Evolution-related amino acids play important role in determining regioselectivity of fatty acid desaturase from <Emphasis Type="Italic">Pichia pastoris</Emphasis>

ω³-fatty acid desaturase and Δ¹²-fatty acid desaturase of Pichia pastoris with distinguishable regioselectivity and high degree of sequence similarity were chosen for regioselectivity research. Chimeras were constructed in which Histidine-rich boxes 1, 2 and the carboxyl terminal region of ω³-fatty acid desaturase were replaced with corresponding region of Δ¹²-fatty acid desaturase. The replacement was found to result in a change of regioselectivity from ωy to x + 3 by functionally characterizing these chimeric enzymes in Saccharomyces cerevisae strain INVScI. Using site-directed mutagenesis, we further demonstrated that seven conserved amino acids of ω³-fatty acid desaturase within the first two Histidine-rich regions are responsible for the regioselectivity switch. Therefore, the regioselectivity of fatty acid desaturases may be better understood by investigating the evolutionary relationships of different fatty acid desaturases. Dongsheng Wei is the partake of first-author’s profits.     

Co-expression of the borage Δ6 desaturase and the Arabidopsis Δ15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean

Two relatively rare fatty acids, γ-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, α-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Δ⁶ desaturase and an Arabidopsis Δ¹⁵ desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Δ⁶ desaturase event with the Δ¹⁵ desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean β-conglycinin promoter. Soybean events that carried only the Δ¹⁵ desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids.     

Functional characterization of a delta 6-desaturase gene from the black seabream (<Emphasis Type="Italic">Acanthopagrus schlegeli</Emphasis>)

Delta 6-fatty acid desaturase (D6DES) is used in the synthesis of polyunsaturated fatty acids (PUFAs) from microorganisms to higher animals, including arachidonic acid (ARA) and eicosapentaenoic acid (EPA). A 1,338 bp full-length cDNA encoding D6DES was cloned from Acanthopagrus schlegeli (AsD6DES) through degenerate- and RACE-PCR methods. A recombinant vector expressing AsD6DES (pYES-AsD6DES) was subsequently constructed and transformed into Saccharomyces cerevisiae to test the enzymatic activity of AsD6DES towards the production of n-6 and n-3 fatty acids. The exogenously expressed AsD6DES produced γ-linolenic acid (18:3 n-6) and stearidonic acid (18:4n-3) at 26 and 36% from exogenous linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3), respectively, indicating that it is essentially a delta 6-fatty acid desaturase.     

Enhanced heterologous production of eicosapentaenoic acid in Escherichia coli cells that co-express eicosapentaenoic acid biosynthesis pfa genes and foreign DNA fragments including a high-performance catalase gene, vktA

Cellular eicosapentaenoic acid (EPA) makes up approximately 3% of total fatty acids in Escherichia coli DH5α, a strain that carries EPA biosynthesis genes (pEPAΔ1). EPA was increased to 12% of total fatty acids when the host cell co-expressed the vector pGBM3::sa1(vktA), which carried the high-performance catalase gene, vktA. Where this vector was co-expressed, the transformant accumulated a large amount of VktA protein. However, the EPA production of cells carrying the vector, that included the insert lacking almost the entire vktA gene, was approximately 6%. This suggests that the retention of a large DNA insert in the vector and the accumulation of the resulting protein, but not the catalytic activity of VktA catalase, would potentially be able to increase the content of EPA.     

Effect of cyanocobalamin and p-toluic acid on the fatty acid composition of Schizochytrium limacinum (Thraustochytriaceae, Labyrinthulomycota)

Changes in the fatty acid composition of docosahexaenoic acid (DHA)-producing Schizochytrium limacinum SR21 were investigated. The addition of cyanocobalamin, which is an active component of vitamin B₁₂, decreased the content of odd-chain fatty acids such as pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0). Cyanocobalamin may upregulate the cobalamin-dependent methylmalonyl-CoA mutase, which converts propionic acid to succinic acid, thereby decreasing the content of odd-chain fatty acids. The addition of p-toluic acid resulted in a decrease in docosapentaenoic acid (DPA, 22:5n-6) content and an increase in eicosapentaenoic acid (EPA, 20:5n-3) content in a dose-dependent manner. Two additional peaks of fatty acids, characterized as Δ4,7,10,14-eicosatetraenoic acid (20:4n-7) and Δ4,7,10,14-docosatetraenoic acid (22:4n-9), were detected.     

