Metabolic engineering of Saccharomyces cerevisiae for production of Eicosapentaenoic Acid, using a novel {Delta}5-Desaturase from Paramecium tetraurelia

Very-long-chain polyunsaturated fatty acids, such as arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), have well-documented importance in human health and nutrition. Sustainable production in robust host organisms that do not synthesize them naturally requires the coordinated expression of several heterologous desaturases and elongases. In the present study we show production of EPA in Saccharomyces cerevisiae using glucose as the sole carbon source through expression of five heterologous fatty acid desaturases and an elongase. Novel Δ5-desaturases from the ciliate protozoan Paramecium tetraurelia and from the microalgae Ostreococcus tauri and Ostreococcus lucimarinus were identified via a BLAST search, and their substrate preferences and desaturation efficiencies were assayed in a yeast strain producing the ω6 and ω3 fatty acid substrates for Δ5-desaturation. The Δ5-desaturase from P. tetraurelia was up-to-2-fold more efficient than the microalgal desaturases and was also more efficient than Δ5-desaturases from Mortierella alpina and Leishmania major. In vivo investigation of acyl carrier substrate specificities showed that the Δ5-desaturases from P. tetraurelia, O. lucimarinus, O. tauri, and M. alpina are promiscuous toward the acyl carrier substrate but prefer phospholipid-bound substrates. In contrast, the Δ5-desaturase from L. major showed no activity on phospholipid-bound substrate and thus appears to be an exclusively acyl coenzyme A-dependent desaturase.     

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.     

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.     

Caenorhabditis elegans Delta12-desaturase FAT-2 is a bifunctional desaturase able to desaturate a diverse range of fatty acid substrates at the Delta12 and Delta15 positions

Caenorhabditis elegans FAT-2 has been characterized as fatty acid Δ12-desaturase able to desaturate C16 and C18 fatty acids. However, in this report we show that when expressed in yeast cells this enzyme can also catalyze Δ15 desaturation. This results in the production of both linoleic acid (ω6 C18:2Δ9,12) and linolenic acid (ω3 C18:3Δ9,12,15) from oleic acid (C18:1Δ9) substrate, and hexadecadienoic acid (ω4 C16:2Δ9,12) and hexadecatrienoic acid (ω1 C16:3Δ9,12,15) from palmitoleic acid (C16:1Δ9) substrate. In addition, this enzyme can also produce C14:2Δ9,12, C15:2Δ9,12, C17:2Δ9,12, and C18:4Δ6,9,12,15 when C14:1Δ9, C15:1Δ9, C17:1Δ9, and C18:3Δ6,9,12 substrates are available in yeast cells. Mass spectrometry analysis of 2,4-dimethyloxazoline modification of fatty acid methyl esters confirms the positions of all newly formed double bonds. These results indicate that when expressed in yeast the C. elegans Δ12-desaturase CeFAT-2 shows a characteristic of a bifunctional Δ12/Δ15-desaturase and has a great deal of elasticity with respect to fatty acid chain length in being able to accept fatty acids ranging from C14 to C18. Interestingly, despite possessing a bifunctional Δ12/Δ15 desaturation activity, phylogenetic analysis suggests that C. elegans Δ12-desaturase CeFAT-2 might have arisen independently from other reported dual Δ12/Δ15-desaturases from fungi and protozoa.     

The role of Δ6-desaturase acyl-carrier specificity in the efficient synthesis of long-chain polyunsaturated fatty acids in transgenic plants

The role of acyl‐CoA‐dependent Δ6‐desaturation in the heterologous synthesis of omega‐3 long‐chain polyunsaturated fatty acids was systematically evaluated in transgenic yeast and Arabidopsis thaliana. The acyl‐CoA Δ6‐desaturase from the picoalga Ostreococcus tauri and orthologous activities from mouse (Mus musculus) and salmon (Salmo salar) were shown to generate substantial levels of Δ6‐desaturated acyl‐CoAs, in contrast to the phospholipid‐dependent Δ6‐desaturases from higher plants that failed to modify this metabolic pool. Transgenic plants expressing the acyl‐CoA Δ6‐desaturases from either O. tauri or salmon, in conjunction with the two additional activities required for the synthesis of C20 polyunsaturated fatty acids, contained higher levels of eicosapentaenoic acid compared with plants expressing the borage phospholipid‐dependent Δ6‐desaturase. The use of acyl‐CoA‐dependent Δ6‐desaturases almost completely abolished the accumulation of unwanted biosynthetic intermediates such as γ‐linolenic acid in total seed lipids. Expression of acyl‐CoA Δ6‐desaturases resulted in increased distribution of long‐chain polyunsaturated fatty acids in the polar lipids of transgenic plants, reflecting the larger substrate pool available for acylation by enzymes of the Kennedy pathway. Expression of the O. tauriΔ6‐desaturase in transgenic Camelina sativa plants also resulted in the accumulation of high levels of Δ6‐desaturated fatty acids. This study provides evidence for the efficacy of using acyl‐CoA‐dependent Δ6‐desaturases in the efficient metabolic engineering of transgenic plants with high value traits such as the synthesis of omega‐3 LC‐PUFAs.     

