Fads2 showed Δ6, Δ5 and Δ8 desaturase capacities within the same enzyme, enabling all desaturation reactions required for ARA, EPA and DHA biosynthesis. Moreover, tambaqui possesses two elongases that are bona fide orthologs of elovl5 and elovl2. Their functional characterization confirmed that they can operate towards a variety of PUFA substrates with chain lengths ranging from 18 to 22 carbons. Overall our results provide compelling evidence that demonstrates that all the desaturase and elongase activities required to convert LA and ALA into ARA, EPA and DHA are present in tambaqui within the three genes studied herein, i.e. fads2, elovl5 and elovl2., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Fatty acid desaturases are rate-limiting enzymes in long-chain polyunsaturated fatty acid biosynthesis. The transcription factor peroxisome proliferator-activated receptor alpha b (PPARαb) regulates lipid metabolism in mammals, however, the mechanism whereby PPARαb regulates fatty acid desaturases is largely unknown in fish. In this study, we report the full length cDNA sequence of Trachinotus ovatus fatty acid desaturase, which encodes a 380 amino acid polypeptide, possessing three characteristic histidine domains. Phylogenetic and gene exon/intron structure analyses showed typical phylogeny: the T. ovatus fatty acid desaturase contained a highly conserved exon/intron architecture. Moreover, functional characterization by heterologous expression in yeast indicated that T. ovatus desaturase was a fatty acid desaturase, with Δ4/Δ5/Δ8 Fad activity. Promoter activity assays indicated that ToFads6 desaturase transcription was positively regulated by PPARαb. Similarly, PPARαb RNA interference decreased ToPPARαb and ToFads6 expression at the mRNA and protein levels in a time-dependent manner. Mutation analyses showed that the M2 binding site of PPARαb was functionally important for protein binding, and transcriptional activity of the ToFads6 promoter was significantly decreased after targeted mutation of M2. Electrophoretic mobile shift assays confirmed that PPARαb interacted with the binding site of the ToFads6 promoter region, to regulate ToFads6 transcription. In summary, PPARαb played a vital role in ToFads6 regulation and may promote the biosynthesis of long-chain polyunsaturated fatty acids by regulating ToFads6 expression.

Polyunsaturated fatty acids (PUFAs) have important industrial, physiological, and nutritional properties. Plants use the sequential activities of FAD2 and FAD3 desaturases to convert 18:1Δ9 to the important PUFA 18:3Δ9,12,15, whereas the fungus Fusarium verticillioides 7600 uses the bifunctional desaturase Fm1 for both reactions. Here, we used a combination of sequence comparisons, structural modeling, and mutagenesis experiments to investigate Fm1's regioselectivity and identified two functionally relevant clusters of residues that contribute to Fm1 activity. We found that cluster I (Leu 153 , Phe 157 , and His 194 ), located near the catalytic iron ions, predominantly affects activity, whereas cluster II (Tyr 280 , His 284 , and Leu 287 ), located in a helix forming the entrance to the substrate-binding pocket, mainly specifies 15-desaturation. Individual or combined substitutions of cluster II residues substantially reduced 15-desaturation. The combination of F157W from cluster I with Y280L, H284V, and L287T from cluster II created an increased-activity variant that almost completely lost the ability to desaturate at C15 and acted almost exclusively as a 12-desaturase. No variants were identified in which 15-desaturation occurred in the absence of 12-desaturation. Fm1 displayed only traces of activity with C16 substrate, but several cluster I variants exhibited increased activity with both 18:1 and 16:1 substrates, converting 16:1Δ9 to 16:3Δ9,12,15, consistent with Fm1 performing sequential v + 3 desaturation reactions at C12 and then C15. We propose that cluster II residues interact with the substrate headgroup when the acyl chain contains both Δ9 and Δ12 double bonds, in which case C15 becomes positioned adjacent to the di-iron site enabling a second v + 3 desaturation.

