Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of Candida tropicalis

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in β-oxidation convert these substrates to long-chain α,ω-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is ω-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C_18:1), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to ω-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C_14:0) much more effectively. Both enzymes, in particular CYP52A17, also oxidized ω-hydroxy fatty acids, ultimately generating the α,ω-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for β-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.     

Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin

The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3^cisΔ9,12,15) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.     

Metabolic Engineering Plant Seeds with Fish Oil-Like Levels of DHA

Background

Omega-3 long-chain (≥C₂₀) polyunsaturated fatty acids (ω3 LC-PUFA) have critical roles in human health and development with studies indicating that deficiencies in these fatty acids can increase the risk or severity of cardiovascular and inflammatory diseases in particular. These fatty acids are predominantly sourced from fish and algal oils, but it is widely recognised that there is an urgent need for an alternative and sustainable source of EPA and DHA. Since the earliest demonstrations of ω3 LC-PUFA engineering there has been good progress in engineering the C₂₀ EPA with seed fatty acid levels similar to that observed in bulk fish oil (∼18%), although undesirable ω6 PUFA levels have also remained high.

Methodology/Principal Findings

The transgenic seed production of the particularly important C₂₂ DHA has been problematic with many attempts resulting in the accumulation of EPA/DPA, but only a few percent of DHA. This study describes the production of up to 15% of the C₂₂ fatty acid DHA in Arabidopsis thaliana seed oil with a high ω3/ω6 ratio. This was achieved using a transgenic pathway to increase the C₁₈ ALA which was then converted to DHA by a microalgal Δ6-desaturase pathway.

Conclusions/Significance

The amount of DHA described in this study exceeds the 12% level at which DHA is generally found in bulk fish oil. This is a breakthrough in the development of sustainable alternative sources of DHA as this technology should be applicable in oilseed crops. One hectare of a Brassica napus crop containing 12% DHA in seed oil would produce as much DHA as approximately 10,000 fish.     

A complete enzymatic capacity for biosynthesis of docosahexaenoic acid (DHA, 22 : 6n–3) exists in the marine Harpacticoida copepod Tigriopus californicus

The long-standing paradigm establishing that global production of Omega-3 (n–3) long-chain polyunsaturated fatty acids (LC-PUFA) derived almost exclusively from marine single-cell organisms, was recently challenged by the discovery that multiple invertebrates possess methyl-end (or ωx) desaturases, critical enzymes enabling the biosynthesis of n–3 LC-PUFA. However, the question of whether animals with ωx desaturases have complete n–3 LC-PUFA biosynthetic pathways and hence can contribute to the production of these compounds in marine ecosystems remained unanswered. In the present study, we investigated the complete enzymatic complement involved in the n–3 LC-PUFA biosynthesis in Tigriopus californicus, an intertidal harpacticoid copepod. A total of two ωx desaturases, five front-end desaturases and six fatty acyl elongases were successfully isolated and functionally characterized. The T. californicus ωx desaturases enable the de novo biosynthesis of C₁₈ PUFA such as linoleic and α-linolenic acids, as well as several n–3 LC-PUFA from n–6 substrates. Functions demonstrated in front-end desaturases and fatty acyl elongases unveiled various routes through which T. californicus can biosynthesize the physiologically important arachidonic and eicosapentaenoic acids. Moreover, T. californicus possess a Δ4 desaturase, enabling the biosynthesis of docosahexaenoic acid via the ‘Δ4 pathway’. In conclusion, harpacticoid copepods such as T. californicus have complete n–3 LC-PUFA biosynthetic pathways and such capacity illustrates major roles of these invertebrates in the provision of essential fatty acids to upper trophic levels.     

Arachidonic Acid-metabolizing Cytochrome P450 Enzymes Are Targets of ω-3 Fatty Acids

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the ω-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these ω-3 epoxides are highly active as antiarrhythmic agents, suppressing the Ca²⁺-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F isoforms ω-hydroxylating AA were less regioselective toward EPA and DHA, catalyzing predominantly ω- and ω minus 1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET/EEQ/EDP ratio from 87:0:13 to 27:18:55 in the heart). These results demonstrate that CYP enzymes efficiently convert EPA and DHA to novel epoxy and hydroxy metabolites that could mediate some of the beneficial cardiovascular effects of dietary ω-3 fatty acids.     