Bioorganic chemistry of plant lipid desaturation

The desaturation of long-chain fatty acids is a ubiquitous biotransformation which plays a critical role in the biosynthesis of plant lipids. Species-specific variations lead to unusual fatty acid signatures. Of particular interest is the unique ability of desaturases to oxidize unactivated hydrocarbon chains in such a chemo-, regio- and stereoselective manner. As part of ongoing research into the structure/activity relationships of this large class of enzymes, useful mechanistic tools have been developed to probe the active site. Recently a combination of stereo- and regiospecific deuterium, sulfur and fluorine labelling has been used to study the mechanism of a soluble plant Δ⁹ desaturase. The study of several membrane-bound desaturases has led to the conclusion that the dehydrogenation (desaturation) process is initiated by a kinetically important hydrogen activation step at the carbon of the incipient double bond which is closest to the acyl terminus of the fatty acid chain. A detailed analysis of a related 1,4-desaturation process has also been carried out.     

A cytochrome b5-containing plastid-located fatty acid desaturase from Chlamydomonas reinhardtii

Monogalactosyldiacylglycerol (MGDG) in Chlamydomonas reinhardtii and other green algae contains hexadeca-4,7,10,13-tetraenoic acid (16:4) in the glycerol sn-2 position. While many genes necessary for the introduction of acyl chain double bonds have been functionally characterized, the Δ4-desaturase remained unknown. Using a phylogenetic comparison, a candidate gene encoding the MGDG-specific Δ4-desaturase from Chlamydomonas (CrΔ4FAD) was identified. CrΔ4FAD shows all characteristic features of a membrane-bound desaturase, including three histidine boxes and a transit peptide for chloroplast targeting. But it also has an N-terminal cytochrome b(5) domain, distinguishing it from other known plastid desaturases. Cytochrome b(5) is the primary electron donor for endoplasmic reticulum (ER) desaturases and is often fused to the desaturase domain in desaturases modifying the carboxyl end of the acyl group. Difference absorbance spectra of the recombinant cytochrome b(5) domain of CrΔ4FAD showed that it is functional in vitro. Green fluorescent protein fusions of CrΔ4FAD localized to the plastid envelope in Chlamydomonas. Interestingly, overproduction of CrΔ4FAD in Chlamydomonas not only increased levels of 16:4 acyl groups in cell extracts but specifically increased the total amount of MGDG. Vice versa, the amount of MGDG was lowered in lines with reduced levels of CrΔ4FAD. These data suggest a link between MGDG molecular species composition and galactolipid abundance in the alga, as well as a specific function for this fatty acid in MGDG.     

A small membrane-peripheral region close to the active center determines regioselectivity of membrane-bound fatty acid desaturases from Aspergillus nidulans

Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-Delta12 desaturase and a processive bifunctional oleoyl-Delta12/linoleoyl-omega3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional Delta12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional Delta12/omega3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model.     

A bifunctional delta-fatty acyl acetylenase/desaturase from the moss Ceratodon purpureus. A new member of the cytochrome b5 superfamily.

Many plant genes have been cloned that encode regioselective desaturases catalyzing the formation of cis-unsaturated fatty acids. However, very few genes have been cloned that encode enzymes catalyzing the formation of the functional groups found in unusual fatty acids (e.g. hydroxy, epoxy or acetylenic fatty acids). Here, we describe the characterization of an acetylenase from the moss Ceratodon purpureus with a regioselectivity differing from the previously described Delta12-acetylenase. The gene encoding this protein, together with a Delta6-desaturase, was cloned by a PCR-based approach with primers derived from conserved regions in Delta5-, Delta6-fatty-acid desaturases and Delta8-sphingolipid desaturases. The proteins that are encoded by the two cloned cDNAs are likely to consist of a N-terminal extension of unknown function, a cytochrome b5-domain, and a C-terminal domain that is similar to acyl lipid desaturases with characteristic histidine boxes. The proteins were highly homologous in sequence to the Delta6-desaturase from the moss Physcomitrella patens. When these two cDNAs were expressed in Saccharomyces cerevisiae, both transgenic yeast cultures desaturated Delta9-unsaturated C16- and C18-fatty acids by inserting an additional Delta6cis-double bond. One of these transgenic yeast clones was also able to introduce a Delta6-triple bond into gamma-linolenic and stearidonic acid. This resulted in the formation of 9,12,15-(Z,Z,Z)-octadecatrien-6-ynoic acid, the main fatty acid found in C. pupureus. These results demonstrate that the Delta6-acetylenase from C. pupureus is a bifunctional enzyme, which can introduce a Delta6cis-double bond into 9,12,(15)-C18-polyenoic acids as well as converting a Delta6cis-double bond to a Delta6-triple bond.     

