Functions of PKS Genes in Lipid Synthesis of <Emphasis Type="Italic">Schizochytrium</Emphasis> sp. by Gene Disruption and Metabolomics Analysis

Schizochytrium sp. is a kind of marine microalgae with great potential as promising sustainable source of polyunsaturated fatty acids (PUFAs). Polyketide synthase-like (PKS synthase) is supposed to be one of the main ways to synthesize PUFAs in Schizochytrium sp. In order to study the exact relationship between PKS and PUFA biosynthesis, chain length factor (CLF) and dehydrogenase (DH) were cloned from the PKS gene cluster in Schizochytrium sp., then disrupted by homologous recombination. The results showed that DH- and CLF-disrupted strains had significant decreases (65.85 and 84.24%) in PUFA yield, while the saturated fatty acid (SFA) proportion in lipids was slightly increased. Meanwhile, the disruption of CLF decreased the C-22 PUFA proportion by 57.51% without effect on C-20 PUFA accumulation while DH-disrupted mutant decreased the production of each PUFA. Combined with analysis of protein prediction, it indicated that CLF gene exerted an enormous function on the carbon chain elongation in PUFA synthesis, especially for the final elongation from C-20 to C-22 PUFAs. Metabolomics analysis also suggested that the disruption of both genes resulted in the decrease of PUFAs but increase of SFAs, thus weakening glycolysis and tricarboxylic acid (TCA) cycle pathways. This study offers a broad new vision to research the mechanism of PUFA synthesis in Schizochytrium sp.     

Cloning and functional characterisation of a fatty acyl elongase from southern bluefin tuna (Thunnus maccoyii)

The synthesis of long chain polyunsaturated fatty acids (LCPUFA), such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), involves fatty acyl desaturase and elongase enzymes. The marine fish species southern bluefin tuna (SBT) can accumulate large quantities of omega-3 (n-3) LCPUFA in its flesh but their capacity to synthesize EPA and DHA is uncertain. A cDNA, sbtElovl5, encoding a putative fatty acyl elongase was amplified from SBT liver tissue. The cDNA included an open reading frame (ORF) encoding 294 amino acids. Sequence comparisons and phylogenetic analyses revealed a high level of sequence conservation between sbtElovl5 and fatty acyl elongase sequences from other fish species. Heterologous expression of the sbtElovl5 ORF in Saccharomyces cerevisiae confirmed that it encoded a fatty acyl elongase capable of elongating C_18/20 polyunsaturated fatty acid (PUFA) substrates, but not C₂₂ PUFA substrates. For the first time in an Elovl5, the substrate competition occurring in nature was investigated. Higher activity towards n-3 PUFA substrates than omega-6 (n-6) PUFA substrates was exhibited, regardless of substrate chain length. The sbtElovl5 preferentially elongated 18:4n-3 and 18:3n-6 rather than 20:5n-3 and 20:4n-6. The sbtElovl5 enzyme also elongated saturated and monounsaturated fatty acids.     

Identification of a novel type of polyunsaturated fatty acid synthase involved in arachidonic acid biosynthesis

Arachidonic acid (ARA) is a polyunsaturated fatty acid (PUFA) and an essential component of membrane lipids. However, the PUFA synthase required for ARA biosynthesis has not been identified in any organism. To identify the PUFA synthase producing ARA, we determined the draft genome sequence of the marine bacterium Aureispira marina, which produces a high level of ARA, and found a gene cluster encoding a putative PUFA synthase for ARA production. Expression of the gene cluster in Escherichia coli induced production of ARA, demonstrating that the gene cluster encodes a PUFA synthase required for ARA biosynthesis.     

