What limits production of unusual monoenoic fatty acids in transgenic plants?

Unusual monounsaturated fatty acids are major constituents (greater than 80%) in seeds of Coriandrum sativum L. (coriander) and Thunbergia alata Bojer, as well as in glandular trichomes (greater than 80% derived products) of Pelargonium x hortorum (geranium). These diverged fatty acid structures are produced via distinct plastidial acyl-acyl carrier protein (ACP) desaturases. When expressed in Arabidopsis thaliana (L.) Heynh. under strong seed-specific promoters the unusual acyl-ACP desaturases resulted in accumulation of unusual monoene fatty acids at 1-15% of seed fatty acid mass. In this study, we have examined several factors that potentially limit higher production of unusual monoenes in transgenic oilseeds. (i) Immunoblots indicated that the introduced desaturases were expressed at levels equivalent to or higher than the endogenous delta9 18:0-ACP desaturase. However, the level of unusual fatty acid produced in transgenic plants was not correlated with the level of desaturase expression. (ii) The unusual desaturases were expressed in several backgrounds, including antisense 18:0-ACP desaturase plants, in fab1 mutants, and co-expressed with specialized ACP or ferredoxin isoforms. None of these experiments led to high production of expected products. (iii) No evidence was found for degradation of the unusual fatty acids during seed development. (iv) Petroselinic acid added to developing seeds was incorporated into triacylglycerol as readily as oleic acid, suggesting no major barriers to its metabolism by enzymes of glycerolipid assembly. (v) In vitro and in situ assay of acyl-ACP desaturases revealed a large discrepancy of activity when comparing unusual acyl-ACP desaturases with the endogenous delta9 18:0-ACP desaturase. The combined results, coupled with the sensitivity of acyl-ACP desaturase activity to centrifugation and low salt or detergent suggests low production of unusual monoenes in transgenic plants may be due to the lack of, or incorrect assemble of, a necessary multi-component enzyme association.     

Stearoyl-acyl carrier protein and unusual acyl-acyl carrier protein desaturase activities are differentially influenced by ferredoxin

Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Delta9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [(14)C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium x hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Delta9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction.     

绿色巴夫藻脂肪酸去饱和酶的克隆和初步研究

在高等植物中,Δ9 脂肪酸去饱和酶引入第一个双键到饱和的脂肪酸链中,导致单不饱和脂肪酸的形成。我们通过RT-PCR、RNA ligase mediated RACE (RLM-RACE) and Overlap-PCR方法从海洋微藻绿色巴夫藻中克隆到一个命名为PvfadA的脂肪酸去饱和酶候选基因。通过将PvfadA基因在大肠杆菌表达系统中成功表达,PvFadA可以特异性地将C18:0脂肪酸转变成C18:1脂肪酸。PvFadA的氨基酸序列中存在一个存在于acyl-ACP去饱和酶的特异性金属离子结合区段(D/E)X2HX-100(D/E)X2H。通过同源模建PvFadA的3D结构显示,其包含了11个α螺旋,其中α3、α4、α6和α7组成了一个4个螺旋桶的核心结构,预测其可能是酶的活性中心。PvFadA的3D结构类似于蓖麻和结核分枝杆菌H37Rv的acyl-ACP去饱和酶。     

Isolation of two putative acyl-acyl carrier protein desaturase enzymes from Kochia scoparia

5-Hexadecenoic acid can be used to produce a semichemical method to control the disease-carrying mosquito Culex quinquefasciatus. This unusual fatty acid is produced in the seed of Kochia scoparia. We have isolated two acyl-acyl carrier protein desaturases from this species and expressed them in Escherichia coli to facilitate functional characterisation.     