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.     

Acyl carriers used as substrates by the desaturases and elongases involved in very long-chain polyunsaturated fatty acids biosynthesis reconstituted in yeast

The health benefits attributed to very long-chain polyunsaturated fatty acids and the long term goal to produce them in transgenic oilseed crops have led to the cloning of all the genes coding for the desaturases and elongases involved in their biosynthesis. The encoded activities have been confirmed in vivo by heterologous expression, but very little is known about the actual acyl substrates involved in these pathways. Using a Delta 6-elongase and front-end desaturases from different organisms, we have reconstituted in Saccharomyces cerevisiae the biosynthesis of arachidonic acid from exogenously supplied linoleic acid in order to identify these acyl carriers. Acyl-CoA measurements strongly suggest that the elongation step involved in polyunsaturated fatty acids biosynthesis is taking place within the acyl-CoA pool. In contrast, detailed analyses of lipids revealed that the two desaturation steps (Delta 5 and Delta 6) occur predominantly at the sn-2 position of phosphatidylcholine when using Delta 5- and Delta 6-desaturases from lower plants, fungi, worms, and algae. The specificity of these Delta 6-desaturases for the fatty acid acylated at this particular position as well as a limiting re-equilibration with the acyl-CoA pool result in the accumulation of gamma-linolenic acid at the sn-2 position of phosphatidylcholine and prevent efficient arachidonic acid biosynthesis in yeast. We confirm by using a similar experimental approach that, in contrast, the human Delta 6-desaturase uses linoleoyl-CoA as substrate, which results in high efficiency of the subsequent elongation step. In addition, we report that Delta 12-desaturases have no specificity toward the lipid polar headgroup or the sn-position.     

Δ6-desaturase: improved methodology and analysis of the kinetics in a multi-enzyme system

A new method of assay for the A6-desaturation of linoleic acid was developed. This method, which uses HPLC for separation of the fatty acid substrate and product, exhibited a lower coefficient of variation (0.3%) than the reported TLC method (3.5%) [l], and avoided the step of methylation of the saponified fatty acid substrate and product. Using this new method of assay, the kinetics of the Δ6-desaturase in a multi-enzyme system were analysed. A number of factors that could have striking effects on desaturase kinetics were investigated, including the effect of (i) endogenous microsomal linoleic acid on total substrate concentration, and (ii) the pre-reaction catalysed by acyl-CoA synthetase and competing reactions catalysed by lysophospholipid acyltransferase and acyl-CoA hydrolase. Endogenous free linoleate in the hepatic microsomes was found to be 2.9 ± l.0 AM (0.5 mg microsomal protein/ml), which was comparable to added substrate concentrations (l.8 to 7.9 μM). The kinetics of the Δ6-desaturase were dissected from the kinetics of the above mentioned pre-reaction and competing reactions through a combination of experimental approaches and computer modeling. From computer modeling, a K_m and V_max of l.5 μM and 0.063 nmol/min were calculated for the Δ6-desaturase, compared to K_m and V_max of 10.7 μM and 0.08 nmol/min calculated directly from data uncorrected for endogenous substrate. It was concluded that lysophospholipid acyltransferase, acyl-CoA synthetase and endogenous linoleic acid significantly affect the kinetic measurements of hepatic microsomal Δ6-desaturase. These results have implications for kinetic analyses of all desaturases in microsomal systems.     

Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the omega3/6 Delta8 -desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Delta9 -specific elongating activity from Isochrysis galbana, a Delta8 -desaturase from Euglena gracilis and a Delta5 -desaturase from Mortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Delta9 -elongating activity, which may bypass rate-limiting steps present in the conventional Delta6 -desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.     

