Molecular cloning and functional analysis of a plastidial ω3 desaturase from Emiliania huxleyi.

Emiliania huxleyi is a marine microalga playing a significant ecological and biogeochemical role in oceans. It can produce several polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and octadecapentaenoic acid (OPA, 18:5-3,6,9,12,15), providing a primary source for nutritionally important ω3 PUFAs in the marine food chain. However, the biosynthesis of these PUFAs in this organism is not well understood. In this study, a full length plastidial ω3 desaturase cDNA (EhN3) was cloned from this alga. Heterologous expression of EhN3 with and without the chloroplast targeting peptide (cTP) in cyanobacterium Synechococcus elongatus showed that it possessed high desaturation activity toward C18-ω6 PUFAs, linoleic acid (LA, 18:2-9,12), γ-linolenic acid (GLA, 18:3-6,9,12), and C20-ω6 PUFAs, dihomo-γ-linolenic acid (DGLA, 20:3-8,11,14) and arachidonic acid (ARA, 20:4-5,8,11,14) that were exogenously supplied. Desaturation efficiency could reach almost 100% in a time course. On the other hand, when expressed in Saccharomyces cerevisiae, EhN3 with and without cTP did not exhibit any activity. Lipid analysis of Synechococcus transformants expressing EhN3 showed that it utilized galactolipids as substrates. Transcriptional expression analysis revealed that the expression of the gene increased while the growth temperature decreased, which was correlated with the increased production of ω3-PUFAs, particularly OPA. This is the first report of a plastidial ω3 desaturase from microalgae that can effectively introduce an ω3 double bond into both C18-ω6 and C20-ω6 PUFAs. EhN3 might also be one of the key enzymes involved in the biosynthesis of OPA in E. huxleyi through the plastidial aerobic pathway.

A chloroplast diacylglycerol lipase modulates glycerolipid pathway balance in Arabidopsis.

Two parallel pathways compartmentalized in the chloroplast and the endoplasmic reticulum contribute to thylakoid lipid synthesis in plants, but how these two pathways are coordinated during thylakoid biogenesis and remodeling remains unknown. We report here the molecular characterization of a homologous ADIPOSE TRIGLYCERIDE LIPASE-LIKE gene, previously referred to as ATGLL. The ATGLL gene is ubiquitously expressed throughout development and rapidly upregulated in response to a wide range of environmental cues. We show that ATGLL is a chloroplast non-regioselective lipase with a hydrolytic activity preferentially towards 16:0 of diacylglycerol (DAG). Comprehensive lipid profiling and radiotracer labeling studies revealed a negative correlation of ATGLL expression and the relative contribution of the chloroplast lipid pathway to thylakoid lipid biosynthesis. Additionally, we show that genetic manipulation of ATGLL expression resulted in changes in triacylglycerol levels in leaves. We propose that ATGLL, through affecting the level of prokaryotic DAG in the chloroplast, plays important roles in balancing the two glycerolipid pathways and in maintaining lipid homeostasis in plants.

Co-expressing Eranthis hyemalis lysophosphatidic acid acyltransferase 2 and elongase improves two very long chain polyunsaturated fatty acid production in Brassica carinata.

Docosadienoic acid (DDA, 22:2-13,16) and docosatrienoic acid (DTA, 22:3-13,16,19) are two very long chain polyunsaturated fatty acids (VLCPUFAs) that are recently shown to possess strong anti-inflammatory and antitumor properties. An ELO type elongase (EhELO1) from wild plant Eranthis hyemalis can synthesize the two fatty acids by sequential elongation of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop Brassica carinata produced a considerable amount of DDA and DTA in transgenic seeds. However, these fatty acids were excluded from the sn-2 position of triacylglycerols (TAGs). To improve the production level and nutrition value of the VLCPUFAs in the transgenic oilseed crop, a cytoplasmic lysophosphatidic acid acyltransferase (EhLPAAT2) for the incorporation of the two fatty acids into the sn-2 position of triacylglycerols was identified from E. hyemalis. RT-PCR analysis showed that it was preferentially expressed in developing seeds where EhELO1 was exclusively expressed in E. hyemalis. Seed specific expression of EhLPAAT2 along with EhELO1 in B. carinata resulted in the effective incorporation of DDA and DTA at the sn-2 position of TAGs, thereby increasing the total amount of DDA and DTA in transgenic seeds. To our knowledge, this is the first plant LPAAT that can incorporate VLCPUFAs into TAGs. Improved production of DDA and DTA in the oilseed crop using EhLPAAT2 and EhELO1 provides a real commercial opportunity for high value agriculture products for nutraceutical uses.