Enzymatic characterisation of high-palmitic acid sunflower (Helianthus annuus L.) mutants

Two high-palmitic acid sunflower (Helianthus annuus L.) mutants, CAS-5 and CAS-12, have been biochemically characterised. The enzymatic activities found to be responsible for the mutant characteristics are β-keto-acyl-acyl carrier protein synthetase II (KASII; EC 2.3.1.41) and acyl-acyl carrier protein thioesterase (EC 3.1.2.14). Our data suggest that the high-palmitic acid phenotype observed in both mutant lines is due to the combined effect of a lower KASII activity and a higher thioesterase activity with respect to palmitoyl-acyl carrier protein (16:0-ACP). The level of the latter enzyme appeared to be insufficient to hydrolyse the produced 16:0-ACP completely. As a consequence of this, three new fatty acids appear: palmitoleic acid (16:1 Δ9), asclepic acid (18:1 Δ11), and palmitolinoleic acid (16:2 Δ9 Δ12). These fatty acids should be synthesised from palmitoyl-ACP or a derivative by the action of the stearoyl-ACP desaturase, fatty acid synthetase II and oleoyl-phosphatidylcholine desaturase, respectively. Received: 11 July 1998 / Accepted: 10 October 1998     

Spore germination in <Emphasis Type="Italic">Mortierella alpina</Emphasis> is associated with a transient depletion of arachidonic acid and induction of fatty acid desaturase gene expression

Mortierella alpina is an oleaginous filamentous fungus whose vegetative mycelium is known to accumulate triglyceride oil containing large amounts of arachidonic acid (ARA 20:4, n − 6). We report that the spores of Mortierella alpina also contain a large proportion of ARA, comprising 50% of total fatty acid. Fatty acid desaturase genes were not expressed in dormant spores but were induced during germination, following a significant drop in the level of ARA (down from 50% of total fatty acid to 12%) prior to germ-tube emergence. We propose that ARA serves as a reserve supply of carbon and energy that is utilised during the early stages of spore germination in Mortierella alpina.     

Improvement of polyunsaturated fatty acids synthesis by the coexpression of CYB5 with desaturase genes in Saccharomyces cerevisiae

Since Saccharomyces cerevisiae contains Δ9 fatty acid desaturase (OLE1) as a sole fatty acid desaturase, it produces saturated and monounsaturated fatty acids of 16- and 18-carbon compounds. We showed earlier that Kluyveromyces lactis Δ12 (KlFAD2) and ω3 (KlFAD3) fatty acid desaturase genes enabled S. cerevisiae to make also polyunsaturated fatty acids (PUFAs), linoleic (18:2n-6), and α-linolenic (18:3n-3) acids. Unlike Δ9 fatty acid desaturase Ole1p, the two added fatty acid desaturases (KlFAD2and KlFAD3) do not contain a cytochrome b5 domain, and we now report on effects of the overexpression of K. lactis and S. cerevisiae cytochrome b5 (CYB5) genes as well as temperature effects on PUFA synthesis. Without extra cytochrome b5, while PUFA synthesis is significant at low temperature (20 °C), it was marginal at 30 °C. Overexpression of cytochrome b5 at 20 °C did not affect the fatty acid synthesis so much, but it significantly enhanced the synthesis of PUFA at 30 °C.     

