Delta-6 desaturase from borage converts linoleic acid to gamma-linolenic acid in HEK293 cells

Gamma-linolenic acid (GLA, 18:3 n6) is an essential polyunsaturated fatty acid of the omega-6 family and is found to be effective in prevention and/or treatment of various health problems. In this study, we evaluated the possibility of increasing γ-linolenic acid contents in mammalian cells using the delta-6 gene from Borago officinalis. The borage Δ6-desaturase gene (sDelta-6) was codon-optimized and introduced into HEK293 cells by lipofectin transfection. Co-expression of GFP with sDelta-6 and RT-PCR analysis indicated that sDelta-6 could be expressed in mammalian cells. Subsequently, the heterologous expression of borage Δ6-desaturase was evaluated by fatty acid analysis. Total cellular lipid analysis of transformed cells fed with linoleic acid (LA 18:2 n6) as a substrate showed that the expression of sDelta-6 resulted in an 228–483% (p < 0.05) increase of GLA when compared with that in the control cells. The highest conversion efficiency of LA into GLA in sDelta-6⁺ cells was 6.9 times higher than that in the control group (11.59% vs. 1.69%; p < 0.05). Our present work demonstrated that the sDelta-6 gene from borage could be functionally expressed in mammalian cells, and could convert LA into GLA. Furthermore, this study may pave the way to generate transgenic livestock that can synthesise GLA.     

A Simple Allele-Specific PCR Assay for Detecting <Emphasis Type="Italic">FAD2</Emphasis> Alleles in Both A and B Genomes of the Cultivated Peanut for High-Oleate Trait Selection

In cultivated tetraploid peanut (2n = 4x = 40, AABB), the conversion of oleic acid to linoleic acid is mainly catalyzed by the Δ¹² fatty acid desaturase (FAD). Two homoeologous genes (FAD2A and FAD2B) encoding for the desaturase are located on the A and B genomes, respectively. Abolishing or reducing the desaturase activity by gene mutation can significantly increase the oleic acid/linoleic acid ratio. F435-derived high-oleate peanut cultivars contain two key mutations within the Δ¹² fatty acid desaturase gene which include a 1-bp substitution of G:C→A:T in the A genome and a 1-bp insertion of A:T in the B genome. Both of these mutations contribute to abolishing or reducing the desaturase activity, leading to accumulation of oleate versus linoleate. Currently, detection of FAD2 alleles can be achieved by a cleaved amplified polymorphic sequence marker for the A genome and a real-time polymerase chain reaction (PCR) marker for the B genome; however, detection of these key mutations has to use different assay platforms. Therefore, a simple PCR assay for detection of FAD2 alleles on both genomes was developed by designing allele-specific primers and altering PCR annealing temperatures. This assay was successfully used for detecting FAD2 alleles in peanut. Gas chromatography (GC) was used to determine fatty acid composition of PCR-assayed genotypes. The results from the PCR assay and GC analysis were consistent. This PCR assay is quick, reliable, economical, and easy to use. Implementation of this PCR assay will greatly enhance the efficiency of germplasm characterization and marker-assisted selection of high oleate in peanut.     

Cloning of FAD synthetase gene from Corynebacterium ammoniagenes and its application to FAD and FMN production

The cloning of a bifunctional FAD synthetase gene, which shows flavokinase and FMN adenylyltransferase activities, from Corynebacterium ammoniagenes was tried by hybridization with synthetic DNAs corresponding to the N-terminal amino acid sequence. The cloned PstI-digested 4.4 × 10³-base (4.4-kb) fragment could not express the FAD synthetase activity in E. coli, but could increase the two activities by the same factor of about 20 in C. amminoagenes. The FAD-synthetase-gene-amplified C. amminoagenes cells were applied to the production of FAD from FMN or riboflavin. The productivity of FAD from FMN was increased four to five times compared with the parent strain, and reached a 90% molar yield. The productivity of FAD from riboflavin was increased about eight times, with a 50% molar yield. The addition of Zn²⁺ to the reaction mixtures for the conversion from riboflavin to FAD brought about the specific inhibition of adenylyltransferase activity and resulted in the accumulation of FMN.     