Cloning and functional analysis of putative malonyl-CoA:acyl-carrier protein transacylase gene from the docosahexaenoic acid-producer Schizochytrium sp. TIO1101

Malonyl-CoA:acyl-carrier protein transacylase (MCAT), which transfers the malonyl group from malonyl-CoA to holo-acyl carrier protein (ACP), is a key enzyme in fatty acid biosynthesis. Schizochytrium sp. TIO1101 is a marine protist with high levels of docosahexaenoic acid accumulation. In this study, the putative fabD gene coding MCAT was isolated from Schizochytrium sp. TIO1101. The Schizochytrium MCAT gene (ScTIOfabD) contained an 1176 bp open reading frame encoding a protein of 391 amino acids. The ScTIOfabD gene exhibited high novelty in nucleotide and amino acid sequence. The highest amino acid identity was only 35 % between ScTIOMCAT and the reported MCATs. Further studies demonstrated that ScTIOMCAT could bind malonyl-CoA directly and transfer malonyl group from malonyl-CoA to the ACP domain in vitro. Phylogenetic analysis suggested that ScTIOMCAT was relative close to MCATs of yeast strains. Overexpression of ScTIOMCAT in Saccharomyces cereviseae significantly increased the MCAT activity, without negative effects on the growth rate of the host strain. In addition, ScTIOMCAT generated 16.8 and 62 % increase in biomass and fatty acid accumulation, respectively, and did not alter the profile of fatty acid. Our results indicated that the novel MCAT gene from Schizochytrium sp. TIO1101 was crucial for fatty acid synthesis and had potential applications for genetic modifications of oil-producing species.     

Culturable diversity and biochemical features of thraustochytrids from coastal waters of Southern China

Thraustochytrids are ubiquitous marine osmo-heterotrophic fungi-like microorganisms with only about 40 identified species till now. In this study, a total of 60 thraustochytrid strains were isolated from marine coastal habitats. Analysis of 18S rRNA gene sequences revealed that they belonged to three genera, i.e., Schizochytrium, Aurantiochytrium, and Thraustochytrium. All of the isolates were found to show considerable cellulolytic and lipolytic activities. Strains of Aurantiochytrium sp. and Thraustochytrium sp. were found to produce the highest levels of extracellular polysaccharides (EPS), which reached 345 μg ml^?1 in the growth media. Fourier transform infrared (FTIR) spectra of the EPS samples derived from two thraustochytrids (PKU#Sed1 and #SW1) displayed peaks for carbohydrates, proteins, lipids, uronic acids, and nucleic acids. Fatty acid profiles of four thraustochytrids comprised of palmitic acid (C16:0) and docosahexaenoic acid (DHA) as their major constituents. Schizochytrium sp. demonstrated the highest DHA production at 44 % of total fatty acids (TFA) with biomass and DHA yield of 7.1 and 1.6 g l^?1, respectively, on the fourth day of growth. All the four isolates exhibited considerable production of palmitic acid (16:0) in their fatty acid profiles ranging from 35 to 50 % TFA. This is the first report on extracellular enzymes, EPS, and DHA production from thraustochytrids isolated from the coastal habitats of China.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and α-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n ? 6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n ? 3 fatty acids (α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n ? 6 fatty acids (linoleic acid and arachidonic acid), the total n ? 3 fatty acyl content was reduced in all the phospholipids examined. In n ? 3 and n ? 6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n ? 9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appear to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca²⁺]_i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n ? 3 and n ? 6 PUFA but not in n ? 9 monounsaturated fatty acid modified cells. In Ca²⁺ free medium, OKT3-induced transient increase in [Ca²⁺]_i, representing Ca²⁺ release from the inositol 1,4,5-triphosphate-sensitive Ca²⁺ stores, were similar in control and UFA modified cells. Using Mn²⁺ entry as an index of plasma membrane Ca²⁺ permeability, the rate of fura-2 fluorescence quenching as a result of Mn²⁺ influx stimulated by OKT3 in n ? 9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n ? 3 and n ? 6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca²⁺ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and n ? 9 monounsaturated fatty acids appears to be important for the maintenance of a functional Ca²⁺ influx mechanism.     