Functional Analysis of an Acyltransferase-Like Domain from Polyunsaturated Fatty Acid Synthase in Thraustochytrium.

Biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFA) such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and docosapentaenoic acid (DPA, 22:5-4,7,10,13,16) in protist Thraustochytrium is catalyzed by a polyunsaturated fatty acids (PUFA) synthase comprising three large subunits, each with multiple catalytic domains. This study used complementation test, in vitro assays, and functional expression to characterize an acyltransferase (AT)-like domain in Subunit-B of a PUFA synthase from Thraustochytrium. Complementation test in Escherichia coli showed that the AT-like domain could not restore the growth phenotype of a temperature-sensitive mutant (∆fabDts) defective in malonyl-CoA:ACP transacylase activity. In vitro assays showed that the AT-like domain possessed thioesterase activity towards a few acyl-CoAs tested where docosahexaenoyl-CoA (DHA-CoA) was the preferred substrate. Expression of this domain in an E. coli mutant (∆fadD) defective in acyl-CoA synthetase activity resulted in the increased accumulation of free fatty acids. Site-directed mutagenesis showed that the substitution of two putative active site residues, serine at 96 (S96) and histidine at 220 (H220), in the AT-like domain significantly reduced its activity towards DHA-CoA and accumulation of free fatty acids in the ∆fadD mutant. These results indicate that the AT-like domain of the PUFA synthase does not function as a malonyl-CoA:ACP transacylase, rather it functions as a thioesterase. It might catalyze the last step of the VLCPUFA biosynthesis by releasing freshly synthesized VLCPUFAs attached to ACP domains of the PUFA synthase in Thraustochytrium.

Distinct functions of two FabA-like dehydratase domains of polyunsaturated fatty acid synthase in the biosynthesis of very long-chain polyunsaturated fatty acids.

Thraustochytrium is a unicellular marine protist for the commercial production of very long-chain polyunsaturated fatty acids (VLCPUFAs). Biosynthesis of these VLCPUFAs in the protist is catalysed by a PUFA synthase comprising three subunits, each with multiple catalytic domains. Among these domains, two tandem FabA-like dehydratase domains (DH1 and DH2) in subunit-C together are responsible for introducing double bonds in VLCPUFAs. Domain swapping analysis in yeast showed that the defective phenotype of a Scfas1 mutant could be complemented by expressing an engineered ScFAS1 gene in which the DH domain was replaced by a single DH1 or mutated DH2 of the two. Heterologous expression of the PUFA synthase in E. coli showed that the mutation of DH1 of the two or deletion of DH1 or substitution of DH1 with DH2 resulted in the complete loss of activity in the biosynthesis of VLCPUFAs. Mutation of DH2 of the two or deletion of the DH2 domain produced a small amount of DPA, but not docosahexaenoic acid (DHA). These results indicate that each of the two FabA-like domains of the PUFA synthase possesses distinct function. DH1 domain is essential for the biosynthesis of VLCPUFAs, but DH2 domain is required for the biosynthesis of DHA.

Molecular mechanisms for biosynthesis and assembly of nutritionally important very long chain polyunsaturated fatty acids in microorganisms.