A novel <Emphasis Type="Italic">FAD2</Emphasis>-<Emphasis Type="Italic">1 A</Emphasis> allele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85% oleic acid content

The alteration of fatty acid profiles in soybean to improve soybean oil quality has been a long-time goal of soybean researchers. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of soybean oil compared to other oils. In the lipid biosynthetic pathway, the enzyme fatty acid desaturase 2 (FAD2) is responsible for the conversion of oleic acid precursors to linoleic acid precursors in developing soybean seeds. Two genes encoding FAD2-1A and FAD2-1B were identified to be expressed specifically in seeds during embryogenesis and have been considered to hold an important role in controlling the seed oleic acid content. A total of 22 soybean plant introduction (PI) lines identified to have an elevated oleic acid content were characterized for sequence mutations in the FAD 2-1A and FAD2-1B genes. PI 603452 was found to contain a deletion of a nucleotide in the second exon of FAD2-1A. These important SNPs were used in developing molecular marker genotyping assays. The assays appear to be a reliable and accurate tool to identify the FAD 2-1A and FAD2-1B genotype of wild-type and mutant plants. PI 603452 was subsequently crossed with PI 283327, a soybean line that has a mutation in FAD2-1B. Interestingly, soybean lines carrying both homozygous insertion/deletion mutation (indel) FAD2-1A alleles and mutant FAD2-1B alleles have an average of 82–86% oleic acid content, compared to 20% in conventional soybean, and low levels of linoleic and linolenic acids. The newly identified indel mutation in the FAD2-1A gene offers a simple method for the development of high oleic acid commercial soybean varieties.     

Overexpression of <Emphasis Type="Italic">FAD2</Emphasis> promotes seed germination and hypocotyl elongation in <Emphasis Type="Italic">Brassica napus</Emphasis>

The enzyme fatty acid desaturase 2 (FAD2) transforms oleic acid (C18:1) to linoleic acid (C18:2) in plants and as such is involved in fatty acid synthesis. It is also involved in plant development and self-defense, such as seed germination, leaf expansion and cold resistance. We have cloned the full coding region of the Brassica napus FAD2 gene and ectopically expressed it in B. napus expressing low levels of FAD2. Overexpression of FAD2 under the control of the CaMV 35S promoter resulted in an up-regulated FAD2 mRNA level in B. napus as expected. Further analysis revealed that the FAD2 transgenic lines varied greatly in terms of their physiological characteristics, such as enhanced seed germination and increased hypocotyl length, compared to non-transgenic plants, suggesting that up-regulated FAD2 can promote seed germination and hypocotyl elongation in B. napus. Our results demonstrate the possible roles of FAD2 in plant development and also provide a platform for further analysis of fatty acid synthesis in plants.     

Metabolism of Hydrocarbons in Microorganisms

The crude extract obtained from Pseudomonas aeruginosa grown on xylene as sole carbon source converted p-xylene-methyl-¹⁴C or toluene-methyl-¹⁴C to p-toluic or benzoic acid, respectively. However, addition of p-methylbenzyl or benzyl alcohol to the reaction mixture resulted in accumulation of p-methylbenzyl or benzyl alcohol. This indicates that in the crude extract, p-xylene or toluene is metabolized via its corresponding alcohol to p-toluic or benzoic acid, respectively. The enzyme system responsible for these reactions required NAD or NADH and FAD, and could be stabilized by the presence of glutathione. When the crude extract was fractionated by the use of DEAE-cellulose chromatography, it was demonstrated that two distinct protein fractions and two cofactors (NADH and FAD) were required for the step of the hydroxylation of p-xylene or toluene to its corresponding alcohol. NAD, NADP or NADPH had very few or little activity. FMN partially replaced FAD.     

Cofactors in sarcosine oxidase from Corynebacterium sp. U-96

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme that was reported to contain 1 mol of covalently bound FAD and 1 mol of non-covalently-bound FAD. This work describes the result of reinvestigation of the cofactors in this enzyme. The enzyme was found to contain 1 mol of non-covalently-bound NAD⁺, 1 mol of non-covalently-bound FAD, and 1 mol of covalent FMN. The covalent FMN was identified by the mass and amino acid sequence analyses of the flavin peptide.     