Recent advances in molecular cloning of fatty acid desaturase genes and the regulation of their expression by dietary vitamin A and retinoic acid

Vitamin A, as an essential micronutrient, is involved in higher animals in embryonic development and postnatal growth, reproduction and maintenance of normal skin, immunity and vision. Recently, studies in vivo and in cell lines have shown that vitamin A and its active metabolite, retinoic acid, regulate the expression of fatty acid desaturases including stearoyl-CoA desaturase and delta-5 desaturase. Whereas the former desaturase catalyzes the formation of monounsaturated from saturated fatty acids, the latter enzyme is involved in the desaturation pathway of dietary essential fatty acids for production of polyunsaturated fatty acids. The reaction products of these desaturases serve as critical regulators in a wide range of physiological processes which include fetal growth and development, reproduction, cell differentiation, immune and inflammatory responses.     

Biochemical characterization of a high-palmitoleic acid Helianthus annuus mutant.

In the present work we carried out analytical and biochemical studies on a new high-n-7 monounsaturated fatty acid sunflower (Helianthus annuus L.) mutant. This new line, which has been selected by classical methods of breeding and mutagenesis, shows contents of unusual acyl chains up to 20% (12% of 16:1DELTA9, 5% of 16:2delta9,12 and 6% of 18:1delta11), whereas those fatty acids are found in negligible amounts in common sunflower cultivars. This characterization involved in vivo incubations with radiolabeled acetate and measurement of the last enzymes involved in the intraplastidial de novo fatty acid synthesis: beta-ketoacyl-ACP synthase II, stearoyl-ACP desaturase (EC 1.14.19.2) and acyl-ACP thioesterases (EC 3.1.2.14). Results indicated that the high-palmitoleic acid phenotype was associated with a concerted reduction in the fatty acid synthase II activity with respect to the control lines and an increase of stearoyl-ACP desaturase activity with respect to the high-palmitate mutant line.     

Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis.

Acyl-lipid desaturases introduce double bonds (unsaturated bonds) at specifically defined positions in fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. The desA, desB and desD genes of Synechocystis sp. PCC 6803 encode acyl-lipid desaturases that introduce double bonds at the delta12, omega3 and delta6 positions of C18 fatty acids respectively. The mutation of each of these genes by insertion of an antibiotic resistance gene cartridge completely eliminated the corresponding desaturation reaction. This system allowed us to manipulate the number of unsaturated bonds in membrane glycerolipids in this organism in a step-wise manner. Comparisons of the variously mutated cells revealed that the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed growth of the cells at low temperature and, moreover, it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage. However, the replacement of tri- and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures.     

Expression and Purification of Integral Membrane Fatty Acid Desaturases

Fatty acid desaturase enzymes perform dehydrogenation reactions leading to the insertion of double bonds in fatty acids, and are divided into soluble and integral membrane classes. Crystal structures of soluble desaturases are available; however, membrane desaturases have defied decades of efforts due largely to the difficulty of generating recombinant desaturase proteins for crystallographic analysis. Mortierella alpina is an oleaginous fungus which possesses eight membrane desaturases involved in the synthesis of saturated, monounsaturated and polyunsaturated fatty acids. Here, we describe the successful expression, purification and enzymatic assay of three M. alpina desaturases (FADS15, FADS12, and FADS9-I). Estimated yields of desaturases with purity >95% are approximately 3.5% (Ca. 4.6 mg/L of culture) for FADS15, 2.3% (Ca. 2.5 mg/L of culture) for FADS12 and 10.7% (Ca. 37.5 mg/L of culture) for FADS9-I. Successful expression of high amounts of recombinant proteins represents a critical step towards the structural elucidation of membrane fatty acid desaturases.     

Plant desaturases: harvesting the fat of the land.

The past few years have witnessed a major upsurge in research towards the goal of modifying the lipid composition of plants. Genes encoding a range of different fatty acid desaturase activities have been cloned, and the evolutionary relationships between and within different classes of enzymes have tentatively been established. The effects of expressing some of these desaturases in heterologous hosts have also been studied, often producing unexpected results which contribute further to our understanding of plant lipid modification. It is to be hoped that, in the near future, the goal of producing unusual and valuable fatty acids in transgenic oilseeds will be achieved on a commercial scale.     