Identification of Δ9-elongation activity from <Emphasis Type="Italic">Thraustochytrium aureum</Emphasis> by heterologous expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The Δ9-elongase isolated from Thraustochytrium aureum, which contains a high level of polyunsaturated fatty acids (PUFAs), was demonstrated to be associated with the synthesis of C20 PUFAs. The TaELO gene contains a 825 bp ORF that encodes a protein of 274 amino acids that shares a high similarity with other PUFA elongases. The expression of the TaELO gene in Pichia pastoris resulted in the elongation of linoleic acid (LA, C18:2; n-6) and α-linolenic acid (ALA, C18:3; n-3) to eicosadienoic acid (EDA, C20:2; n-6) and eicosatrienoic acid (ETrA, C20:3; n-3), respectively. The endogenous conversion rate of LA and ALA to EDA and ETrA was 32.68 and 38.57%, respectively. In addition, TaELO was also able to synthesize eicosenoic acid (C20:1; n-9) from oleic acid (OA, C18:1; n-9), even though the conversion level was low (2.81%). Furthermore, TaELO was able to carry out the 6Δ-elongation of γ-linolenic acid (GLA, C18:3; n-6) to dihomo-γ-linolenic acid (DGLA, C20:3; n-6) and Δ5-elongation of eicosapentaenoic acid (EPA, C20:5; n-3) to docosapentaenoic acid (DPA, C22:5; n-3). The conversion rate of GLA to DGLA and EPA to DPA were 93 and 28.36%, respectively. The TaELO protein was confirmed to have multifunctional activities, such as Δ9, Δ6, and Δ5-elongations as well as the elongation of monounsaturated fatty acid.     

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.     

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.     

Cloning of a Novel Omega-6 Desaturase from Flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) and Its Functional Analysis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The Δ12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Δ12 desaturase gene amplified from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids—putative membrane-bound Δ12 desaturase protein. Sequence comparisons show that the novel sequence has 85% similarity with previously reported flax Δ12 desaturase at amino acid level and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that are universally present among plant desaturases. The signature amino acid sequence ‘YNNKL’ was also found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However, exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2.     

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

Nannochloropsis oceanica is an oleaginous microalga rich in ω3 long‐chain polyunsaturated fatty acids (LC‐PUFAs) content, in the form of eicosapentaenoic acid (EPA). We identified the enzymes involved in LC‐PUFA biosynthesis in N. oceanica CCMP1779 and generated multigene expression vectors aiming at increasing LC‐PUFA content in vivo. We isolated the cDNAs encoding four fatty acid desaturases (FAD) and determined their function by heterologous expression in S. cerevisiae. To increase the expression of multiple fatty acid desaturases in N. oceanica CCMP1779, we developed a genetic engineering toolkit that includes an endogenous bidirectional promoter and optimized peptide bond skipping 2A peptides. The toolkit also includes multiple epitopes for tagged fusion protein production and two antibiotic resistance genes. We applied this toolkit, towards building a gene stacking system for N. oceanica that consists of two vector series, pNOC‐OX and pNOC‐stacked. These tools for genetic engineering were employed to test the effects of the overproduction of one, two or three desaturase‐encoding cDNAs in N. oceanica CCMP1779 and prove the feasibility of gene stacking in this genetically tractable oleaginous microalga. All FAD overexpressing lines had considerable increases in the proportion of LC‐PUFAs, with the overexpression of Δ12 and Δ5 FAD encoding sequences leading to an increase in the final ω3 product, EPA.     

Changes in desaturase activity and the fatty acid composition of microsomal membranes from liver tissue of thermally-acclimating rainbow trout

Summary Rainbow trout (Salmo gairdneri) were acclimated to either 5 or 20°C, and then transferred to the opposite temperature, and changes in the fatty acid composition of liver microsomal membranes and the activities of the hepatic Δ9, Δ6, and Δ5 desaturases were measured at intervals of up to one month post-transfer. Inital changes (days 0–3) in fatty acid composition were: (1) an increase in the proportion of saturates and a decrease in the proportion of polyunsaturates during warm acclimation, and (2) a decrease in the proportion of saturates during cold acclimation. The activity of the Δ6 desaturase approximately doubled immediately following the changes in temperature, but alterations in Δ9 and Δ5 desaturase activities required at least 3 days to occur. The results indicate that desaturase enzymes do not play a major role in the initial adaptation of membrane fatty acid composition to changes in temperature. However, the desaturase enzymes may be involved in the later stages (3–28 days) of the acclimatory process. The proportion of monoenes was well correlated with Δ9 desaturase activity during both transfers, and appeared to be adjusted as required to offset changes in the proportion of polyunsaturates. Supported by National Science Foundation Grant PCM-8301757 to J.R.H.     

