Synthesis of oleyl oleate wax esters in Arabidopsis thaliana and Camelina sativa seed oil.

Seed oil composed of wax esters with long-chain monoenoic acyl moieties represents a high-value commodity for industry. Such plant-derived sperm oil-like liquid wax esters are biodegradable and can have excellent properties for lubrication. In addition, wax ester oil may represent a superior substrate for biodiesel production. In this study, we demonstrate that the low-input oil seed crop Camelina sativa can serve as a biotechnological platform for environmentally benign wax ester production. Two biosynthetic steps catalysed by a fatty alcohol-forming acyl-CoA reductase (FAR) and a wax ester synthase (WS) are sufficient to achieve wax ester accumulation from acyl-CoA substrates. To produce plant-derived sperm oil-like liquid wax esters, the WS from Mus musculus (MmWS) or Simmondsia chinensis (ScWS) were expressed in combination with the FAR from Mus musculus (MmFAR1) or Marinobacter aquaeolei (MaFAR) in seeds of Arabidopsis thaliana and Camelina sativa. The three analysed enzyme combinations Oleo3:mCherry:MmFAR1∆c/Oleo3:EYFP:MmWS, Oleo3:mCherry:MmFAR1∆c/ScWS and MaFAR/ScWS showed differences in the wax ester molecular species profiles and overall biosynthetic performance. By expressing MaFAR/ScWS in Arabidopsis or Camelina up to 59% or 21% of the seed oil TAGs were replaced by wax esters, respectively. This combination also yielded wax ester molecular species with highest content of monounsaturated acyl moieties. Expression of the enzyme combinations in the Arabidopsis fae1 fad2 mutant background high in oleic acid resulted in wax ester accumulation enriched in oleyl oleate (18:1/18:1 > 60%), suggesting that similar values may be obtained with a Camelina high oleic acid line.

Synthesis of ascorbyl oleate by transesterification of olive oil with ascorbic acid in polar organic media catalyzed by immobilized lipases.

The reaction of transesterification between oils (e.g., olive oil) and ascorbic acid in polar anhydrous media (e.g., tert-amyl alcohol) catalyzed by immobilized lipases for the preparation of natural liposoluble antioxidants (e.g., ascorbyl oleate) was studied. Three commercial lipases were tested: Candida antarctica B lipase (CALB), Thermomyces lanuginosus lipase (TLL) and Rhizomucor miehei lipase (RML). Each lipase was immobilized by three different protocols: hydrophobic adsorption, anionic exchange and multipoint covalent attachment. The highest synthetic yields were obtained with CALB adsorbed on hydrophobic supports (e.g., the commercial derivative Novozym 435). The rates and yields of the synthesis of ascorbyl oleate were higher when using the solvent dried with molecular sieves, at high temperatures (e.g. 45°C) and with a small excess of oil (2 mol of oil per mol of ascorbic acid). The coating of CALB derivatives with polyethyleneimine (PEI) improved its catalytic behavior and allowed the achievement of yields of up to 80% of ascorbyl oleate in less than 24h. CALB adsorbed on a hydrophobic support and coated with PEI was 2-fold more stable than a non-coated derivative and one hundred-fold more stable than the best TLL derivative. The best CALB derivative exhibited a half-life of 3 days at 75°C in fully anhydrous media, and this derivative maintained full activity after 28 days at 45°C in dried tert-amyl alcohol.

Hydroxyl octadecenoic acids biosynthesized by crown galls of Panax quinquefolium induced by artermisinic acid.

OBJECTIVE: To investigate the effect of artermisinic acid on the secondary metabolites production of Panax quinquefolium crown galls. METHODS: Artemisinic acid was added into the suspended cells of Panax quinquefolium crown galls and co-culture for two days. Products were isolated with chromatographic method. RESULTS: Three hydroxyl octadecenoic acids [9,12,13-trihydroxy-10-octadecenoic acid (1), 11,12,13-trihydroxy-9-octadecenoic acid (2) and 11-hydroxy-12,13-epoxy-9-octadecenoic acid (3)] were isolated from crown galls of Panax quinquefolium. CONCLUSION: Artermisinic acid as one of the new type of phytohormones that might induce the production of 13-lipoxygenases in crown galls of Panax quinquefolium.