The ω-3 fatty acid desaturase (ω3Des) is a key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs). However, the enzyme exhibits a significant preference towards different fatty acid substrates. To examine the molecular mechanism of its substrate specificity, a series of site-directed mutants were constructed based on the membrane topology model and functionally characterised by heterologous expression in Saccharomyces cerevisiae. Our results revealed that the W106F and V137T mutations markedly decreased the enzyme activity which indicated that these two residues were associated with substrate recognition. In contrast, the A44S, M156I and W291M mutations showed significant increments (30 to 40%) of the conversion rate for AA substrate desaturation, which suggests that these residues play a pivotal role in desaturation of longer chain-length substrates. Through homology modelling of 3-dimensional structures and molecular docking of FADS15, we propose that the critical residues that bind to the CoA groups may affect substrate localisation and govern substrate preference and chain-length specificity. Our work increases the understanding of the structure-function relationships of the microbial membrane-bound desaturases. The growing knowledge of the molecular mechanism will also aid in the efficient production of value-added fatty acids.

Background: Provision of long-chain polyunsaturated fatty acids (LC-PUFA) in vertebrates occurs through the diet or via endogenous production from C 18 precursors through consecutive elongations and desaturations. It has been postulated that the abundance of LC-PUFA in the marine environment has remarkably modulated the gene complement and function of Fads in marine teleosts. In vertebrates two fatty acyl desaturases, namely Fads1 and Fads2, encode ∆5 and ∆6 desaturases, respectively. To fully clarify the evolutionary history of LC-PUFA biosynthesis in vertebrates, we investigated the gene repertoire and function of Fads from species placed at key evolutionary nodes., Results: We demonstrate that functional Fads1Δ5 and Fads2∆6 arose from a tandem gene duplication in the ancestor of vertebrates, since they are present in the Arctic lamprey. Additionally, we show that a similar condition was retained in ray-finned fish such as the Senegal bichir and spotted gar, with the identification of fads1 genes in these lineages. Functional characterisation of the isolated desaturases reveals the first case of a Fads1 enzyme with ∆5 desaturase activity in the Teleostei lineage, the Elopomorpha. In contrast, in Osteoglossomorpha genomes, while no fads1 was identified, two separate fads2 duplicates with ∆6 and ∆5 desaturase activities respectively were uncovered., Conclusions: We conclude that, while the essential genetic components involved LC-PUFA biosynthesis evolved in the vertebrate ancestor, the full completion of the LC-PUFA biosynthesis pathway arose uniquely in gnathostomes.

Little is known about the structure-function relationship of membrane-bound lipid desaturases. Using a domain-swapping strategy, we found that the N terminus (comprising the two first transmembrane segments) region of Bacillus cereus DesA desaturase improves Bacillus subtilis Des activity. In addition, the replacement of the first two transmembrane domains from Bacillus licheniformis inactive open reading frame (ORF) BL02692 with the corresponding domain from DesA was sufficient to resurrect this enzyme. Unexpectedly, we were able to restore the activity of ORF BL02692 with a single substitution (Cys40Tyr) of a cysteine localized in the first transmembrane domain close to the lipid-water interface. Substitution of eight residues (Gly90, Trp104, Lys172, His228, Pro257, Leu275, Tyr282, and Leu284) by site-directed mutagenesis produced inactive variants of DesA. Homology modeling of DesA revealed that His228 is part of the metal binding center, together with the canonical His boxes. Trp104 shapes the hydrophobic tunnel, whereas Gly90 and Lys172 are probably involved in substrate binding/recognition. Pro257, Leu275, Tyr282, and Leu284 might be relevant for the structural arrangement of the active site or interaction with electron donors. This study reveals the role of the N-terminal region of Δ5 phospholipid desaturases and the individual residues necessary for the activity of this class of enzymes., (Copyright © 2018 Sastre et al.)