Genetic regulation of unsaturated fatty acid composition in C. elegans

Delta-9 desaturases, also known as stearoyl-CoA desaturases, are lipogenic enzymes responsible for the generation of vital components of membranes and energy storage molecules. We have identified a novel nuclear hormone receptor, NHR-80, that regulates delta-9 desaturase gene expression in Caenorhabditis elegans. Here we describe fatty acid compositions, lifespans, and gene expression studies of strains carrying mutations in nhr-80 and in the three genes encoding delta-9 desaturases, fat-5, fat-6, and fat-7. The delta-9 desaturase single mutants display only subtle changes in fatty acid composition and no other visible phenotypes, yet the fat-5;fat-6;fat-7 triple mutant is lethal, revealing that endogenous production of monounsaturated fatty acids is essential for survival. In the absence of FAT-6 or FAT-7, the expression of the remaining desaturases increases, and this ability to compensate depends on NHR-80. We conclude that, like mammals, C. elegans requires adequate synthesis of unsaturated fatty acids and maintains complex regulation of the delta-9 desaturases to achieve optimal fatty acid composition.     

Elongation of polyunsaturated fatty acids in trypanosomatids

Leishmania major synthesizes polyunsaturated fatty acids by using Delta6, Delta5 and Delta4 front-end desaturases, which have recently been characterized [Tripodi KE, Buttigliero LV, Altabe SG & Uttaro AD (2006) FEBS J273, 271-280], and two predicted elongases specific for C18 Delta6 and C20 Delta5 polyunsaturated fatty acids, respectively. Trypanosoma brucei and Trypanosoma cruzi lack Delta6 and Delta5 desaturases but contain Delta4 desaturases, implying that trypanosomes use exogenous polyunsaturated fatty acids to produce C22 Delta4 fatty acids. In order to identify putative precursors of these C22 fatty acids and to completely describe the pathways for polyunsaturated fatty acid biosynthesis in trypanosomatids, we have performed a search in the three genomes and identified four different elongase genes in T. brucei, five in T. cruzi and 14 in L. major. After a phylogenetic analysis of the encoded proteins together with elongases from a variety of other organisms, we selected four candidate polyunsaturated fatty acid elongases. Leishmania major CAJ02037, T. brucei AAX69821 and T. cruzi XP_808770 share 57-52% identity, and group together with C20 Delta5 polyunsaturated fatty acid elongases from algae. The predicted activity was corroborated by functional characterization after expression in yeast. T. brucei elongase was also able to elongate Delta8 and Delta11 C20 polyunsaturated fatty acids. L. major CAJ08636, which shares 33% identity with Mortierella alpinaDelta6 elongase, showed a high specificity for C18 Delta6 polyunsaturated fatty acids. In all cases, a preference for n6 polyunsaturated fatty acids was observed. This indicates that L. major has, as predicted, Delta6 and Delta5 elongases and a complete pathway for polyunsaturated fatty acid biosynthesis. Trypanosomes contain only Delta5 elongases, which, together with Delta4 desaturases, allow them to use eicosapentaenoic acid and arachidonic acid, a precursor that is relatively abundant in the host, for C22 polyunsaturated fatty acid biosynthesis.     

A Determinant of Substrate Specificity Predicted from the Acyl-Acyl Carrier Protein Desaturase of Developing Cat''s Claw Seed

Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Δ9) plus cis-vaccenic acid (18:1Δ11) in its seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds. The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Δ9–18:0)-ACP desaturase from castor (Ricinis communis L.) and other species. Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Δ9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Δ9–18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases. Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Δ9–18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP. The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Δ9–18:0-ACP desaturase.     