Lipids from heterotrophic microbes: advances in metabolism research.

Heterotrophic oleaginous microorganisms are capable of producing over 20% of their weight in single cell oils (SCOs) composed of triacylglycerols (TAGs). These TAGs contain fatty acids, such as palmitic, stearic and oleic acids, that are well-suited for biodiesel applications. Although some of these microbes are able to accumulate SCOs while growing on inexpensive agro-industrial biomass, the competition with plant oil resources means that a significant increase in productivity is desired. The present review aims to summarize recent details in lipid metabolism research and engineering (e.g. direct fatty acid ethyl ester production), as well as culture condition optimization and innovations, such as solid-state or semi-solid-state fermentation, that can all contribute to higher productivity and further advancement of the field.

Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait.

BACKGROUND: The alteration of fatty acid profiles in soybean [Glycine max (L.) Merr.] to improve soybean oil quality is an important and evolving theme in soybean research to meet nutritional needs and industrial criteria in the modern market. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of the oil. Commodity soybean oil typically contains 20% oleic acid and the target for high oleic acid soybean oil is approximately 80% of the oil; previous conventional plant breeding research to raise the oleic acid level to just 50-60% of the oil was hindered by the genetic complexity and environmental instability of the trait. The objective of this work was to create the high oleic acid trait in soybeans by identifying and combining mutations in two delta-twelve fatty acid desaturase genes, FAD2-1A and FAD2-1B. RESULTS: Three polymorphisms found in the FAD2-1B alleles of two soybean lines resulted in missense mutations. For each of the two soybean lines, there was one unique amino acid change within a highly conserved region of the protein. The mutant FAD2-1B alleles were associated with an increase in oleic acid levels, although the FAD2-1B mutant alleles alone were not capable of producing a high oleic acid phenotype. When existing FAD2-1A mutations were combined with the novel mutant FAD2-1B alleles, a high oleic acid phenotype was recovered only for those lines which were homozygous for both of the mutant alleles. CONCLUSIONS: We were able to produce conventional soybean lines with 80% oleic acid in the oil in two different ways, each requiring the contribution of only two genes. The high oleic acid soybean germplasm developed contained a desirable fatty acid profile, and it was stable in two production environments. The presumed causative sequence polymorphisms in the FAD2-1B alleles were developed into highly efficient molecular markers for tracking the mutant alleles. The resources described here for the creation of high oleic acid soybeans provide a framework to efficiently develop soybean varieties to meet changing market demands.

Culture conditions stimulating high gama-Linolenic acid accumulation by Spirulina platensis / Condições de cultura simulando o levado acúmulo de ácido gama-linolênico por Spirulina platensis

Gamma-linolenic acid (GLA) production by Spirulina platensis under different stress-inducing conditions was studied. Submerged culture studies showed that low temperature (25ºC), strong light intensity (6 klux) and primrose oil supplement (0.8 percentw/v) induced 13.2 mg/g, 14.6 mg/g and 13.5 mg linolenic acid per gram dry cell weight respectively. A careful observation of fatty acid profile of the cyanobacteria shows that, oleic acid and linoleic acid, in experiments with varying growth temperature and oil supplements respectively, helped in accumulating excess γ-linolenic acid. In addition, cultures grown at increasing light regimes maintained the γ-linolenic acid to the total fatty acid ratio(GLA/TFA) constant, despite any change in γ-linolenic acid content of the cyanobacteria.

Estudou-se a produção de ácido γ-linolênico por Spirulina platensis em diferentes condições de estresse. Culturas submersas indicaram que temperatura baixa (25ºC), forte intensidade de luz (6 klux) e suplementação com óleo de prímula (0,8 por cento p/v) induziram a produção de ácido linolênico de 13,2 mg/g, 14,6 mg/g e 13,5 mg/g peso seco, respectivamente. Uma observação cuidadosa do perfil de ácidos graxos da cianobacteria indica que os ácidos oléico e linoléico, em experimentos com diferentes temperaturas de crescimento e suplementos de óleo, auxiliaram no acúmulo de excesso de ácido γ-linolênico. Além disso, as culturas obtidas em intensidades crescentes de luz mantiveram a relação ácido γ-linolênico/ácidos graxos totais constante, independentemente de qualquer mudança no conteúdo de ácido γ-linolênico da cianobactéria.