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6n-3), arachidonic acid (ARA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5-n3) are nutritionally important for humans and animals. De novo biosynthesis of these fatty acids mainly occurs in microorganisms and goes through either an aerobic pathway catalyzed by type I/II fatty acid synthase, desaturases and elongases or an anaerobic pathway catalyzed by a polyunsaturated fatty acid synthase. After synthesis, VLCPUFAs must be incorporated into glycerolipids for storage through acyl assembly processes. Understanding the mechanisms for the biosynthesis of VLCPUFAs and their incorporation into glycerolipids is important not only for developing a renewable, sustainable and environment-friendly source of these fatty acids in microorganisms, but also, for designing effective strategies for metabolic engineering of these fatty acids in heterologous systems. This review highlights recent findings which have increased our understanding of biosynthesis of VLCPUFAs and their incorporation into glycerolipids in microorganisms. Future directions in improving the production of VLCPUFAs in native microbial producers are also discussed along with transgenic production of these fatty acids in oleaginous microorganisms and oilseed crops for food and feed uses.

The Biosynthetic Pathway of Major Avenanthramides in Oat.

Avenanthramides are a group of N-cinnamoylanthranilic acids, with health-promoting properties mainly found in oat (Avena sativa L.). However, the biosynthetic mechanism for the main three types of avenanthramides (Avn-A, Avn-B and Avn-C) is not completely understood. In the present study, we report molecular identification and functional characterization of three different types of genes from oat encoding 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) and a caffeoyl-CoA O-methyltransferase (CCoAOMT) enzymes, all involved in the biosynthesis of these avenanthramides. In vitro enzymatic assays using the proteins expressed in Escherichia coli showed that oat 4CL could convert p-coumaric acid, caffeic acid and ferulic acid to their CoA thioesters. Oat HHTs were only responsible for the biosynthesis of Avn-A and Avn-C using hydroxyanthranilic acid as an acyl acceptor and p-coumaroyl-CoA and caffeoyl-CoA as an acyl donor, respectively. Avn-B was synthesized by a CCoAOMT enzyme through the methylation of Avn-C. Collectively, these results have elucidated the molecular mechanisms for the biosynthesis of three major avenanthramides in vitro and paved the way for metabolic engineering of the biosynthetic pathway in heterologous systems to produce nutraceutically important compounds and make possible genetic improvement of this nutritional trait in oat through marker-assisted breeding.

Enhancing oil production in Arabidopsis through expression of a ketoacyl-ACP synthase domain of the PUFA synthase from Thraustochytrium.

BACKGROUND: Plant seed oil is an important bioresource for human food and animal feed, as well as industrial bioproducts. Therefore, increasing oil content in seeds has been one of the primary targets in the breeding programs of oilseed crops. Thraustochytrium is a marine protist that can produce a high level of very long-chain polyunsaturated fatty acids (VLCPUFAs) using a PUFA synthase, a polyketide synthase-like fatty acid synthase with multiple catalytic domains. Our previous study showed that a KS domain from the synthase could complement an Escherichia coli mutant defective in ß-ketoacyl-ACP synthase I (FabB) and increase the total fatty acid production. In this study, this KS domain from the PUFA synthase was further functionally analyzed in Arabidopsis thaliana for the capacity of oil production. RESULTS: The plastidial expression of the KS domain could complement the defective phenotypes of a KASI knockout mutant generated by CRISPR/Cas9. Seed-specific expression of the domain in wild-type Arabidopsis significantly increased seed weight and seed oil, and altered the unsaturation level of fatty acids in seeds, as well as promoted seed germination and early seedling growth. CONCLUSIONS: The condensation process of fatty acid biosynthesis in plants is a limiting step, and overexpression of the KS domain from a PUFA synthase of microbial origin offers a new strategy to increase oil production in oilseed plants.

An engineered oilseed crop produces oil enriched in two very long chain polyunsaturated fatty acids with potential health-promoting properties.

Very long chain polyunsaturated fatty acids (VLCPUFAs) are well recognized for their health benefits in humans and animals. Here we report that identification and characterization of a gene (EhELO1) encoding the first functional ELO type elongase (3-ketoacyl-CoA synthase) in higher plants that is involved in the biosynthesis of two VLCPUFAs docosadienoic acid (DDA, 22:2n-6) and docosatrienoic acid (DTA, 22:3n-3) that possess potential health-promoting properties. Functional analysis of the gene in yeast indicated that this novel enzyme could elongate a wide range of polyunsaturated fatty acids with 18-22 carbons and effectively catalyze the biosynthesis of DDA and DTA by the sequential elongations of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop Brassica carinata showed that the transgenic plants produced the level of DDA and DTA at approximately 30% of the total fatty acids in seeds, and the amount of the two fatty acids remained stable over four generations. The oilseed crop producing a high and sustained level of DDA and DTA provides an opportunity for high value agricultural products for nutritional and medical uses.