High contents of 24:6(n-3) and 20:1(n-13) fatty acids in the brittle star Amphiura elandiformis from Tasmanian coastal sediments

The presence of long-chain polyunsaturated fatty acids (PUFA; ca. 9% of total fatty acids) in marine sediments near Dover, southern Tasmania, Australia prompted a search for their likely source. Analysis of a number of different species of benthic fauna isolated from these sediments revealed that the brittle star Amphiura elandiformis contained abundant PUFA including high contents of the uncommon long-chain fatty acid 24:6(n-3), but much smaller amounts of the more common animal PUFA 22:6(n-3). This is the first report of the lipid composition of this animal. Identifications of the unsaturated fatty acids were confirmed by formation of DMOX derivatives which gave characteristic and easily interpreted mass spectra. The 24:6(n-3) PUFA has been identified in some genera of brittle stars, but not others. It is rarely found in significant amounts in other marine animals. DMDS adducts were used to identify the positions of double bonds in the monounsaturated fatty acids. The major 20:1 isomer was identified as the rarely reported 20:1(n-13) fatty acid. The two fatty acids 20:1(n-13) and 24:6(n-3) may be useful biomarkers in food-web studies for identifying a brittle star diet and for recognising contributions of organic detritus from this benthic animal to marine sediments.     

Exploring a new,soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock

Performance of a new lipase from Novozymes (Callera Trans L) was studied for fatty acid methylesters (FAMEs) production. In order to reduce the costs of the industrial enzymatic biodiesel production process, the enzyme was used in its soluble form instead of the common immobilized preparations. Cost reduction was also achieved by using crude (non-degummed) soybean oil as a cheaper raw material. The effect of water content during Callera Trans L-catalyzed FAMEs production was explored from evaluation of free fatty acids (FFAs), tri- di or monoacylglycerides (TAGs, DAGs, MAGs) variation during 24 h reaction. An excellent 96% FAMEs release was achieved when low (3–5%) water concentrations were used in the conversion of crude soybean oil. Time course HPLC analysis of the reaction products suggests that the soluble enzyme proceeds through a mechanism of methylester formation based on a first hydrolysis step that releases FFAs, DAGs or MAGs, followed by esterification of FFAs with methanol for FAMEs production.     

Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis

Using chemical mutagenesis, mutants of Hansenula polymorpha that were defective in fatty acid synthesis were selected based on their growth requirements on saturated fatty acid mixtures. One mutant (S7) was incapable of synthesizing polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. A genetic analysis demonstrated that the S7 strain had a double lesion affecting fatty acid synthesis and Δ¹²-desaturation. A segregant with a defect in PUFA synthesis (H69-2C) displayed normal growth characteristics in the temperature range of 20–42 °C through a modulation of the cellular fatty acid composition. Compared with the parental strain, this yeast mutant had increased sensitivity at low and high temperatures (15 and 48 °C, respectively) with an increased tolerance to oxidative stress. The responses to ethanol stress were similar for the parental and PUFA-defective strains. Myristic acid was also determined to play an essential role in the cell growth of H. polymorpha. These findings suggest that both the type of cellular fatty acids and the composition of fatty acids might be involved in the stress responsive mechanisms in this industrially important yeast.     

Thraustochytrid Marine Protists: Production of PUFAs and Other Emerging Technologies

