Expression of meadowfoam Des5 and FAE1 genes in yeast and in transgenic soybean somatic embryos,and their roles in fatty acid modification

Meadowfoam (Limnanthes spp.) species are unique in that their seeds are rich in the unusual fatty acids Δ⁵-eicosenoic acid (C20:1^Δ5) and the diene, C22:2^{Δ5, Δ13}. Previously the cloning of Δ⁵ desaturase (Des5) and fatty acid elongase 1 (FAE1) meadowfoam genes and their expression in soybean were reported. Here, we present the first successful expression of the Limnanthes Des5 in yeast, resulting in the desaturation of C16:0, C18:0 and C20:0 to their corresponding cis Δ⁵ isomers. In soybean (Glycine max L.), Limnanthes Des5/FAE1 double transformant somatic embryos fed with radiolabeled C14:0 or C16:0 could elongate these substrates to C18:0, C20:0 and C22:0 and C24:0. However, radiolabeled C18:1^Δ9 or C20:1^Δ11 were not elongated to their respective monounsaturated very long-chain products, confirming that the cloned Limnanthes FAE1 homolog gene product was specific for elongating saturated fatty acids. To understand better the biosynthetic pathway for C22:2^{Δ5, Δ13}, soybean somatic embryos transformed with the Des5 cDNA were fed in culture with 〚1-¹⁴C〛C 22:1^Δ13 fatty acid, which resulted in the biosynthesis of 〚1-¹⁴C〛-labeled C22:2^{Δ5, Δ13}. Cell-free preparations enriched with detergent-solubilized Δ⁵ desaturase activity extracted from both developing meadowfoam seeds and from Des5 transgenic soybean embryos, produced ¹⁴C-22:2^{Δ5, Δ13} when supplied with 〚1-¹⁴C〛 C22:1-CoA. Thus, both the in vivo and in vitro experiments showed that the biosynthesis of C22:2^{Δ5, Δ13} can occur in somatic soybean embryos transformed with the Limnanthes Des5 cDNA, and confirmed that the pathway for C22:2 biosynthesis in meadowfoam involves further desaturation of erucoyl-CoA by a Δ⁵-regiospecific desaturase.     

Revealing the complementation of ferredoxin by cytochrome b 5 in the Spirulina-∆6-desaturation reaction by N-terminal fusion and co-expression of the fungal-cytochrome b 5 domain and Spirulina-∆6-acyl-lipid desaturase

Spirulina-acyl-lipid desaturases are integral membrane proteins found in thylakoid and plasma membranes. These enzymes catalyze the fatty acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. It has been reported that the cyanobacterial desaturases use ferredoxin as an electron donor, whereas the acyl-lipid desaturase in plant cytoplasm and the acyl-CoA desaturase of animals and fungi use cytochrome b ₅. The low level of ferredoxin present in Escherichia coli cells leads to an inability to synthesize GLA when the cells are transformed with the Spirulina-∆⁶ desaturase, desD, and grown in the presence of the reaction substrate, linoleic acid. In this study, Spirulina-∆⁶ desaturase, encoded by the desD gene, was N-terminally fused and co-expressed with the cytochrome b ₅ domain from Mucor rouxii. The product, GLA, made heterologously in E. coli and Saccharomyces cerevisiae, was then detected and analyzed. The results revealed the production of GLA by Spirulina-∆⁶ desaturase fused or co-expressed with cytochrome b ₅ in E. coli cells, in which GLA production by this gene cannot occur in the absence of cytochrome b ₅. Moreover, the GLA production ability in the E. coli host cells was lost after the single substitution mutation was introduced to H52 in the HPGG motif of the cytochrome b ₅ domain. These results revealed the complementation of the ferredoxin requirement by the fusion or co-expression of the fungal-cytochrome b ₅ domain in the desaturation process of Spirulina-∆⁶ desaturase. Furthermore, the free form of cytochrome b ₅ domain can also enhance GLA production by the Spirulina-desD gene in yeast cells.     