Lipid Classes and Fatty Acids in Ophryotrocha cyclops,a Dorvilleid from Newfoundland Aquaculture Sites

A new opportunistic annelid (Ophryotrocha cyclops) discovered on benthic substrates underneath finfish aquaculture sites in Newfoundland (NL) may be involved in the remediation of organic wastes. At those aquaculture sites, bacterial mats and O. cyclops often coexist and are used as indicators of organic enrichment. Little is known on the trophic strategies used by these annelids, including whether they might consume bacteria or other aquaculture-derived wastes. We studied the lipid and fatty acid composition of the annelids and their potential food sources (degraded flocculent organic matter, fresh fish pellets and bacterial mats) to investigate feeding relationships in these habitats and compared the lipid and fatty acid composition of annelids before and after starvation. Fish pellets were rich in lipids, mainly terrestrially derived C₁₈ fatty acids (18:1ω9, 18:2ω6, 18:3ω3), while bacterial samples were mainly composed of ω7 fatty acids, and flocculent matter appeared to be a mixture of fresh and degrading fish pellets, feces and bacteria. Ophryotrocha cyclops did not appear to store excessive amounts of lipids (13%) but showed a high concentration of ω3 and ω6 fatty acids, as well as a high proportion of the main fatty acids contained in fresh fish pellets and bacterial mats. The dorvilleids and all potential food sources differed significantly in their lipid and fatty acid composition. Interestingly, while all food sources contained low proportions of 20:5ω3 and 20:2ω6, the annelids showed high concentrations of these two fatty acids, along with 20:4ω6. A starvation period of 13 days did not result in a major decrease in total lipid content; however, microscopic observations revealed that very few visible lipid droplets remained in the gut epithelium after three months of starvation. Ophryotrocha cyclops appears well adapted to extreme environments and may rely on lipid-rich organic matter for survival and dispersal in cold environments.     

Mitochondrial 2,4-dienoyl-CoA Reductase Deficiency in Mice Results in Severe Hypoglycemia with Stress Intolerance and Unimpaired Ketogenesis

The mitochondrial β-oxidation system is one of the central metabolic pathways of energy metabolism in mammals. Enzyme defects in this pathway cause fatty acid oxidation disorders. To elucidate the role of 2,4-dienoyl-CoA reductase (DECR) as an auxiliary enzyme in the mitochondrial β-oxidation of unsaturated fatty acids, we created a DECR–deficient mouse line. In Decr^−/− mice, the mitochondrial β-oxidation of unsaturated fatty acids with double bonds is expected to halt at the level of trans-2, cis/trans-4-dienoyl-CoA intermediates. In line with this expectation, fasted Decr^−/− mice displayed increased serum acylcarnitines, especially decadienoylcarnitine, a product of the incomplete oxidation of linoleic acid (C_18:2), urinary excretion of unsaturated dicarboxylic acids, and hepatic steatosis, wherein unsaturated fatty acids accumulate in liver triacylglycerols. Metabolically challenged Decr^−/− mice turned on ketogenesis, but unexpectedly developed hypoglycemia. Induced expression of peroxisomal β-oxidation and microsomal ω-oxidation enzymes reflect the increased lipid load, whereas reduced mRNA levels of PGC-1α and CREB, as well as enzymes in the gluconeogenetic pathway, can contribute to stress-induced hypoglycemia. Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure. This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.     

Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C₁₆ to C₂₀ fatty acids preferentially. It converted oleic acid (C_18:1) more efficiently than stearic acid (C_18:0) and linoleic acid (C_18:2) and much more effectively than α-linolenic acid (C_18:3). No products were detected when C₁₀ to C₁₂ fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C₁₄ to C₂₀ saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps.     

A rapid method for isolating glandular trichomes

A physical method is described for the rapid isolation of plant trichomes, with emphasis on stalked glandular types. The technique involved breaking frozen trichomes with powdered dry ice and collection of glandular heads by sieving from larger tissue fragments. This method was applied to several plants that bear similar stalked trichomes: geranium (Pelargonium), potato (Solanum tuberosum), tomato (Lycopersicon esculentum), squash (Cucurbita pepo), and velvetleaf (Abutilon theophrasti). The tissue preparation was of sufficient quality without further purification for biochemical and molecular studies. The preparation maintained the biochemical integrity of the trichomes for active enzymes and usable nucleic acids. A large quantity of tissue can be harvested; for example, 351 milligrams dry weight of glandular trichomes were harvested from geranium pedicels in 12 hours. The utility of the technique was demonstrated by examining the fatty acid composition of tall glandular trichomes of geraniums, Pelargonium ×hortorum L.H. Bailey. These purified cells contained high concentrations of unusual ω5-unsaturated fatty acids, proportionally 23.4% of total fatty acids in the trichomes. When the trichomes were removed, the supporting tissue contained no ω5-fatty acids, thereby unequivocally localizing ω5-fatty acids to the trichomes. Because ω5-fatty acids are unique precursors for the biosynthesis of ω5-anacardic acids, we conclude that anacardic acid synthesis must occur in the glandular trichomes.     