Thraustochytrids, the heterotrophic, marine, straminipilan protists, are now established candidates for commercial production of the omega-3 polyunsaturated fatty acid (ω-3 PUFA), docosahexaenoic acid (DHA), that is important in human health and aquaculture. Extensive screening of cultures from a variety of habitats has yielded strains that produce at least 50% of their biomass as lipids, and DHA comprising at least 25% of the total fatty acids, with a yield of at least 5 g L⁻¹. Most of the lipids occur as triacylglycerols and a lesser amount as phospholipids. Numerous studies have been carried out on salinity, pH, temperature, and media optimization for DHA production. Commercial production is based on a fed batch method, using high C/N ratio that favors lipid accumulation. Schizochytrium DHA is now commercially available as nutritional supplements for adults and as feeds to enhance DHA levels in larvae of aquaculture animals. Thraustochytrids are emerging as a potential source of other PUFAs such as arachidonic acid and oils with a suite of PUFA profiles that can have specific uses. They are potential sources of asataxanthin and carotenoid pigments, as well as other lipids. Genes of the conventional fatty acid synthesis and the polyketide-like PUFA synthesis pathways of thraustochytrids are attracting attention for production of recombinant PUFA-containing plant oils. Future studies on the basic biology of these organisms, including biodiversity, environmental adaptations, and genome research are likely to point out directions for biotechnology explorations. Potential areas include enzymes, polysaccharides, and secondary metabolites.     

An active site mutant of Escherichia coli cyclopropane fatty acid synthase forms new non-natural fatty acids providing insights on the mechanism of the enzymatic reaction

We have produced and purified an active site mutant of the Escherichia coli cyclopropane fatty acid synthase (CFAS) by replacing the strictly conserved G236 within cyclopropane synthases, by a glutamate residue, which corresponds to E146 of the homologous mycolic acid methyltransferase, Hma, producing hydroxymethyl mycolic acids. The G236E CFAS mutant had less than 1% of the in vitro activity of the wild type enzyme. We expressed the G236E CFAS mutant in an E. coli (DE3) strain in which the chromosomal cfa gene had been deleted. After extraction of phospholipids and conversion into the corresponding fatty acid methyl esters (FAMEs), we observed the formation of cyclopropanated FAMEs suggesting that the mutant retained some of the normal activity in vivo. However, we also observed the formation of new C17 methyl-branched unsaturated FAMEs whose structures were determined using GC/MS and NMR analyses. The double bond was located at different positions 8, 9 or 10, and the methyl group at position 10 or 9. Thus, this new FAMEs are likely arising from a 16:1 acyl chain of a phospholipid that had been transformed by the G236E CFAS mutant in vivo. The reaction catalyzed by this G236E CFAS mutant thus starts by the methylation of the unsaturated acyl chain at position 10 or 9 yielding a carbocation at position 9 or 10 respectively. It follows then two competing steps, a normal cyclopropanation or hydride shift/elimination events giving different combinations of alkenes. This study not only provides further evidence that cyclopropane synthases (CSs) form a carbocationic intermediate but also opens the way to CSs engineering for the synthesis of non-natural fatty acids.     

Efficient Phagocytosis Requires Triacylglycerol Hydrolysis by Adipose Triglyceride Lipase

Macrophage phagocytosis is an essential biological process in host defense and requires large amounts of energy. To date, glucose is believed to represent the prime substrate for ATP production in macrophages. To investigate the relative contribution of free fatty acids (FFAs) in this process, we determined the phagocytosis rates in normal mouse macrophages and macrophages of adipose triglyceride lipase (ATGL)-deficient mice. ATGL was shown to be the rate-limiting enzyme for the hydrolysis of lipid droplet-associated triacylglycerol (TG) in many tissues. Here, we demonstrate that Atgl^−/− macrophages fail to efficiently hydrolyze cellular TG stores leading to decreased cellular FFA concentrations and concomitant accumulation of lipid droplets, even in the absence of exogenous lipid loading. The reduced availability of FFAs results in decreased cellular ATP concentrations and impaired phagocytosis suggesting that fatty acids must first go through a cycle of esterification and re-hydrolysis before they are available as energy substrate. Exogenously added glucose cannot fully compensate for the phagocytotic defect in Atgl^−/− macrophages. Hence, phagocytosis was also decreased in vivo when Atgl^−/− mice were challenged with bacterial particles. These findings imply that phagocytosis in macrophages depends on the availability of FFAs and that ATGL is required for their hydrolytic release from cellular TG stores. This novel mechanism links ATGL-mediated lipolysis to macrophage function in host defense and opens the way to explore possible roles of ATGL in immune response, inflammation, and atherosclerosis.     