A series of linear dipeptide derivatives ( 4 - 10 ) were prepared and evaluated as antimicrobial agents via the synthesis of N -(2-(2-hydrazinyl-2-oxoethylamino)-2-oxoethyl) nicotinamide ( 4 ). Compound 4 was reacted with 4-chlorobenzaldehyde or 4-hydroxybenzaldehyde, to give the hydrazones 5 and 6 , respectively. On the other hand, Compound 4 was coupled with phenylisocyanate or methylisothiocyanate to give Compounds 7 and 8 , respectively. The latter compounds ( 7 and 8 ) were coupled with chloroacetic acid to give oxazolidine ( 9 ) and thiazolidine ( 10 ), respectively. The newly synthesized dipeptide compounds were confirmed by means of their spectral data. The antimicrobial activity of the newly synthesized compounds 4 - 10 was evaluated by agar well diffusion, and they showed good activity. Compounds 4 , 5 , and 9 gave the most promising activity in this study. Most of the tested compounds possessed MIC values ranging from 50 to 500 µg/mL. Furthermore, docking studies were carried out on enoyl reductase from E. coli and cytochrome P450 14 α-sterol demethylase (Cyp51) from Candida albicans active sites. The MolDock scores of the seven tested compounds ranged between -117 and -171 and between -107 and -179, respectively., Competing Interests: The authors declare no conflicts of interest.

Fatty acid desaturases (FADs) catalyze the introduction of a double bond into acyl chains. Two FAD groups have been identified in plants: acyl-acyl carrier proteins (ACPs) and acyl-lipid or membrane-bound FAD. The former catalyze the conversion of 18:0 to 18:1 and to date have only been identified in plants. The latter are found in eukaryotes and bacteria and are responsible for multiple desaturations. In this study, we identified 82 desaturase gene and protein sequences from 10 grass species deposited in GenBank that were analyzed using bioinformatic approaches. Subcellular localization predictions of desaturase family revealed their localization in plasma membranes, chloroplasts, endoplasmic reticula, and mitochondria. The in silico mapping showed multiple chromosomal locations in most species. Furthermore, the presence of the characteristic histidine domains, the predicted motifs, and the finding of transmembrane regions strongly support the protein functionality. The identification of putative regulatory sites in the promotor and the expression profiles revealed the wide range of pathways in which fatty acid desaturases are involved. This study is an updated survey on desaturases of grasses that provides a comprehensive insight into diversity and evolution. This characterization is a necessary first step before considering these genes as candidates for new biotechnological approaches.

Omega-3 fatty acid desaturase (ω-3 FAD, D15D) is a key enzyme for α-linolenic acid (ALA) biosynthesis. Both chia (Salvia hispanica) and perilla (Perilla frutescens) contain high levels of ALA in seeds. In this study, the ω-3 FAD gene family was systematically and comparatively cloned from chia and perilla. Perilla FAD3, FAD7, FAD8 and chia FAD7 are encoded by single-copy (but heterozygous) genes, while chia FAD3 is encoded by 2 distinct genes. Only 1 chia FAD8 sequence was isolated. In these genes, there are 1 to 6 transcription start sites, 1 to 8 poly(A) tailing sites, and 7 introns. The 5'UTRs of PfFAD8a/b contain 1 to 2 purine-stretches and 2 pyrimidine-stretches. An alternative splice variant of ShFAD7a/b comprises a 5'UTR intron. Their encoded proteins harbor an FA_desaturase conserved domain together with 4 trans-membrane helices and 3 histidine boxes. Phylogenetic analysis validated their identity of dicot microsomal or plastidial ω-3 FAD proteins, and revealed some important evolutionary features of plant ω-3 FAD genes such as convergent evolution across different phylums, single-copy status in algae, and duplication events in certain taxa. The qRT-PCR assay showed that the ω-3 FAD genes of two species were expressed at different levels in various organs, and they also responded to multiple stress treatments. The functionality of the ShFAD3 and PfFAD3 enzymes was confirmed by yeast expression. The systemic molecular and functional features of the ω-3 FAD gene family from chia and perilla revealed in this study will facilitate their use in future studies on genetic improvement of ALA traits in oilseed crops.