Metabolic engineering to produce γ-linolenic acid in <Emphasis Type="Italic">Brassica napus</Emphasis> using a Δ6-desaturase from pike eel

We generated γ-linolenic acid (GLA, C18:3^Δ6,9,12)-producing transgenic Brassica napus transformed with McD6DES, the Δ6-desaturase gene identified from pike eel (Muraenesox cinereus) under the control of the seed-specific vicilin promoter. Seed-specific expression of McD6DES in B. napus produced up to 8.4% of GLA by creating a double bond at the sixth position from the carboxyl end of linoleic acid (LA, C18:2^Δ9,12) in seeds. These results demonstrate that McD6DES expression enables to reconstitute in polyunsaturated fatty acid biosynthetic pathways, highlighting the potential of GLA biosynthesis as a target for metabolic engineering of oilseed crops.     

Identification and characterization of new Δ-17 fatty acid desaturases

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

Identification of the substrate recognition region in the Δ6-fatty acid and Δ8-sphingolipid desaturase by fusion mutagenesis

Δ⁸-sphingolipid desaturase and Δ⁶-fatty acid desaturase share high protein sequence identity. Thus, it has been hypothesized that Δ⁶-fatty acid desaturase is derived from Δ⁸-sphingolipid desaturase; however, there is no direct proof. The substrate recognition regions of Δ⁶-fatty acid desaturase and Δ⁸-sphingolipid desaturase, which aid in understanding the evolution of these two enzymes, have not been reported. A blackcurrant Δ⁶-fatty acid desaturase and a Δ⁸-sphingolipid desaturase gene, RnD6C and RnD8A, respectively, share more than 80 % identity in their coding protein sequences. In this study, a set of fusion genes of RnD6C and RnD8A were constructed and expressed in yeast. The Δ⁶- and Δ⁸-desaturase activities of the fusion proteins were characterized. Our results indicated that (1) the exchange of the C-terminal 172 amino acid residues can lead to a significant decrease in both desaturase activities; (2) amino acid residues 114–174, 206–257, and 258–276 played important roles in Δ⁶-substrate recognition, and the last two regions were crucial for Δ⁸-substrate recognition; and (3) amino acid residues 114–276 of Δ⁶-fatty acid desaturase contained the substrate recognition site(s) responsible for discrimination between ceramide (a substrate of Δ⁸-sphingolipid desaturase) and acyl-PC (a substrate of Δ⁶-fatty acid desaturase). Substituting the amino acid residues 114-276 of RnD8A with those of RnD6C resulted in a gain of Δ⁶-desaturase activity in the fusion protein but a loss in Δ⁸-sphingolipid desaturase activity. In conclusion, several regions important for the substrate recognition of Δ8-sphingolipid desaturase and Δ⁶-fatty acid desaturase were identified, which provide clues in understanding the relationship between the structure and function in desaturases.     

The composition of fatty acids in the endosperm and embryo lipids of <Emphasis Type="Italic">Pinus sibirica</Emphasis> and <Emphasis Type="Italic">P. sylvestris</Emphasis> seeds

The fatty acid (FA) composition of storage lipids in the seed endosperms and embryos of two pine species, Pinus sibirica and P. sylvestris, and possible biosynthetic pathways of these acids were studied by the GLC method. Linoleic acid predominated in the embryo and endosperm lipids of both P. sibirica (43.5 and 42.6%) and P. sylvestris (44.8 and 46.8%); this was evidently determined by a high expression of the gene encoding stearoyl-Δ9 acyl-lipid desaturase and the fad2 gene encoding microsomal ω6 acyl-lipid desaturase. P. sibirica lipids of the embryo and endosperm contained more oleic acid (22.0 and 24.0%, respectively) than corresponding P. sylvestris lipids (18.7 and 14%). Storage lipids of conifer seeds contain Δ5-unsaturated FAs: taxoleic (18:2Δ5,9), ephedrenic (18:2Δ5,11), pinoleenic (18:3Δ5,9,12), skiadonic (18:3Δ5,11, 14), and coniferonic (18:4Δ5,9,12,15). In the endosperm and embryos of P. sylvestris, the content of pinolenic acid was higher (22.1 and 19.6%) than in P. sibirica seeds (19.1 and 18.6%).     