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.

Identification and functional analysis of new peroxygenases in oat.

MAIN CONCLUSION: Two new peroxygenases for the biosynthesis of epoxy fatty acids in oat were identified and functionally analyzed by heterologous expression along with rationally designed site-directed mutagenesis. Oat (Avena sativa L.) contains a large family of peroxygenases, a group of heme-containing monooxygenases catalyzing hydroperoxide-dependent epoxidation of unsaturated fatty acids. Here, we report identification and functional analysis of two new peroxygenases AsPXG2 and AsPXG3 from oat. The open reading frame (ORF) of AsPXG2 contains 702 bps encoding a polypeptide of 233 amino acids, while the ORF of AsPXG3 is 627 bps coding for 208 amino acids. Both AsPXG2 and AsPXG3 comprise a single transmembrane domain, conserved histidines for heme binding and a conserved EF-hand motif for calcium binding, but they only share about 50% amino acid sequence identity with each other. When expressed in Escherichia coli and Pichia pastoris, AsPXG3 showed high epoxidation activity, while AsPXG2 exhibited no activity in E. coli and low activity in P. pastoris. AsPXG3 could effectively epoxidize both mono- and polyunsaturated fatty acids with linolenic acid being the most preferred substrate. Site-directed mutagenesis was employed to investigate the structure-function relationship of oat peroxygenase on 12 conserved residues of AsPXG3. Replacement of two conserved histidines, the ligands to the prosthetic heme group of the peroxygenase, by alanine resulted in complete loss of activity. Substitution of three conserved residues surrounding the two histidines resulted in reduction of the enzymatic activity by more than 80%. These results imply that these conserved residues might be located in or near the catalytic pocket, where the two histidine residues coordinate the heme group and the surrounding residues define the shape and size of the pocket for interaction with the heme as well as two substrates.

Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli.

Thraustochytrium sp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes in Escherichia coli The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both E. colifabB and fabF mutants. Overexpression of these domains in wild-type E. coli led to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains in E. coli are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in ThraustochytriumIMPORTANCE Very-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a megaenzyme with multiple subunits, each with multiple catalytic domains. Furthermore, the fundamental mechanism for this enzyme to synthesize these fatty acids still remains unknown. This report started with dissecting the embedded KS domains of the PUFA synthase from marine protist Thraustochytrium sp. strain ATCC 26185 and then expressing them in wild-type E. coli and mutants defective in condensation of acyl-ACP with malonyl-ACP. Successful complementation of the mutants and improved fatty acid production in the overexpression experiments indicate that these KS domains can effectively function as stand-alone enzymes in E. coli This result has paved the way for further studying of molecular mechanisms of the PUFA synthase for the biosynthesis of VLCPUFAs.

Biosynthetic mechanism of very long chain polyunsaturated fatty acids in Thraustochytrium sp. 26185.

Thraustochytrium, a unicellular marine protist, has been used as a commercial source of very long chain PUFAs (VLCPUFAs) such as DHA (22:6n-3). Our recent work indicates coexistence of a Δ4-desaturation-dependent pathway (aerobic) and a polyketide synthase-like PUFA synthase pathway (anaerobic) to synthesize the fatty acids in Thraustochytrium sp. 26185. Heterologous expression of the Thraustochytrium PUFA synthase along with a phosphopantetheinyl transferase in Escherichia coli showed the anaerobic pathway was highly active in the biosynthesis of VLCPUFAs. The amount of Δ4 desaturated VLCPUFAs produced reached about 18% of the total fatty acids in the transformant cells at day 6 in a time course of the induced expression. In Thraustochytrium, the expression level of the PUFA synthase gene was much higher than that of the Δ4 desaturase gene, and also highly correlated with the production of VLCPUFAs. On the other hand, Δ9 and Δ12 desaturations in the aerobic pathway were either ineffective or absent in the species, as evidenced by the genomic survey, heterologous expression of candidate genes, and in vivo feeding experiments. These results indicate that the anaerobic pathway is solely responsible for the biosynthesis for VLCPUFAs in Thraustochytrium.

Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance.

Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a "push" (synthesis) and "pull" (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the Δ12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses.

Structure determinants for the substrate specificity of acyl-CoA Δ9 desaturases from a marine copepod.

In contrast to soluble acyl-ACP desaturases from plants, little is known about the structure-guiding principle underlying substrate specificity and regioselectivity of membrane-bound acyl-CoA desaturases from animals, mainly due to lack of the three-dimensional structure information. Here we report identification of two homologous membrane-bound acyl-CoA Δ9 desaturases (ChDes9-1 and ChDes9-2) from the marine copepod Calanus hyperboreus that accumulates more than 90% of total storage lipids in the form of wax esters. ChDes9-2 is a common Δ9 desaturase with substrate specificity to long chain fatty acid 18:0, while ChDes9-1 is a new type of Δ9 desaturase introducing a Δ9 double bond into a wide range of very long chain fatty acids ranging from 20:0 to 26:0. Reciprocal domain swapping and site-directed mutagenesis guided by the membrane topology revealed that presence or absence of an amphipathic and bulky residue, tyrosine, in the middle of the second transmembrane domain was important in determining the substrate specificity of the two desaturases. To examine the mechanistic structure for the substrate specificity, tyrosine-scanning mutagenesis was employed to systematically substitute the residues in the transmembrane domain of the very long chain desaturase. The results showed that the transmembrane domain formed an α-helix structure probably involved in formation of the substrate-binding pocket and the corresponding residue of the tyrosine likely resided at the critical position within the pocket mediating the interaction with the substrates, thereby specifying the chain length of the substrates.

Phytophthora infestans cholinephosphotransferase with substrate specificity for very-long-chain polyunsaturated fatty acids.

The effective flux between phospholipids and neutral lipids is critical for a high level of biosynthesis and accumulation of very-long-chain polyunsaturated fatty acids (VLCPUFAs), such as arachidonic acid (ARA; 20:4n-6), eicosapentaenoic acid (EPA; 20:5n-3), and docosahexaenoic acid (DHA; 22:6n-3). Here we describe a cDNA (PiCPT1) from Phytophthora infestans, a VLCPUFA-producing oomycete, that may have a role in acyl trafficking between diacylglycerol (DAG) and phosphatidylcholine (PC) during the biosynthesis of VLCPUFAs. The cDNA encodes a polypeptide of 393 amino acids with a conserved CDP-alcohol phosphotransferase motif and approximately 27% amino acid identity to the Saccharomyces cerevisiae cholinephosphotransferase (ScCPT1). In vitro assays indicate that PiCPT1 has high cholinephosphotransferase (CPT) activity but no ethanolaminephosphotransferase (EPT) activity. Substrate specificity assays show that it prefers VLCPUFA-containing DAGs, such as ARA DAG and DHA DAG, as substrates. Real-time PCR analysis reveals that expression of PiCPT1 was upregulated in P. infestans organisms fed with exogenous VLCPUFAs. These results lead us to conclude that PiCPT1 is a VLCPUFA-specific CPT which may play an important role in shuffling VLCPUFAs from DAG to PC in the biosynthesis of VLCPUFAs in P. infestans.

The front-end desaturase: structure, function, evolution and biotechnological use.

Very long chain polyunsaturated fatty acids such as arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are essential components of cell membranes, and are precursors for a group of hormone-like bioactive compounds (eicosanoids and docosanoids) involved in regulation of various physiological activities in animals and humans. The biosynthesis of these fatty acids involves an alternating process of fatty acid desaturation and elongation. The desaturation is catalyzed by a unique class of oxygenases called front-end desaturases that introduce double bonds between the pre-existing double bond and the carboxyl end of polyunsaturated fatty acids. The first gene encoding a front-end desaturase was cloned in 1993 from cyanobacteria. Since then, front-end desaturases have been identified and characterized from a wide range of eukaryotic species including algae, protozoa, fungi, plants and animals including humans. Unlike front-end desaturases from bacteria, those from eukaryotes are structurally characterized by the presence of an N-terminal cytochrome b5-like domain fused to the main desaturation domain. Understanding the structure, function and evolution of front-end desaturases, as well as their roles in the biosynthesis of very long chain polyunsaturated fatty acids offers the opportunity to engineer production of these fatty acids in transgenic oilseed plants for nutraceutical markets.