Changes in fatty acids and sterols during batch growth of <Emphasis Type="Italic">Pavlova viridis</Emphasis> in photobioreactor

Changes in the composition of fatty acids and sterols of Pavlova viridis cultured in an air-lift photobioreactor were studied using gas chromatography-mass spectrometry (GC-MS). The results show that radical changes in fatty acid and sterol contents and compositions occurred during growth phase transitions: the total lipid increased along with the culture age, from 166.4 mg g⁻¹ (late exponential phase) to 232.7 mg g⁻¹ (linear phase), and increased further to be 235.1 mg g⁻¹ in the stationary phase. Polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA), decreased along with the culture time, PUFAs, and EPA contents maximized in the late exponential phase to become 46.2 mg g⁻¹ and 22.1 mg g⁻¹ respectively; there was no significant change in docosahexaenoic acid (DHA) content during the whole growth phase, although it reached the peak in the linear phase with 3.5 mg g⁻¹. As for the sterols, two unique sterols with two hydroxyl groups, termed pavlovols, were observed. 4α,24-Dimethylcholestan-3β,4β-diol, one of the pavlovols, increased almost 2-fold from the late exponential phase (2.5 mg g⁻¹) to the stationary phase (4.3 mg g⁻¹). On the contrary, the contents of stigmasterol and sitosterol decreased with culture age, with the maximum content of 2.4 mg g⁻¹ and 3.1 mg g⁻¹, both obtained in the late exponential phase, respectively. The results indicate that growth phase control could be used as a methodology to optimize the total lipid, EPA, PUFA, and sterol contents with the potential for both aquaculture feeds and nutraceutical applications, especially for further research into unique pavlovols.     

Dietary supplementation with docosahexaenoic acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition

Treatment with the ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exerts cardioprotective effects, and suppresses Ca²⁺-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the ω-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca²⁺-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of ω-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca²⁺-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca²⁺ retention capacity and decreased Ca²⁺-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca²⁺-induced MPTP opening.     

Effects of reduced diameter of bag cultures on content of essential fatty acids and cell density in a continuous algal production system

Cell density and fatty acid (FA) content of Pavlova lutheri and Chaetoceros muelleri were analysed in a continuous algal production system (250-L bags) with reduced diameter. The cell density and FA content and composition in the algal production system were determined in replicate bags over a period of 5 weeks. The results showed that the cell density and essential FAs increased during the experiment for both species. After 5 weeks the mean cell numbers had increased to 6.0 ± 0.3 × 10⁶ cells mL⁻¹ in the P. lutheri bags and 6.0 ± 0.4 × 10⁶ cells mL⁻¹ in the C. muelleri bags. The content of total FAs increased significantly (p < 0.05) in all of the bags during the experiment. At the end of the experiment the mean total FA content were 2.7 ± 0.3 pg cell⁻¹ in the P. lutheri bags and 1.8 ± 0.1 pg cell⁻¹ in the C. muelleri bags. Maximum total FA content registered was 3.0 pg cell⁻¹ in one of the P. lutheri bags. The content of the essential FAs (ARA, EPA, DHA) increased over time in both of the species. At the end of the experiment the content of EPA (0.6 ± 0.1 pg cell⁻¹) and DHA (0.3 ± 0.0 pg cell⁻¹) were highest in the P. lutheri bags, while ARA (0.1 ± 0.0 pg cell⁻¹) was highest in C. muelleri. EPA and DHA constituted 22% and 11%, respectively, of total FA content in P. lutheri, while ARA constituted 6% of total FA content in C. muelleri. The results from this experiment indicate that flagellates such as P. lutheri perform better in narrow bags with improved light conditions, while diatoms like C. muelleri perform better in wider bags under light limitation. Implications for bivalve hatcheries are discussed.