High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds

Gamma linolenic acid (GLA; C18:3Δ6,9,12 cis), also known as γ-Linolenic acid, is an important essential fatty acid precursor for the synthesis of very long chain polyunsaturated fatty acids and important pathways involved in human health. GLA is synthesized from linoleic acid (LA; C18:2Δ9,12 cis) by endoplasmic reticulum associated Δ6-desaturase activity. Currently sources of GLA are limited to a small number of plant species with poor agronomic properties, and therefore an economical and abundant commercial source of GLA in an existing crop is highly desirable. To this end, the seed oil of a high LA cultivated species of safflower (Carthamus tinctorius) was modified by transformation with Δ6-desaturase from Saprolegnia diclina resulting in levels exceeding 70% (v/v) of GLA. Levels around 50% (v/v) of GLA in seed oil was achieved when Δ12-/Δ6-desaturases from Mortierella alpina was over-expressed in safflower cultivars with either a high LA or high oleic (OA; C18:1Δ9 cis) background. The differences in the overall levels of GLA suggest the accumulation of the novel fatty acid was not limited by a lack of incorporation into the triacylgylcerol backbone (>66% GLA achieved), or correlated with gene dosage (GLA levels independent of gene copy number), but rather reflected the differences in Δ6-desaturase activity from the two sources. To date, these represent the highest accumulation levels of a newly introduced fatty acid in a transgenic crop. Events from these studies have been propagated and recently received FDA approval for commercialization as Sonova?400.     

Truncation Mutants Highlight a Critical Role for the N- and C-termini of the Spirulina Δ6 Desaturase in Determining Regioselectivity

The results of our previous study on heterologous expression in Escherichia coli of the gene desD, which encodes Spirulina Δ⁶ desaturase, showed that co-expression with an immediate electron donor—either cytochrome b ₅ or ferredoxin—was required for the production of GLA (γ-linolenic acid), the product of the reaction catalyzed by Δ⁶ desaturase. Since a system for stable transformation of Spirulina is not available, studies concerning Spirulina-enzyme characterization have been carried out in heterologous hosts. In this present study, the focus is on the role of the enzyme’s N- and C-termini, which are possibly located in the cytoplasmic phase. Truncated enzymes were expressed in E. coli by employing the pTrcHisA expression system. The truncation of the N- and C-terminus by 10 (N10 and C10) and 30 (N30 and C30) amino acids, respectively, altered the enzyme’s regioselective mode from one that measures from a preexisting double bond to that measuring from the methyl end of the substrate.     

Two Fatty Acid Elongases Possessing C18-Δ6/C18-Δ9/C20-Δ5 or C16-Δ9 Elongase Activity in Thraustochytrium sp. ATCC 26185

Thraustochytrids, unicellular eukaryotic marine protists, accumulate polyunsaturated fatty acids. Here, we report the molecular cloning and functional characterization of two fatty acid elongase genes (designated tselo1 and tselo2), which could be involved in the desaturase/elongase (standard) pathway in Thraustochytrium sp. ATCC 26185. TsELO1, the product of tselo1 and classified into a Δ6 elongase group by phylogenetic analysis, showed strong C18-Δ6 elongase activity and relatively weak C18-Δ9 and C20-Δ5 activities when expressed in the budding yeast Saccharomyces cerevisiae. TsELO2, classified into a Δ9 elongase subgroup, showed only C16-Δ9 activity. When expressed in Aurantiochytrium limacinum mh0186 using a thraustochytrid-derived promoter and a terminator, TsELO1 exhibited almost the same specificity as expressed in the yeast but TsELO2 showed weak C18-Δ9 activity, in addition to its main C16-Δ9 activity. These results suggest that TsELO1 functions not only as a C18-Δ6 and a C20-Δ5 elongase in the main route but also as a C18-Δ9 elongase in the alternative route of standard pathway, while TsELO2 functions mainly as a C16-Δ9 elongase generating vaccenic acid (C18:1n?7) in thraustochytrids. This is the first report describing a fatty acid elongase harboring C16-Δ9 activity in thraustochytrids.     