Oxygenation of polyunsaturated fatty acids and oxidative stress within blood platelets

The oxygenation metabolism of arachidonic acid (ArA) has been early described in blood platelets, in particular with its conversion into the potent labile thromboxane A₂ that induces platelet aggregation and vascular smooth muscle cells contraction. In addition, the primary prostaglandins D₂ and E₂ have been mainly reported as inhibitors of platelet function. The platelet 12-lipoxygenase (12-LOX) product, i.e. the hydroperoxide 12-HpETE, appears to stimulate platelet ArA metabolism at the level of its release from membrane phospholipids through phospholipase A₂ (cPLA₂) and cyclooxygenase (COX-1) activities, the first enzymes in prostanoid production cascade. Also, 12-HpETE may regulate the oxygenation of other polyunsaturated fatty acids (PUFA) by platelets, especially that of eicosapentaenoic acid (EPA). On the other hand, the reduced product of 12-HpETE, 12-HETE, is able to antagonize TxA₂ action. This is even more obvious for the 12-LOX end-products from docosahexaenoic acid (DHA), 11- and 14-HDoHE. In addition, 12-HpETE plays a key role in platelet oxidative stress as observed in pathophysiological conditions, but may be regulated by DHA with a bimodal way according to its concentration. Other oxygenated products of PUFA, especially omega-3 PUFA, produced outside platelets may affect platelet functions as well.     

Kinetically guided,ratiometric tuning of fatty acid biosynthesis

Cellular metabolism is a nonlinear reaction network in which dynamic shifts in enzyme concentration help regulate the flux of carbon to different products. Despite the apparent simplicity of these biochemical adjustments, their influence on metabolite biosynthesis tends to be context-dependent, difficult to predict, and challenging to exploit in metabolic engineering. This study combines a detailed kinetic model with a systematic set of in vitro and in vivo analyses to explore the use of enzyme concentration as a control parameter in fatty acid synthesis, an essential metabolic process with important applications in oleochemical production. Compositional analyses of a modeled and experimentally reconstituted fatty acid synthase (FAS) from Escherichia coli indicate that the concentration ratio of two native enzymes—a promiscuous thioesterase and a ketoacyl synthase—can tune the average length of fatty acids, an important design objective of engineered pathways. The influence of this ratio is sensitive to the concentrations of other FAS components, which can narrow or expand the range of accessible chain lengths. Inside the cell, simple changes in enzyme concentration can enhance product-specific titers by as much as 125-fold and elicit shifts in overall product profiles that rival those of thioesterase mutants. This work develops a kinetically guided approach for using ratiometric adjustments in enzyme concentration to control the product profiles of FAS systems and, broadly, provides a detailed framework for understanding how coordinated shifts in enzyme concentration can afford tight control over the outputs of nonlinear metabolic pathways.     

Interaction of rat liver microsomes containing saturated or unsaturated fatty acids with fatty acid binding protein: Peroxidation effect

In the studies described here rat liver microsomes containing labeled palmitic, stearic, oleic or linoleic acids were incubated with fatty acid binding protein (FABP) and the rate of removal of¹⁴C-labeled fatty acids from the membrane by the soluble protein was measured using a model system. More unsaturated than saturated fatty acids were removed from native liver microsomes incubated with similar amounts of FABP. Thein vitro peroxidation of microsomal membranes mediated by ascorbate-Fe⁺⁺, modified its fatty acid composition with a considerable decrease of the peroxidizability index. These changes in the microsomes facilitated the removal of oleic and linoeic acids by FABP, but the removal of palmitic and stearic acids was not modified. This effect is proposed to result from a perturbation of membrane structure following peroxidation with release of free fatty acids from susceptible domains.Abbreviations BSA bovine serum albumin - FABP fatty acid binding protein     

Synthesis of multimethyl-branched fatty acids by avian and mammalian fatty acid synthetase and its regulation by malonyl-CoA decarboxylase in the uropygial gland