Decrease in Membrane Phospholipid Unsaturation Induces Unfolded Protein Response

Various kinds of fatty acids are distributed in membrane phospholipids in mammalian cells and tissues. The degree of fatty acid unsaturation in membrane phospholipids affects many membrane-associated functions and can be influenced by diet and by altered activities of lipid-metabolizing enzymes such as fatty acid desaturases. However, little is known about how mammalian cells respond to changes in phospholipid fatty acid composition. In this study we showed that stearoyl-CoA desaturase 1 (SCD1) knockdown increased the amount of saturated fatty acids and decreased that of monounsaturated fatty acids in phospholipids without affecting the amount or the composition of free fatty acid and induced unfolded protein response (UPR), evidenced by increased expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) mRNAs and splicing of Xbox-binding protein 1 (XBP1) mRNA. SCD1 knockdown-induced UPR was rescued by various unsaturated fatty acids and was enhanced by saturated fatty acid. Lysophosphatidylcholine acyltransferase 3 (LPCAT3), which incorporates preferentially polyunsaturated fatty acids into phosphatidylcholine, was up-regulated in SCD1 knockdown cells. Knockdown of LPCAT3 synergistically enhanced UPR with SCD1 knockdown. Finally we showed that palmitic acid-induced UPR was significantly enhanced by LPCAT3 knockdown as well as SCD1 knockdown. These results suggest that a decrease in membrane phospholipid unsaturation induces UPR.     

Substrate Specificity and Regioselectivity of Δ12 and ω3 Fatty Acid Desaturases from Saccharomyces kluyveri

Δ12 and ω3 fatty acid desaturases are key enzymes in the synthesis of polyunsaturated fatty acids (PUFAs), which are important constituents of membrane glycerolipids and also precursors to signaling molecules in many organisms. In this study, we determined the substrate specificity and regioselectivity of the Δ12 and ω3 fatty acid desaturases from Saccharomyces kluyveri (Sk-FAD2 and Sk-FAD3). Based on heterologous expression in Saccharomyces cerevisiae, it was found that Sk-FAD2 converted C16–20 monounsaturated fatty acids to diunsaturated fatty acids by the introduction of a second double bond at the ν+3 position, while Sk-FAD3 recognized the ω3 position of C18 and C20. Furthermore, fatty acid analysis of major phospholipids suggested that Sk-FAD2 and Sk-FAD3 have no strong substrate specificity toward the lipid polar head group or the sn-positions of fatty acyl groups in phospholipids.     

Analysis of docosahexaenoic acid biosynthesis in Crypthecodinium cohnii by 13C labelling and desaturase inhibitor experiments

The lipids of the heterotrophic microalga Crypthecodinium cohnii contain the omega-3 polyunsaturated fatty acid (PUFA) and docosahexaenoic acid (22:6) to a level of over 30%. The pathway of 22:6 synthesis in C. cohnii is unknown. The ability of C. cohnii to use 13C-labelled externally supplied precursor molecules for 22:6 biosynthesis was tested by 13C NMR analysis. Furthermore, the presence of desaturases (typical for aerobic PUFA synthesis) was studied by the addition of specific desaturase inhibitors in the growth medium. The addition of 1-(13)C acetate or 1-(13)C butyrate in the growth medium resulted in 22:6 with only the odd carbon atoms enriched. Apparently, two-carbon units were used as building blocks for 22:6 synthesis and butyrate was first split into two-carbon units prior to incorporation in 22:6. When 1-(13)C oleic acid was added to the growth medium, 1-(13)C oleic acid was incorporated into the lipids of C. cohnii but was not used as a precursor for the synthesis of 22:6. Specific desaturase inhibitors (norflurazon and propyl gallate) inhibited lipid accumulation in C. cohnii. The fatty acid profile, however, was not altered. In contrast, in the arachidonic acid-producing fungus, Mortierella alpina, these inhibitors not only decreased the lipid content but also altered the fatty acid profile. Our results can be explained by the presence of three tightly regulated separate systems for the fatty acid production by C. cohnii, namely for (1). the biosynthesis of saturated fatty acids, (2). the conversion of saturated fatty acids to monounsaturated fatty acids and (3). the de novo synthesis of 22:6 with desaturases involved.     