Polyunsaturated fatty acids (PUFAs) are essential lipids for cell function, normal growth, and development, serving as key structural components of biological membranes and modulating critical signal transduction events. Omega-3 (n-3) long chain PUFAs (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect against inflammatory diseases and enhance brain development and function. This had led to a marked increase in demand for fish and fish oils in human diets, supplements, and aquaculture and created a need for new, sustainable n-3 LC-PUFA sources. We have studied for the first time homogenous preparations of the membrane-type ω6 and ω3 fatty acid desaturases from the fungus Mortierella alpina, as a model system to produce PUFAs. These desaturases possess a di-iron metal center and are selective for 18:1 n-9 and 18:2 n-6 acyl-CoA substrates, respectively. Sequence alignments and membrane topology predictions support that these enzymes have unique cap regions that may include the rearrangement and repositioning of the active site, especially when compared to the mammalian stearoyl-coenzyme A desaturase-1 (SCD1) and the related sphingolipid α-hydroxylase (Scs7p) that act upon different substrates.

Background: Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains. Extensive studies of fatty acid desaturases have been done in many plants. However, less is known about the diversity of this gene family in peanut (Arachis hypogaea L.), an important oilseed crop that is cultivated worldwide., Results: In this study, twelve novel AhFADs genes were identified and isolated from peanut. Quantitative real-time PCR analysis indicated that the transcript abundances of AhFAB2-2 and AhFAD3-1 were higher in seeds than in other tissues examined, whereas the AhADS and AhFAD7-1 transcripts were more abundant in leaves. AhFAB2-3, AhFAD3-2, AhFAD4, AhSLD-4, and AhDES genes were highly expressed in flowers, whereas AhFAD7-2, AhSLD-2, and AhSLD-3 were expressed most strongly in stems. During seed development, the expressions of AhFAB2-2, AhFAD3-1, AhFAD7-1, and AhSLD-3 gradually increased in abundance, reached a maximum expression level, and then decreased. The AhFAB2-3, AhFAD3-2, AhFAD4, AhADS, and AhDES transcript levels remained relatively high at the initial stage of seed development, but decreased thereafter. The AhSLD-4 transcript level remained relatively low at the initial stage of seed development, but showed a dramatic increase in abundance at the final stage. The AhFAD7-2 and AhSLD-2 transcript levels remained relatively high at the initial stage of seed development, but then decreased, and finally increased again. The AhFAD transcripts were differentially expressed following exposure to abiotic stresses or abscisic acid. Moreover, the functions of one AhFAD6 and four AhSLD genes were confirmed by heterologous expression in Synechococcus elongates or Saccharomyces cerevisiae., Conclusions: The present study provides valuable information that improves understanding of the biological roles of FAD genes in fatty acid synthesis, and will help peanut breeders improve the quality of peanut oil via molecular design breeding.

Numerous tools to generate phylogenetic estimates are available, but there is no single protocol that will produce an accurate phylogenetic tree for any dataset. Here, we investigated some of those tools, paying particular attention to different alignment algorithms, in order to produce a phylogeny for the integral membrane fatty acid desaturase (FAD) family. Herein, we report a novel streamlined protocol which utilises peptide pattern recognition (PPR). This protocol can theoretically be applied universally to generate accurate multiple sequence alignments and improve downstream phylogenetic analyses. Applied to the desaturases, the protocol generated the first detailed phylogenetic estimates for the family since 2003, which suggested they may have evolved from three functionally distinct desaturases and further, that desaturases evolved first in cyanobacteria. In addition to the phylogenetic outputs, we mapped PPR sequence motifs onto an X-ray protein structure to provide insights into biochemical function and demonstrate the complementarity of PPR and phylogenetics., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Dihydroceramide desaturase 1 (Des1) catalyzes the last step of ceramide synthesis de novo, thus regulating the physiologically relevant balance between dihydrosphingolipids and sphingolipids. Here we report on the configurational preference of Des1 towards isomeric Δ 6 -unsaturated dihydroceramide analogs and the discovery of a potent Des1 inhibitor.