The effect of solids dilution rate and oil source on trans C18:1 and conjugated linoleic acid production by ruminal microbes in continuous culture.

The objective of this study was to evaluate the effect of solids dilution rate (SDR) and oil source [soybean oil (SBO) or linseed oil (LSO)] on the ruminal production of trans C18:1 and conjugated linoleic acid (CLA). A dual-flow continuous culture system consisting of 4 fermenters was used in a 4 x 4 Latin square design with a factorial arrangement of treatments over 4 consecutive periods of 10 d each. Treatment diets (50:50 forage to concentrate) were fed at 120 g/d of dry matter (DM) in 3 equal portions. The concentrate mix contained 1% fish oil and either 2% SBO or 2% LSO on a DM basis. Treatments were as follows: 1) SBO at 6%/h SDR, 2) SBO at 3%/h SDR, 3) LSO at 6%/h SDR, and 4) LSO at 3%/h SDR. The oil source by SDR interaction was not significant for trans C18:1 and CLA fatty acids. The concentrations of trans C18:1 and vaccenic acid were greater in effluents when diets were supplemented with SBO vs. LSO (37.11 vs. 34.09 and 32.71 vs. 29.70 mg/g of DM, respectively) and at high SDR than low SDR (37.60 vs. 33.61 and 32.72 vs. 29.61 mg/g of DM, respectively). The concentration of cis-9, trans-11 CLA in effluents was also greater with SBO than LSO (0.81 vs. 0.40 mg/g of DM) supplementation and at high SDR than low SDR (0.68 vs. 0.54 mg/g of DM). Biohydrogenation of linoleic acid and linolenic acid increased at higher SDR within each oil treatment. Based on these results, SBO supplementation at high SDR enhances ruminal production of vaccenic acid, and therefore could potentially enhance cis-9, trans-11 CLA in milk fat through synthesis by Delta9-desaturase.

A multifunctional acyl-acyl carrier protein desaturase from Hedera helix L. (English ivy) can synthesize 16- and 18-carbon monoene and diene products.

A desaturase with 83% sequence identity to the coriander delta(4)-16:0-ACP desaturase was isolated from developing seeds of Hedera helix (English ivy). Expression of the ivy desaturase in Arabidopsis resulted in the accumulation of 16:1delta(4) and its expected elongation product 18:1delta(6) (petroselinic acid). Expression in Escherichia coli resulted in the accumulation of soluble, active protein that was purified to apparent homogeneity. In vitro assays confirmed delta(4) desaturation with 16:0-ACP; however, with 18:0-acyl acyl carrier protein (ACP) desaturation occurred at the delta(9) position. The ivy desaturase also converted 16:1delta(9)-ACP and 18:1delta(9)-ACP to the corresponding delta(4,9) dienes. These data suggest at least two distinct substrate binding modes, one placing C4 at the diiron active site and the other placing C9 at the active site. In the latter case, 18:0 would likely bind in an extended conformation as described for the castor desaturase with 9-carbons accommodated in the cavity beyond the dirron site. However, delta(4) desaturation would require the accommodation of 12 carbons for C16 substrates or 14 carbons for C18 substrates. The amino acids lining the substrate binding cavity of ivy and castor desaturases are conserved except for T117R and P179I (castor/ivy). Paradoxically, both substitutions, when introduced into the castor desaturase, favored the binding of shorter acyl chains. Thus, it seems likely that delta(4) desaturation would require a non-extended, perhaps U-shaped, substrate conformation. A cis double bond may facilitate the initiation of such a non-extended conformation in the monounsaturated substrates. The multifunctional properties of the ivy desaturase make it well suited for further dissection of the determinants of regiospecificity.

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.

Transgenic production of epoxy fatty acids by expression of a cytochrome P450 enzyme from Euphorbia lagascae seed.

Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Delta(12)-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Delta(12)-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developing E. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to the CYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Delta(9,15)) in cultures supplied with alpha-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Delta(9,15). Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Delta(12)-epoxy group of vernolic acid.

An unusual seed-specific 3-ketoacyl-ACP synthase associated with the biosynthesis of petroselinic acid in coriander.

Petroselinic acid (18:1 delta6) is the major component of the seed oil of Umbelliferae species such as coriander (Coriandrum sativum) as well as Araliaceae and Garryaceae species. This unusual fatty acid is synthesized in plastids by the delta4 desaturation of palmitoyl-acyl carrier protein (16:0-ACP) and subsequent elongation of delta4-hexadecenoyl (16:1 delta4)-ACP. To characterize the enzymatic nature of the elongation reaction, an in vitro assay was developed with 16:1 delta4-ACP and 16:0-ACP as substrates. Extracts from developing coriander seeds elongated 16:1 delta4-ACP in a competitive assay at rates ten-fold greater than that with 16:0-ACP. In contrast, extracts from castor seeds, which do not synthesize petroselinic acid, displayed a strong preference for the elongation of 16:0-ACP rather than 16:1 A4-ACP. In addition, the elongation of 16:1 A4-ACP and 16:0-ACP by coriander seed extracts was strongly inhibited by cerulenin at concentrations as low as 10 microM. This finding suggested that the elongation of 16:1 A4-ACP and 16:0-ACP in coriander seed is catalyzed by a 3-ketoacyl-ACP synthase (KAS) 1-type enzyme(s), rather than a KAS II-type activity that is typically associated with 16:0-ACP elongation. Consistent with this, a cDNA for a diverged form of KAS I was isolated from a cDNA library prepared from developing coriander seed. Using a variety of heterologous probing techniques, no KAS II-type cDNAs could be identified in this library. Multiple alignment of KAS amino acid sequences indicated that, although the polypeptide corresponding to the coriander cDNA is more closely related to KAS I. its active site motif deviates from those found in both KAS I and KAS II enzymes. Also suggestive of a possible role in petroselinic acid synthesis, antibodies raised to the recombinant protein recognize an abundant 45 kDa polypeptide in coriander endosperm that is not detected in coriander leaves. These antibodies also recognize a major band of similar size in developing seeds of English ivy (Hedera helix), an Araliaceae species that also accumulates petroselinic acid in a seed-specific manner.

An epoxy alcohol synthase pathway in higher plants: biosynthesis of antifungal trihydroxy oxylipins in leaves of potato.

[1-14C]Linoleic acid was incubated with a whole homogenate preparation of potato leaves (Solanum tuberosum L., var. Bintje). The methyl-esterified product was subjected to straight-phase high-performance liquid chromatography and was found to contain four major radioactive oxidation products, i.e., the epoxy alcohols methyl 10(S),11(S)-epoxy-9(S)-hydroxy-12(Z)-octadecenoate (14% of the recovered radioactivity) and methyl 12(R), 13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoate (14%), and the trihydroxy derivatives methyl 9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoate (18%)and methyl 9(S), 12(S),13(S)-trihydroxy-10(E)-octadecenoate (30%). The structures and stereochemical configurations of these oxylipins were determined by chemical and spectral methods using the authentic compounds as references. Incubations performed in the presence of glutathione peroxidase revealed that lipoxygenase activity of potato leaves generated the 9- and 13-hydroperoxides of linoleic acid in a ratio of 95:5. Separate incubations of these hydroperoxides showed that linoleic acid 9(S)-hydroperoxide was metabolized into epoxy alcohols by particle-bound epoxy alcohol synthase activity, whereas the 13-hydroperoxide was metabolized into alpha- and gamma-ketols by a particle-bound allene oxide synthase. It was concluded that the main pathway of linoleic acid metabolism in potato leaves involved 9-lipoxygenase-catalyzed oxygenation into linoleic acid 9(S)-hydroperoxide followed by rapid conversion of this hydroperoxide into epoxy alcohols and a slower, epoxide hydrolase-catalyzed conversion of the epoxy alcohols into trihydroxy-octadecenoates. Trihydroxy derivatives of linoleic and linolenic acids have previously been reported to be growth-inhibitory to plant-pathogenic fungi, and a role of the new pathway of linoleic acid oxidation in defense reactions against pathogens is conceivable.

Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation.

Acetylenic bonds are present in more than 600 naturally occurring compounds. Plant enzymes that catalyze the formation of the Delta12 acetylenic bond in 9-octadecen-12-ynoic acid and the Delta12 epoxy group in 12,13-epoxy-9-octadecenoic acid were characterized, and two genes, similar in sequence, were cloned. When these complementary DNAs were expressed in Arabidopsis thaliana, the content of acetylenic or epoxidated fatty acids in the seeds increased from 0 to 25 or 15 percent, respectively. Both enzymes have characteristics similar to the membrane proteins containing non-heme iron that have histidine-rich motifs.

Production of hormones by fungi.

Twenty species of filamentous fungi were tested for their gibberellin and sterol production, with emphasis on the action of some factors on these products. While all cultures produced both gibberellin and sterol in variable amounts, Aspergillus flavus, A. niger, A. ochraceus, Cladosporium cladosporioides, Gibberella zeae, Penicillium funiculosum, P. italicum and P. rubrum were the best producers. The isolates of A. niger isolated from soil and P. italicum isolated from citrus fruit were selected for further studies. Gibberellin was highly excreted by A. niger in medium fortified with 0.3:0.3 mol nitrogen-phosphorus ratio. On the other hand, sterol was accumulated in P. italicum by vegetable oils more than fruit oils. However, in shaken culture, the organic and olive oils retarded sterol formation with enhanced accumulation of fatty acids.

Phospholipase activity in synovial fluid from patients with rheumatoid arthritis, osteoarthritis and crystal-associated arthritis.

Phospholipase activity was assayed in cell-free synovial fluid (SF) from patients with rheumatoid arthritis (RA, n = 28), osteoarthritis (OA, n = 10), and crystal-associated arthritis (C, n = 7) by measuring the release of either [14C]oleic acid or [3H]arachidonic acid from radiolabeled E. coli phospholipids. Activity measured by oleic acid release was not significantly different between the three groups of patients (RA = 571 +/- 43.3, OA = 460 +/- 54.7 and C = 718 +/- 162.6 pmol/min/mg). Arachidonic acid release was significantly (p less than 0.005) less in OA (31 +/- 7.3) than RA (61 +/- 4.7) which was similar to C (58 +/- 17.6 pmol/min/mg). Arachidonic acid release correlated significantly with the SF white blood cell count (r = 0.483, p less than 0.01). This study shows the importance of the type of substrate used to measure phospholipase activity and indicates that differences in the capacity to release arachidonic acid may exist between RA and OA disease states.

Synthesis and utilization of fatty acids by wild-type and fatty acid auxotrophs of Caulobacter crescentus.

The fatty acid composition of the dimorphic bacterium Caulobacter crescentus was found to consist primarily of 16- and 18-carbon fatty acids, both saturated and monounsaturated, in agreement with the findings of Chow and Schmidt (J. Gen. Microbiol. 83:359-373, 1974). In addition, two minor but as yet unidentified fatty acids were detected. Chromatographic mobilities suggested that these fatty acids may be a cyclopropane and a branched-chain fatty acid. In addition, we demonstrated that the fatty acid composition of wild-type C. crescentus can be altered by growing the cells in medium supplemented with any one of a variety of unsaturated fatty acids. Linoleic acid, a diunsaturated fatty acid which is not synthesized by C. crescentus, was incorporated into phospholipids without apparent modification. In addition, we found that C. crescentus, like Escherichia coli, synthesizes vaccenic acid (18:1 delta 11,cis) rather than oleic acid (18:1 delta 9,cis). This result allowed us to deduce that the mechanism of fatty acid desaturation in C. crescentus is anaerobic, as it is in E. coli. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. Neither of these mutants resembled the E. coli unsaturated fatty acid auxotrophs, which have defined enzymatic lesions in fatty acid biosynthesis. Rather, the mutants appeared to have defects relating to the complex coordination of membrane biogenesis and cell cycle events in C. crescentus.