Fatty acid synthetase, partially purified by gel filtration with Sepharose 4B from goose liver, showed the same relative rate of incorporation of methylmalonyl-CoA (compared to malonyl-CoA) as that observed with the purified fatty acid synthetase from the uropygial gland. In the presence of acetyl-CoA, methylmalonyl-CoA was incorporated mainly into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8,10-pentamethyl-dodecanoic acid by the enzyme from both sources. Methylmalonyl-CoA was a competitive inhibitor with respect to malonyl-CoA for the enzyme from the gland just as previously observed for fatty acid synthetase from other animals. Furthermore, rabbit antiserum prepared against the gland enzyme cross-reacted with the liver enzyme, and Ouchterlony double-diffusion analyses showed complete fusion of the immunoprecipitant lines. The antiserum inhibited both the synthesis of n-fatty acids and branched fatty acids catalyzed by the synthetase from both liver and the uropygial gland. These results suggest that the synthetases from the two tissues are identical and that branched and n-fatty acids are synthesized by the same enzyme. Immunological examination of the 105,000g supernatant prepared from a variety of organs from the goose showed that only the uropygial gland contained a protein which cross-reacted with the antiserum prepared against malonyl-CoA decarboxylase purified from the gland. Thus, it is concluded that the reason for the synthesis of multimethyl-branched fatty acids by the fatty acid synthetase in the gland is that in this organ the tissue-specific and substrate-specific decarboxylase makes only methylmalonyl-CoA available to the synthetase. Fatty acid synthetase, partially purified from the mammary gland and the liver of rats, also catalyzed incorporation of [methyl-¹⁴C]methylmalonyl-CoA into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8-tetramethylundecanoic acid with acetyl-CoA and propionyl-CoA, respectively, as the primers. Evidence is also presented that fatty acids containing straight and branched regions can be generated by the fatty acid synthetase from the rat and goose, from methylmalonyl-CoA in the presence of malonyl-CoA or other precursors of n-fatty acids. These results provide support for the hypothesis that, under the pathological conditions which result in accumulation of methylmalonyl-CoA, abnormal branched acids can be generated by the fatty acid synthetase.     

Overexpression of acetyl-CoA synthetase increased the biomass and fatty acid proportion in microalga Schizochytrium

High acetate accumulation was produced during glucose fermentation in high cell density cultures, which is harmful to cell growth. In order to reduce the negative impact of acetate accumulation on the fermentation products, we introduced the Escherichia coli acetyl-CoA synthetase (ACS) gene into the marine microalga Schizochytrium sp. TIO1101, generating genetically modified ACS transformants. The results of PCR and blotting analyses showed that the exogenous ACS gene was incorporated into the genome and successfully expressed. The engineered Schizochytrium increased the pH value and reduced the acetate concentration in the final fermentation medium significantly. Furthermore, the ACS transformants exhibited faster growth and glucose consumption rates than the wild-type strain. The biomass and fatty acid proportion of ACS transformants increased by 29.9 and 11.3 %, respectively. Taken together, the data suggest that ACS overexpression in Schizochytrium might improve the utilization of carbon resource and decrease the production of acetate byproduct. These results demonstrate that application of ACS in metabolic genetic engineering could improve the properties of Schizochytrium significantly.     

Effect of selected plant species within biodiverse pasture on in vitro fatty acid biohydrogenation and tissue fatty acid composition of lamb