Background: Eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (AA, C20:4n-6) are long-chain polyunsaturated fatty acids (LCPUFAs) with relevant roles in the organism. EPA and DHA are synthesized from the precursor alpha-linolenic acid (ALA, C18:3n-3), whereas AA is produced from linoleic acid (LA, C18:2n-6) through the action of Δ5 and Δ6-desaturases. High-fat diet (HFD) decreases the activity of both desaturases and LCPUFA accretion in liver and other tissues. Hydroxytyrosol (HT), a natural antioxidant, has an important cytoprotective effects in different cells and tissues., Methods: Male mice C57BL/6 J were fed a control diet (CD) (10% fat, 20% protein, 70% carbohydrates) or a HFD (60% fat, 20% protein, 20% carbohydrates) for 12 weeks. Animals were daily supplemented with saline (CD) or 5 mg HT (HFD), and blood and the studied tissues were analyzed after the HT intervention. Parameters studied included liver histology (optical microscopy), activity of hepatic desaturases 5 and 6 (gas-liquid chromatography of methyl esters derivatives) and antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase by spectrophotometry), oxidative stress indicators (glutathione, thiobarbituric acid reactants, and the antioxidant capacity of plasma), gene expression assays for sterol regulatory element-binding protein 1c (SREBP-1c) (qPCR and ELISA), and LCPUFA profiles in liver, erythrocyte, brain, heart, and testicle (gas-liquid chromatography)., Results: HFD led to insulin resistance and liver steatosis associated with SREBP-1c upregulation, with enhancement in plasma and liver oxidative stress status and diminution in the synthesis and storage of n-6 and n-3 LCPUFAs in the studied tissues, compared to animals given control diet. HT supplementation significantly reduced fat accumulation in liver and plasma as well as tissue metabolic alterations induced by HFD. Furthermore, a normalization of desaturase activities, oxidative stress-related parameters, and tissue n-3 LCPUFA content was observed in HT-treated rats over control animals., Conclusions: HT supplementation prevents metabolic alterations in desaturase activities, oxidative stress status, and n-3 LCPUFA content in the liver and extrahepatic tissues of mice fed HFD.