Nonesterified fatty acids and lipid peroxidation

Oxygen free radicals damage cells through peroxidation of membrane lipids. Gastrointestinal mucosal membranes were found to be resistant to in vitro lipid peroxidation as judged by malonaldehyde and conjugated diene production and arachidonic acid depletion. The factor responsible for this in this membrane was isolated and chemically characterised as the nonesterified fatty acids (NEFA), specifically monounsaturated fatty acid, oleic acid. Authentic fatty acids when tested in vitro using liver microsomes showed similar inhibition. The possible mechanism by which NEFA inhibit peroxidation is through iron chelation and iron-fatty acid complex is incapable of inducing peroxidation. Free radicals generated independent of iron was found to induce peroxidaton of mucosal membranes. Gastrointestinal mucosal membranes were found to contain unusually large amount of NEFA. Circulating albumin is known to contain NEFA which was found to inhibit iron induced peroxidation whereas fatty acid free albumin did not have any effect. Addition of individual fatty acids to this albumin restored its inhibitory capacity among which monounsaturated fatty acids were more effective. These studies have shown that iron induced lipid peroxidation damage is prevented by the presence of nonesterified fatty acids.     

Regulation of mammalian desaturases by myristic acid: N-terminal myristoylation and other modulations

Myristic acid, the 14-carbon saturated fatty acid (C14:0), usually accounts for small amounts (0.5%-1% weight of total fatty acids) in animal tissues. Since it is a relatively rare molecule in the cells, the specific properties and functional roles of myristic acid have not been fully studied and described. Like other dietary saturated fatty acids (palmitic acid, lauric acid), this fatty acid is usually associated with negative consequences for human health. Indeed, in industrialized countries, its excessive consumption correlates with an increase in plasma cholesterol and mortality due to cardiovascular diseases. Nevertheless, one feature of myristoyl-CoA is its ability to be covalently linked to the N-terminal glycine residue of eukaryotic and viral proteins. This reaction is called N-terminal myristoylation. Through the myristoylation of hundreds of substrate proteins, myristic acid can activate many physiological pathways. This review deals with these potentially activated pathways. It focuses on the following emerging findings on the biological ability of myristic acid to regulate the activity of mammalian desaturases: (i) recent findings have described it as a regulator of the Δ4-desaturation of dihydroceramide to ceramide; (ii) studies have demonstrated that it is an activator of the Δ6-desaturation of polyunsaturated fatty acids; and (iii) myristic acid itself is a substrate of some fatty acid desaturases. This article discusses several topics, such as the myristoylation of the dihydroceramide Δ4-desaturase, the myristoylation of the NADH-cytochrome b5 reductase which is part of the whole desaturase complex, and other putative mechanisms.     

The role of cytochrome b5 fusion desaturases in the synthesis of polyunsaturated fatty acids

The biosynthetic pathway of polyunsaturated fatty acids (PUFAs) has been the subject of much interest over the last few years. Significant progress has been made in the identification of the enzymes required for PUFA synthesis; in particular, the fatty acid desaturases which are central to this pathway have now all been identified. These "front-end" desaturases are all members of the cytochrome b(5) fusion desaturase superfamily, since they contain an N-terminal domain that is orthologous to the microsomal cytochrome b(5). Examination of the primary sequence relationships between the various PUFA-specific cytochrome b(5) fusion desaturases and related fusion enzymes allows inferences regarding the evolution of this important enzyme class. More importantly, this knowledge helps underpin our understanding of polyunsaturated fatty acid biosynthesis.     

植物脂肪酸去饱和酶及其编码基因研究进展

脂肪酸去饱和酶是催化脂肪酸链特定位置形成双键和产生不饱和脂肪酸的酶类。植物脂肪酸去饱和酶主要有5种(FAD2、FAD3、FAD6、FAD7和FAD8), 可分为ω-3型 (FAD2、FAD6)和ω-6型(FAD3、FAD7、FAD8)两大类。其编码基因(FAD2、FAD3、FAD6、FAD7和FAD8)在植物中一般有多个拷贝。同种基因在不同植物中拷贝数不同, 同一植物中相同基因的不同拷贝间在序列特征、表达调控和功能等方面也存在显著差异。本文根据国内外对脂肪酸去饱和酶基因及编码产物的研究现状, 分别从它们的分类、拷贝数、结构、作用机制、表达调控等方面的研究进展进行了详细的分类阐述。