The effect of botanical diversity on supply of polyunsaturated fatty acids (PUFA) to ruminants in vitro, and the fatty acid (FA) composition of muscle in lambs was investigated. Six plant species, commonly grown as part of UK herbal ley mixtures (Trifolium pratense, Lotus corniculatus, Achillea millefolium, Centaurea nigra, Plantago lanceolata and Prunella vulgaris), were assessed for FA profile, and in vitro biohydrogenation of constituent PUFA, to estimate intestinal supply of PUFA available for absorption by ruminants. Modelling the in vitro data suggested that L. corniculatus and P. vulgaris had the greatest potential to increase 18:3n-3 supply to ruminants, having the highest amounts escaping in vitro biohydrogenation. Biodiverse pastures were established using the six selected species, under-sown in a perennial ryegrass-based sward. Lambs were grazed (~50 days) on biodiverse or control pastures and the effects on the FA composition of musculus longissimus thoracis (lean and subcutaneous fat) and musculus semimembranosus (lean) were determined. Biodiverse pasture increased 18:2n-6 and 18:3n-3 contents of m. semimembranosus (+14.8 and +7.2 mg/100 g tissue, respectively) and the subcutaneous fat of m. longissimus thoracis (+158 and +166 mg/100 g tissue, respectively) relative to feeding a perennial ryegrass pasture. However, there was no effect on total concentrations of saturated FA in the tissues studied. It was concluded that enhancing biodiversity had a positive impact on muscle FA profile reflected by increased levels of total PUFA.     

Biochemical characterization of a chloroplast localized fatty acid reductase from Arabidopsis thaliana

Primary long-chain fatty alcohols are present in a variety of phyla. In eukaryotes, the production of fatty alcohols is catalyzed by fatty acyl-CoA reductase (FAR) enzymes that convert fatty acyl-CoAs or acyl-ACPs into fatty alcohols. Here, we report on the biochemical properties of a purified plant FAR, Arabidopsis FAR6 (AtFAR6). In vitro assays show that the enzyme preferentially uses 16 carbon acyl-chains as substrates and produces predominantly fatty alcohols. Free fatty acids and fatty aldehyde intermediates can be released from the enzyme, in particular with suboptimal chain lengths and concentrations of the substrates. Both acyl-CoA and acyl-ACP could serve as substrates. Transient expression experiments in Nicotiana tabacum showed that AtFAR6 is a chloroplast localized FAR. In addition, expression of full length AtFAR6 in Nicotiana benthamiana leaves resulted in the production of C16:0-alcohol within this organelle. Finally, a GUS reporter gene fusion with the AtFAR6 promoter showed that the AtFAR6 gene is expressed in various tissues of the plant with a distinct pattern compared to that of other Arabidopsis FARs, suggesting specialized functions in planta.     

Functional analysis of the dehydratase domains of a PUFA synthase from Thraustochytrium in Escherichia coli

Thraustochytrium sp. 26185, a unicellular marine protist, synthesizes docosahexaenoic acid, an omega-3 very long chain polyunsaturated fatty acid (VLC-PUFAs), by a polyunsaturated fatty acid (PUFA) synthase comprising three large subunits with multiple catalytic dehydratase (DH) domains critical for introducing double bonds at the specific position of fatty acids. To investigate functions of these DH domains, one DH domain from subunit-A and two DH domains from subunit-C of the PUFA synthase were dissected and expressed as stand-alone enzymes in Escherichia coli. The results showed that all these DH domains could complement the defective phenotype of a E. coli FabA temperature sensitive mutant, despite they have only modest sequence similarity with FabA, indicating they can function as 3-hydroxyacyl-ACP dehydratase for the biosynthesis of unsaturated fatty acids in E. coli. Site-directed mutagenesis analysis confirmed the authenticity of active site residues in these domains. In addition, overexpression of the three domains in a wild type E. coli strain resulted in the substantial alteration of fatty acid profiles including productions and ratio of unsaturated to saturated fatty acids. A combination of evidences from sequence comparison, functional expression, and mutagenesis analysis suggest that the DH domain from subunit-A is similar to DH domains from polyketide synthases, while the DH domains from subunit-C are more comparable to E. coli FabA in catalytic functions. Successful complementation and functional expression of the embedded DH domains from the PUFA synthase in E. coli is an important step towards for elucidating the molecular mechanism in the biosynthesis of VLC-PUFAs in Thraustochytrium.