Background: Some oleaginous fungi can produce large amounts of polyunsaturated fatty acids (PUFAs) which serve many physiological functions. Numerous desaturases are critical for the synthesis of PUFAs. This study aimed to investigate the regulation of lipid production and desaturase gene expression in Mortierella sp. AGED in response to different environmental factors, and the relationships between lipid production and desaturase gene expression., Results: The fatty acid composition and mRNA levels of desaturase genes were significantly changed under low temperatures. With the exception of Δ5-desaturase, the transcript levels of all desaturase genes increased at a temperature of 20 °C. Changes in content of lipid and PUFAs responding to low temperature were consistent with desaturase gene expression. Time course studies on gene expression showed that mRNA levels of four desaturase genes increased rapidly after transferring the cells to low temperature. Ethanol (1.5% v/v) increased the transcript levels of Δ9-, Δ6- and Δ5-desaturase genes significantly and of Δ12-desaturase gene slightly. Different metal ions such as Ca 2+ , Zn 2+ and Fe 3+ could stimulate PUFA synthesis and up-regulate desaturase gene transcription, while Cu 2+ inhibited desaturase gene expression and lipid accumulation., Conclusion: This study should enable us to understand the regulatory mechanism of desaturase gene expression and lipid synthesis. It is helpful to improve PUFA productivity in Mortierella sp. AGED. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Silver barb (Puntius gonionotus) is considered a promising medium carp species for freshwater aquaculture in Asia. This study in silver barb was carried out to evaluate the effects of total or partial substitution of dietary fish oil (FO) with linseed oil (LO) on growth, nutrient utilization, whole-body composition, muscle and liver fatty acid composition. Fish (12.1±0.4g of initial body weight) were fed for 60days with five experimental iso-proteinous, iso-lipidic and iso-caloric diets in which FO (control diet) was replaced by 33.3%, 50%, 66.7% and 100% LO. Final weight, weight gain, percent weight gain, SGR decreased linearly (p<0.001) with increasing LO levels in the diets. Dietary LO substitution levels did not significantly (p>0.05) affect the feed conversion ratio (FCR), protein efficiency ratio (PER) and whole body proximate composition. Furthermore, enhanced level of LO increased α-linolenic acid (ALA; 18:3n3) and linoleic acid (LA; 18:2n6) and decreased eicosapentaenoic acid (EPA; 20:5n3) and docosahexaenoic acid (DHA; 22:6n3) in muscle and liver. To understand the molecular mechanism of long chain-polyunsaturated fatty acid (LC-PUFA) biosynthesis, we cloned and characterized the fatty acyl Δ6 desaturase (Δ6 fad) cDNA and investigated its expression in various organs/tissues following replacement of FO with LO in the diet. The full-length Δ6 fad cDNA was 2056bp encoding 444 amino acids and was widely expressed in various organs/tissues. Replacement of FO with LO increased the expression of Δ6 fad mRNA in liver, muscle and intestine but no significant difference was found in the brain., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Arctic Chlamydomonas sp. is a dominant microalgal strain in cold or frozen freshwater in the Arctic region. The full-length open reading frame of the omega-6 fatty acid desaturase gene (AChFAD6) was obtained from the transcriptomic database of Arctic Chlamydomonas sp. from the KOPRI culture collection of polar micro-organisms. Amino acid sequence analysis indicated the presence of three conserved histidine-rich segments as unique characteristics of omega-6 fatty acid desaturases, and three transmembrane regions transported to plastidic membranes by chloroplast transit peptides in the N-terminal region. The AChFAD6 desaturase activity was examined by expressing wild-type and V254A mutant (Mut-AChFAD6) heterologous recombinant proteins. Quantitative gas chromatography indicated that the concentration of linoleic acids in AChFAD6-transformed cells increased more than 3-fold [6.73 ± 0.13 mg g -1 dry cell weight (DCW)] compared with cells transformed with vector alone. In contrast, transformation with Mut-AChFAD6 increased the concentration of oleic acid to 9.23 ± 0.18 mg g -1 DCW, indicating a change in enzymatic activity to mimic that of stearoyl-CoA desaturase. These results demonstrate that AChFAD6 of Arctic Chlamydomonas sp. increases membrane fluidity by enhancing denaturation of C18 fatty acids and facilitates production of large quantities of linoleic fatty acids in prokaryotic expression systems.

We report n-6 monounsaturated primary alcohols (C 26:1 , C 28:1 , and C 30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4's principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation's effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

The maintenance of a fluid lipid bilayer is key for membrane integrity and cell viability. We are only beginning to understand how eukaryotic cells sense and maintain the characteristic lipid compositions and bulk membrane properties of their organelles. One of the key factors determining membrane fluidity and phase behavior is the proportion of saturated and unsaturated acyl chains in membrane lipids. Saccharomyces cerevisiae is an ideal model organism to study the regulation of the lipid acyl chain composition via the OLE pathway. The OLE pathway comprises all steps involved in the regulated mobilization of the transcription factors Mga2 and Spt23 from the endoplasmic reticulum (ER), which then drive the expression of OLE1 in the nucleus. OLE1 encodes for the essential Δ9-fatty acid desaturase Ole1 and is crucial for de novo biosynthesis of unsaturated fatty acids (UFAs) that are used as lipid building blocks. This review summarizes our current knowledge of the OLE pathway, the best-characterized, eukaryotic sense-and-control system regulating membrane lipid saturation, and identifies open questions to indicate future directions.