Characterization of three novel desaturases involved in the delta-6 desaturation pathways for polyunsaturated fatty acid biosynthesis from Phytophthora infestans

Phytophthora infestans is the causative agent of potato blight that resulted in the great famine in Ireland in the nineteenth century. This microbe can release large amounts of the C20 very long-chain polyunsaturated fatty acids arachidonic acid (ARA; 20:4Δ^{5, 8, 11, 14}) and eicosapentaenoic acid (EPA; 20:5Δ^{5, 8, 11, 14, 17}) upon invasion that is known to elicit a hypersensitive response to their host plant. In order to identify enzymes responsible for the biosynthesis of these fatty acids, we blasted the recently fully sequenced P. infestans genome and identified three novel putatively encoding desaturase sequences. These were subsequently functionally characterized by expression in Saccharomyces cerevisiae and confirmed that they encode desaturases with Δ12, Δ6 and Δ5 activity, designated here as PinDes12, PinDes6 and PinDes5, respectively. This, together with the combined fatty acid profiles and a previously identified Δ6 elongase activity, implies that the ARA and EPA are biosynthesized predominantly via the Δ6 desaturation pathways in P. infestans. Elucidation of ARA and EPA biosynthetic mechanism may provide new routes to combating this potato blight microbe directly or by means of conferring resistance to important crops.     

Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002

Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C₁₈ fatty acids increased in cells grown at lower temperature for all lipid classes, and ω3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the ω3 position of C₁₈ fatty acids increased gradually during a 12-h period after a temperature shift-down to 22°C. However, the mRNA levels of the desA (Δ12 desaturase), desB (ω3 desaturase) and desC (Δ9 desaturase) genes increased within 15 min after a temperature shift-down to 22°C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38°C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22°C and 38°C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium.     

INTRACRANIAL CONVERSION OF LINOLEIC ACID TO ARACHIDONIC ACID: EVIDENCE FOR LACK OF Δ8 DESATURASE IN THE BRAIN

Eleven-day old rats were given intracranial injection of [1-¹⁴C]linoleic acid (all cis 9,12 octadecadienoic acid) and sacrificed after 8 h. Analysis of brain fatty acids showed that 16:0, 18:2, 20:2,20:3 and 20:4 were labeled. Separation by AgN03:Si02 TLC plates followed by reductive ozonolysis characterized thc polyunsaturated fatty acids as 18:2 (Δ9,12), 20:2 (Δ11,14), 20:3 (Δ8,11,14) and 20:4 (Δ5,8,11,14). A smaller amount of 18:3 (Δ6,9,12) was also identified. This initially suggested 20:2 (A1 1,14) as an intermediate in the optional pathway of biosynthesis of arachidonate. However, when [l-14C]eicosadienoic acid (Δ1 1,141 itself was injected in the brain it was converted to 20:3 (Δ5,11,14) (a non-methylene interrupted double bond system) rather than the expected 20:3 (Δ8,11,14). Only a small amount of arachidonate was formed from 20:2 (Δ11,14). Thus it was concluded that 20:2 (Δ11,14) was not an intermediate in the pathways of arachidonate biosynthesis due to lack of Δ⁵ desaturase in thc brain which agrees with the findings of SPKECRER & LEE (1975) in rat liver.     