The Sunflower High-oleic Mutant Ol Carries Variable Tandem Repeats of FAD2-1, a Seed-specific Oleoyl-phosphatidyl Choline Desaturase

Ol, a chemically induced, incompletely dominant mutation, greatly increases oleic acid and is correlated with greatly reduced expression of a seed-specific oleoyl-phosphatidyl choline desaturase (FAD2-1) in developing seeds of sunflower (Helianthus annuus L.). FAD2-1 is duplicated in high-oleic (mutant) strains and cosegregates with Ol. Codominant RFLP markers have been developed for FAD2-1 and are diagnositic for the Ol mutation; however, the structure of the mutant FAD2-1 locus is unknown and polymorphic sequence-tagged-site (STS) DNA markers have not been developed for FAD2-1. The mutant was discovered to carry tandem repeats of FAD2-1 separated by a 2.67 kb intergenic region. The upstream repeat (FAD2-1U) carries a 1.69 kb intron in the 5′UTR, whereas the downstream repeat (FAD2-1D) is missing the first 1.54 kb of the 5′UTR and intron. Other than the deletion in FAD2-1D, no DNA polymorphisms were identified between wildtype and mutant FAD2-1 alleles among elite oilseed inbred lines. We developed dominant INDEL markers diagnostic for presence or absence of the Ol mutation (tandem FAD2-1 repeats) by targeting DNA sequences upstream of FAD2-1D, identified 49 SNPs and five INDELs (two haplotypes) in DNA sequences downstream of FAD2-1 in the wildtype and FAD2-1U in the mutant, identified polymorphic [AT]_n and [GT]_n repeats in the 3′UTR of FAD2-1, and developed codominant SSR and INDEL markers for FAD2-1. Novel FAD2-1 alleles found in exotic low-oleic genotypes could be introgressed into elite low-oleic genotypes to facilitate marker-assisted selection of Ol in mid- and high-oleic sunflower breeding programs.     

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a “sparing effect” on proline in the mutant.The incorporation of L-[U-¹⁴C]proline and L-[³H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologuewas incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.     

Metaphosphate-dependent phosphorylation of riboflavin to FMN by Corynebacterium ammoniagenes

Using the bifunctional FAD synthetase from Corynebacterium ammoniagenes, which has the two sequential activities of flavokinase and FMN adenylyl-transferase in FAD biosynthesis, a method of production of the intermediate FMN without any accumulation of FAD was investigated. Various phosphate polymers having no adenylyl moiety were tested for their ability to phosphorylate riboflavin to FMN, using a crude enzyme from C. ammoniagenes/pKH46, which is an FAD-synthetase-gene-dosed strain. Only metaphosphate, other than ATP, could phosphorylate riboflavin to FMN, but FAD did not accumulate at all. The conditions for the conversion of riboflavin to FMN were optimized. The metaphosphate-dependent phosphorylation reaction required Mg²⁺ as the most effective divalent cation. The best concentrations were 10 mM for MgCl₂ and 3mg/ml for metaphosphate. The riboflavin added to the reaction mixture was almost completely converted into FMN after 6 h incubation in the presence of high concentrations of the enzyme preparation.     

Histone acetyltransferase general control non‐repressed protein 5 (GCN5) affects the fatty acid composition of Arabidopsis thaliana seeds by acetylating fatty acid desaturase3 (FAD3)

Seed oils are important natural resources used in the processing and preparation of food. Histone modifications represent key epigenetic mechanisms that regulate gene expression, plant growth and development. However, histone modification events during fatty acid (FA) biosynthesis are not well understood. Here, we demonstrate that a mutation of the histone acetyltransferase GCN5 can decrease the ratio of α‐linolenic acid (ALA) to linoleic acid (LA) in seed oil. Using RNA‐Seq and ChIP assays, we identified FAD3, LACS2, LPP3 and PLAIIIβ as the targets of GCN5. Notably, the GCN5‐dependent H3K9/14 acetylation of FAD3 determined the expression levels of FAD3 in Arabidopsis thaliana seeds, and the ratio of ALA/LA in the gcn5 mutant was rescued to the wild‐type levels through the overexpression of FAD3. The results of this study indicated that GCN5 modulated FA biosynthesis by affecting the acetylation levels of FAD3. We provide evidence that histone acetylation is involved in FA biosynthesis in Arabidopsis seeds and might contribute to the optimization of the nutritional structure of edible oils through epigenetic engineering.     