The present study was conducted to explore the mechanisms leading to differences among fishes in the ability to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs). Replacement of fish oil with vegetable oil caused varied degrees of increase in 18-carbon fatty acid content and decrease in n-3 LC-PUFA content in the muscle and liver of rainbow trout (Oncorhynchus mykiss), Japanese seabass (Lateolabrax japonicus) and large yellow croaker (Larimichthys crocea), suggesting that these fishes have differing abilities to biosynthesize LC-PUFAs. Fish oil replacement also led to significantly up-regulated expression of FADS2 and SREBP-1 but different responses of the two PPAR-α homologues in the livers of these three fishes. An in vitro experiment indicated that the basic transcription activity of the FADS2 promoter was significantly higher in rainbow trout than in Japanese seabass or large yellow croaker, which was consistent with their LC-PUFA biosynthetic abilities. In addition, SREBP-1 and PPAR-α up-regulated FADS2 promoter activity. These regulatory effects varied considerably between SREBP-1 and PPAR-α, as well as among the three fishes. Taken together, the differences in regulatory activities of the two transcription factors targeting FADS2 may be responsible for the different LC-PUFA biosynthetic abilities in these three fishes that have adapted to different ambient salinity.

Sea urchins are broadly recognised as a delicacy and their quality as food for humans is highly influenced by their diet. Lipids in general and the long-chain polyunsaturated fatty acids (LC-PUFA) in particular, are essential nutrients that determine not only the nutritional value of sea urchins but also guarantee normal growth and reproduction in captivity. The contribution of endogenous production (biosynthesis) of LC-PUFA in sea urchins remained unknown. Using Paracentrotus lividus as our model species, we aimed to characterise both molecularly and functionally the repertoire of fatty acyl desaturases (Fads), key enzymes in the biosynthesis of LC-PUFA, in sea urchins. Three Fads, namely FadsA, FadsC1 and FadsC2, were characterised. The phylogenetic analyses suggested that the repertoire of Fads within the Echinodermata phylum varies among classes. On one hand, orthologues of the P. lividus FadsA were found in other echinoderm classes including starfishes, brittle stars and sea cucumbers, thus suggesting that this desaturase is virtually present in all echinoderms. Contrarily, the FadsC appears to be sea urchin-specific desaturase. Finally, a further desaturase termed as FadsB exists in starfishes, brittle stars and sea cucumbers, but appears to be missing in sea urchins. The functional characterisation of the P. lividus Fads confirmed that the FadsA was a Δ5 desaturase with activity towards saturated and polyunsaturated fatty acids (FA). Moreover, our experiments confirmed that FadsA plays a role in the biosynthesis of non-methylene interrupted FA, a group of compounds typically found in marine invertebrates. On the other hand, both FadsC desaturases from P. lividus showed Δ8 activity. The present results demonstrate that P. lividus possesses desaturases that account for all the desaturation reactions required to biosynthesis the physiological essential eicosapentaenoic and arachidonic acids through the so-called "Δ8 pathway"., Competing Interests: The authors have declared that no competing interests exist.

The Brazilian teleost Arapaima gigas is an iconic species of the Amazon. In recent years a significant effort has been put into the farming of arapaima to mitigate overfishing threats. However, little is known regarding the nutritional requirements of A. gigas in particular those for essential fatty acids including the long-chain polyunsaturated fatty acids (LC-PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The ability to biosynthesize LC-PUFA is dependent upon the gene repertoire of fatty acyl desaturases (Fads) and elongases (Elovl), as well as their fatty acid specificities. In the present study we characterized both molecularly and functionally an orthologue of the desaturase fatty acid desaturase 2 (fads2) from A. gigas. The isolated sequence displayed the typical desaturase features, a cytochrome b 5 -domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. Functional characterization of A. gigas fads2 showed that, similar to other teleosts, the A. gigas fads2 exhibited a predominant Δ6 activity complemented with some capacity for Δ8 desaturation. Given that A. gigas belongs to one of the oldest teleostei lineages, the Osteoglossomorpha, these findings offer a significant insight into the evolution LC-PUFA biosynthesis in teleosts., (Copyright © 2016 Elsevier Inc. All rights reserved.)