Functional Expression of <Emphasis Type="Italic">Spirulina</Emphasis>-Δ<Superscript>6</Superscript> Desaturase Gene in Yeast, <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Spirulina-acyl-lipid desaturases are membrane-bound enzymes found in thylakoid and plasma membranes. These enzymes carry out the fatty acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. In this study, Spirulina-Δ⁶ desaturase encoded by the desD gene was heterologously expressed and characterized in Saccharomyces cerevisiae. We then conducted site-directed mutagenesis of the histidine residues in the three histidine boxes to determine the role of these amino acid residues in the enzyme function. Our results showed that while four mutants showed complete loss of Δ⁶-desaturase activity and two mutants showed only trace of the activity, the enzyme activity could be partially restored by chemical rescue using exogenously provided imidazole. This study reveals that the histidine residues (which have imidazole as their functional group) in the conserved clusters play a critical role in Δ⁶-desaturase activity, possibly by providing a di-iron catalytic center. In our previous study, this enzyme was expressed in Escherichia coli. The results reveal that the enzyme can function only in the presence of an exogenous cofactor, ferredoxin, provided in vitro. This evidence suggests that baker’s yeast has a cofactor that can complement ferredoxin, thought to act as an electron donor for the Δ⁶ desaturation in cyanobacteria, including Spirulina. The electron donor of the Spirulina-Δ⁶ desaturation in yeast is more likely to be cytochrome b₅, which is absent in E. coli. This means that the enzyme expressed in S. cerevisiae can catalyze the biosynthesis of the product, GLA, in vivo.     

Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid

Main conclusion

Arabidopsis was engineered to produce 21.2 % punicic acid in the seed oil. Possible molecular factors limiting further accumulation of the conjugated fatty acid were investigated. Punicic acid (18:3Δ^{9cis,11trans,13cis} ) is a conjugated linolenic acid isomer and is a main component of Punica granatum (pomegranate) seed oil. Medical studies have shown that punicic acid is a nutraceutical with anti-cancer and anti-obesity properties. It has been previously demonstrated that the conjugated double bonds in punicic acid are produced via the catalytic action of fatty acid conjugase (FADX), which is a homolog of the oleate desaturase. This enzyme catalyzes the conversion of the Δ¹²-double bond of linoleic acid (18:2Δ^9cis,12cis ) into conjugated Δ^11trans and Δ^13cis -double bonds. Previous attempts to produce punicic acid in transgenic Arabidopsis thaliana seeds overexpressing P. granatum FADX resulted in a limited accumulation of punicic acid of up to 4.4 %, accompanied by increased accumulation of oleic acid (18:1?^9cis ), suggesting that production of punicic acid in some way inhibits the activity of oleate desaturase (Iwabuchi et al. 2003). In the current study, we applied a new strategy to enhance the production of punicic acid in a high linoleic acid A. thaliana fad3/fae1 mutant background using the combined expression of P. granatum FADX and FAD2. This approach led to the accumulation of punicic acid at the level of 21 % of total fatty acids and restored the natural proportion of oleic acid observed in the A. thaliana fad3/fae1 mutant. In addition, we provide new insights into the high oleate phenotype and describe factors limiting the production of punicic acid in genetically engineered plants.     

Biosynthesis of triacylglycerol in Thunbergia alata: Additional evidence for involvement of phosphatidylcholine in unusual monoenoic oil production

The seed oil of Thunbergia alata has an unusual fatty acid composition which consists of more than 80 % 16:1^Δ6. This fatty acid is produced in the plastid by the action of a Δ6 palmitoyl (16:0)-ACP desaturase. To examine the biosynthesis of triacylglycerol (TAG) containing high concentrations of this unusual monoenoic fatty acid, endosperm dissected from developing T. alata seeds was labeled with [1-¹⁴C]-acetate. At early time points (5–15 min), the predominant labeled lipid was PC whereas at later time points (greater than 30 min) TAG became the major labeled lipid. Analysis of the acyl group composition of each lipid revealed that radiolabeled 16:1^Δ6 was highest at early time points in PC while at later time points, it was found to be highest in TAG. Further analysis of the distribution of labeled acyl groups within PC indicated that 16:1^Δ6 at the sn-2 position comprised the majority (55–78 %) of total labeled acyl groups whereas 16:1^Δ6 at the sn-1 position constituted only a small fraction (12–15 %) of the total labeled acyl groups. In contrast, unlabeled PC contained lower amounts of 16:1^Δ6 (16 %) at the sn-2 position. These results are consistent with previous studies suggesting a flux of novel monoenoic acids through PC during TAG biosynthesis, and furthermore imply a stereospecific flux through the sn-2 position of PC.