Delta-8 desaturation activity varies among fatty acyl desaturases of teleost fish: high activity in delta-6 desaturases of marine species

The benefits of dietary fish and fish oil are derived from n-3 long-chain polyunsaturated fatty acids (LC-PUFA) that have beneficial effects in a range of human diseases and pathologies such as cardiovascular and other inflammatory disorders, neural development and neurological pathologies. The precursor of n-3 LC-PUFA, 18:3n-3 does not have the same beneficial effects prompting interest in the pathways of endogenous synthesis of LC-PUFA in vertebrates. The LC-PUFA biosynthesis pathway classically involves Δ6 and Δ5 fatty acyl desaturases (Fad), but it was recently shown that Δ6 Fad in mammals also displayed Δ8 activity demonstrating a possible alternative "Δ8-pathway" for the synthesis of LC-PUFA. Our primary hypothesis was that Δ8 desaturase activity would be a common feature of vertebrate Δ6 Fads, and so the aim of the present study was to determine the ability of teleostei Fads for Δ8 desaturation activity. To this end, cDNAs for Fads from a range of freshwater, diadromous and marine teleost fish species were assayed for Δ8 activity in the heterologous yeast expression system. In summary, the present study has demonstrated that Δ8 desaturation activity was also a characteristic of fish orthologs, although the activity varied notably between freshwater/diadromous and marine fish species, with the latter possessing Fads2-like proteins with Δ8 activity far higher than mammalian FADS2. The data showed that, generally, the fish Fad are technically υ-3 desaturases, with new double bonds introduced 3C beyond a pre-existing double bond. However, the ability of zebrafish and rabbitfish Fads, previously characterised as Δ6/Δ5 bifunctional desaturases, to introduce non-methylene interrupted double bonds in 20:3n-3 and 20:2n-6 suggested that a novel combination of regioselectivity modes operates within these enzymes.     

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.     

The Effect of Dietary Sterol on the Activity of Fatty Acid Desaturases Isolated from Tetrahymena setosa

Tetrahymena setosa has a nutritional requirement for micro amounts of sterol, a requirement which is also satisfied by relatively large amounts of either intact phospholipids or a mixture of unsaturated fatty acids normally found in these ciliates. Three microsomal fatty acyl-CoA desaturases have been isolated from T. setosa and partially characterized. These enzymes which can account for the formation of the majority of the ciliate's unsaturated fatty acids, include: a Δ9, a Δ12 and a Δ6 desaturase which catalyze the transformation of stearoyl-CoA to oleic acid, of oleoyl-CoA to linoleic acid and of linoleoyl-CoA to ?-linolenic acid, respectively. The stearoyl CoA desaturase required NAD (or NADP), ATP and free CoA; the Δ6 and Δ12 desaturases required NADP, but not ATP or CoA. Cellular levels of the three desaturases were highest in mid-logarithmic phase cells and lowest in stationary phase cells. In order to determine if there was a relationship between the sterol requirement and the ability of the organism to desaturate, T. setosa was grown in a synthetic medium supplemented with either cholesterol or a phospholipid which permits growth in the absence of cholesterol, or with both phospholipid and cholesterol. Cells grown with phospholipid alone had only half as much stearoyl-CoA and oleoyl-CoA desaturase activity as cells of identical culture age grown either on cholesterol alone or on cholesterol plus phospholipid.     

Requirements of delta 9 and delta 12 fatty acid desaturation in Neurospora

Microsomes prepared from the wild-type strain and lipid auxotrophs of Neurospora were analyzed for delta 9 - (stearoyl-CoA) and delta 12 - (oleoyl-CoA) desaturase activities. The wild-type delta 9-desaturase was found to have a 20-fold higher specific activity and 2-fold lower activation energy than the delta 12-desaturase. In addition, delta 12-desaturase had higher Km app values for oleoyl-CoA and for NADH than the equivalent values for delta 9-desaturase. These properties were correlated with a rate-limiting role of delta 12-desaturase in the production of 18:2, the major fatty acid of Neurospora. The delta 12-desaturase also exhibited a higher tolerance to pH changes and to cyanide than did the delta 9-desaturase. Both activities could be measured in the same reaction mixture using stearoyl-CoA as the substrate, indicating a coupling of the two enzymes. Enrichment of cellular membranes of the wild-type Neurospora with 18:0 and 18:1, 18:2, 18:3 fatty acids led to the conclusion that the presence of excess substrate in the membrane induces activation of the appropriate desaturase. These experiments also suggested that the membrane fluidity, as determined by the degree of unsaturation of membrane fatty acids, may influence the activities of the desaturating enzymes. Perturbation of the polar head groups of the membrane phospholipids indicated that the correct composition of anionic phospholipids is an absolute requirement for the function of both desaturases. These studies show that the activities of the delta 9-desaturase and the delta 12-desaturase are regulated by a variety of factors and that the delta 12-desaturase is subjected to less stringent controls than the delta 9-desaturase.