A peroxygenase pathway involved in the biosynthesis of epoxy fatty acids in oat.

While oat (Avena sativa) has long been known to produce epoxy fatty acids in seeds, synthesized by a peroxygenase pathway, the gene encoding the peroxygenase remains to be determined. Here we report identification of a peroxygenase cDNA AsPXG1 from developing seeds of oat. AsPXG1 is a small protein with 249 amino acids in length and contains conserved heme-binding residues and a calcium-binding motif. When expressed in Pichia pastoris and Escherichia coli, AsPXG1 catalyzes the strictly hydroperoxide-dependent epoxidation of unsaturated fatty acids. It prefers hydroperoxy-trienoic acids over hydroperoxy-dienoic acids as oxygen donors to oxidize a wide range of unsaturated fatty acids with cis double bonds. Oleic acid is the most preferred substrate. The acyl carrier substrate specificity assay showed phospholipid and acyl-CoA were not effective substrate forms for AsPXG1 and it could only use free fatty acid or fatty acid methyl esters as substrates. A second gene, AsLOX2, cloned from oat codes for a 9-lipoxygenase catalyzing the synthesis of 9-hydroperoxy-dienoic and 9-hydroperoxy-trienoic acids, respectively, when linoleic (18:2-9c,12c) and linolenic (18:3-9c,12c,15c) acids were used as substrates. The peroxygenase pathway was reconstituted in vitro using a mixture of AsPXG1 and AsLOX2 extracts from E. coli. Incubation of methyl oleate and linoleic acid or linolenic acid with the enzyme mixture produced methyl 9,10-epoxy stearate. Incubation of linoleic acid alone with a mixture of AsPXG1 and AsLOX2 produced two major epoxy fatty acids, 9,10-epoxy-12-cis-octadecenoic acid and 12,13-epoxy-9-cis-octadecenoic acid, and a minor epoxy fatty acid, probably 12,13-epoxy-9-hydroxy-10-transoctadecenoic acid. AsPXG1 predominately catalyzes intermolecular peroxygenation.

Molecular analysis of ∆6 desaturase and ∆6 elongase from Conidiobolus obscurus in the biosynthesis of eicosatetraenoic acid, a ω3 fatty acid with nutraceutical potentials.

Conidiobolus obscurus, an entomopathogenic fungus able to infect aphids, was previously reported to produce substantial amounts of very long chain polyunsaturated fatty acids (VLCPUFAs) that may mediate the insect infection. However, the genes involved in the biosynthesis of these VLCPUFAs from the order Entomophthorales have yet to be identified. Using degenerate reverse transcriptase-polymerase chain reaction and rapid amplification of the cDNA end methods, we cloned a ∆6 desaturase cDNA (CoD6) and a ∆6 elongase cDNA (CoE6) from C. obscurus. Expression of CoD6 and CoE6 in Saccharomyces cerevisiae revealed CoD6 could introduce a Δ6 double bond into α-linolenic acid (18:3n-3), and CoE6 preferentially elongated 18-carbon Δ6 desaturated fatty acid stearidonic acid (18:4n-3). When the fungus was grown under a temperature shift from 20 °C to 10 °C, the transcript level of CoD6 and CoE6 increased, whereas when the fungal culture was shifted from 20 °C to 30 °C, the transcript level of both genes decreased. The entire eicosatetraenoic acid biosynthetic pathway was reconstituted in yeast using four genes, CoD6 and CoE6 from C. obscurus, CpDes12 (a Δ12 desaturase) and CpDesX (a ω3 desaturase) from Claviceps purpurea. Yeast transformants expressing the four genes produced ten new fatty acids including the final product eicosatetraenoic acid (ETA). This represents the reconstitution of the entire ETA pathway in yeast without supplementation of any exogenous fatty acids.