Significant enhancement of fatty acid composition in seeds of the allohexaploid,Camelina sativa,using CRISPR/Cas9 gene editing

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. thaliana T₂ and T₃ generation seeds exhibiting these types of altered fatty acid profiles were homozygous for disrupted FAD2 alleles. In the allohexaploid, Camelina, guide RNAs were designed that simultaneously targeted all three homoeologous FAD2 genes. This strategy that significantly enhanced oil composition in T₃ and T₄ generation Camelina seeds was associated with a combination of germ‐line mutations and somatic cell mutations in FAD2 genes in each of the three Camelina subgenomes.     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20–60°C) and protein concentration (5–100 µmol L^?1). The time courses of loss of enzyme activity in 100 mmol L^?1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k_a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k_b) and binding (k_?b) as well as the irreversible aggregation of the apo-enzyme (k_agg) were less sensitive to changes in temperature, their activation energy (E_a) being about 52 kJ mol^?1 in comparison with an E_a value of 185 kJ mol^?1 for k_a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5′-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^?1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

Modification of fatty acid composition in tomato (Lycopersicon esculentum) by expression of a borage delta6-desaturase

The improvement of nutritional quality is one potential application for the genetic modification of plants. One possible target for such manipulation is the modification of fatty acid metabolism. In this work, expression of a borage Δ⁶-desaturase cDNA in tomato (Lycopersicon esculentum L.) has been shown to produce γ-linolenic acid (GLA; 18:3 Δ^6,9,12) and octadecatetraenoic acid (OTA; 18:4 Δ^6,9,12,15) in transgenic leaf and fruit tissue. This genetic modification has also, unexpectedly, resulted in a reduction in the percentage of linoleic acid (LA 18:2 Δ^9,12) and a concomitant increase in the percentage of α-linolenic acid (ALA; 18:3 Δ^9,12,15) in fruit tissue. These changes in fatty acid composition are thought to be beneficial for human health.     

Construction and Functional Analysis of CRISPR/Cas9 Vector of <i>FAD2</i> Gene Family in Soybean

Soybean oleic acid content is one of the important indexes to evaluate the quality of soybean oil. In the synthesis pathway of soybean fatty acids, the FAD2 gene family is the key gene that regulates the production of linoleic acid from soybean oleic acid. In this study, CRISPR/Cas9 gene editing technology was used to regulate FAD2 gene expression. Firstly, the CRISPR/Cas9 single knockout vectors GmFAD2-1B and GmFAD2-2C and double knockout vectors GmFAD2-2A-3 were constructed. Then, the three vectors were transferred into the recipient soybean variety Jinong 38 by Agrobacterium-mediated cotyledon node transformation, and the mutant plants were obtained. Functional analysis and comparison of the mutant plants of the T2 and T3 generations were carried out. The results showed that there was no significant difference in agronomic traits between the CRISPR/Cas9 single and double knockout vectors and the untransformed CRISPR/Cas9 receptor varieties. The oleic acid content of the plants that knocked out the CRISPR/Cas9 double gene vector was significantly higher than that of the single gene vector.     

Membrane lipid polyunsaturation mediated by FATTY ACID DESATURASE 2 (FAD2) is involved in endoplasmic reticulum stress tolerance in Arabidopsis thaliana

Unsaturation of membrane glycerolipid classes at their hydrophobic fatty acid tails critically affects the physical nature of the lipid molecule. In Arabidopsis thaliana, 7 fatty acid desaturases (FADs) differently desaturate each glycerolipid class in plastids and the endoplasmic reticulum (ER). Here, we showed that polyunsaturation of ER glycerolipids is required for the ER stress response. Through systematic screening of FAD mutants, we found that a mutant of FAD2 resulted in a hypersensitive response to tunicamycin, a chemical inducer of ER stress. FAD2 converts oleic acid to linoleic acid of the fatty acyl groups of ER‐synthesized phospholipids. Our functional in vivo reporter assay revealed the ER localization and distinct tissue‐specific expression patterns of FAD2. Moreover, glycerolipid profiling of both mutants and overexpressors of FAD2 under tunicamycin‐induced ER stress conditions, along with phenotypic screening of the mutants of the FAD family, suggested that the ratio of monounsaturated fatty acids to polyunsaturated fatty acids, particularly 18:1 to 18:2 species, may be an important factor in allowing the ER membrane to cope with ER stress. Therefore, our results suggest that membrane lipid polyunsaturation mediated by FAD2 is involved in ER stress tolerance in Arabidopsis.