Fish Oil Attenuates Omega-6 Polyunsaturated Fatty Acid-Induced Dysbiosis and Infectious Colitis but Impairs LPS Dephosphorylation Activity Causing Sepsis

Clinically, excessive ω-6 polyunsaturated fatty acid (PUFA) and inadequate ω-3 PUFA have been associated with enhanced risks for developing ulcerative colitis. In rodent models, ω-3 PUFAs have been shown to either attenuate or exacerbate colitis in different studies. We hypothesized that a high ω-6: ω-3 PUFA ratio would increase colitis susceptibility through the microbe-immunity nexus. To address this, we fed post-weaned mice diets rich in ω-6 PUFA (corn oil) and diets supplemented with ω-3 PUFA (corn oil+fish oil) for 5 weeks. We evaluated the intestinal microbiota, induced colitis with Citrobacter rodentium and followed disease progression. We found that ω-6 PUFA enriched the microbiota with Enterobacteriaceae, Segmented Filamentous Bacteria and Clostridia spp., all known to induce inflammation. During infection-induced colitis, ω-6 PUFA fed mice had exacerbated intestinal damage, immune cell infiltration, prostaglandin E2 expression and C. rodentium translocation across the intestinal mucosae. Addition of ω-3 PUFA on a high ω-6 PUFA diet, reversed inflammatory-inducing microbial blooms and enriched beneficial microbes like Lactobacillus and Bifidobacteria, reduced immune cell infiltration and impaired cytokine/chemokine induction during infection. While, ω-3 PUFA supplementation protected against severe colitis, these mice suffered greater mortality associated with sepsis-related serum factors such as LPS binding protein, IL-15 and TNF-α. These mice also demonstrated decreased expression of intestinal alkaline phosphatase and an inability to dephosphorylate LPS. Thus, the colonic microbiota is altered differentially through varying PUFA composition, conferring altered susceptibility to colitis. Overall, ω-6 PUFA enriches pro-inflammatory microbes and augments colitis; but prevents infection-induced systemic inflammation. In contrast, ω-3 PUFA supplementation reverses the effects of the ω-6 PUFA diet but impairs infection-induced responses resulting in sepsis. We conclude that as an anti-inflammatory agent, ω-3 PUFA supplementation during infection may prove detrimental when host inflammatory responses are critical for survival.     

NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min⁻¹ mg⁻¹ and 10.0 ± 0.5 nmol min⁻¹ mg⁻¹, respectively. The highest k_cat/K_m value of ω3 and ω6 desaturase was 0.41 µM⁻¹ min⁻¹ and 0.09 µM⁻¹ min⁻¹ when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.     

Cloning and Characterization of Three Fatty Alcohol Oxidase Genes from Candida tropicalis Strain ATCC 20336

Candida tropicalis (ATCC 20336) converts fatty acids to long-chain dicarboxylic acids via a pathway that includes among other reactions the oxidation of ω-hydroxy fatty acids to ω-aldehydes by a fatty alcohol oxidase (FAO). Three FAO genes (one gene designated FAO1 and two putative allelic genes designated FAO2a and FAO2b), have been cloned and sequenced from this strain. A comparison of the DNA sequence homology and derived amino acid sequence homology between these three genes and previously published Candida FAO genes indicates that FAO1 and FAO2 are distinct genes. Both genes were individually cloned and expressed in Escherichia coli. The substrate specificity and K_m values for the recombinant FAO1 and FAO2 were significantly different. Particularly striking is the fact that FAO1 oxidizes ω-hydroxy fatty acids but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols but not ω-hydroxy fatty acids. Analysis of extracts of strain H5343 during growth on fatty acids indicated that only FAO1 was highly induced under these conditions. FAO2 contains one CTG codon, which codes for serine (amino acid 177) in C. tropicalis but codes for leucine in E. coli. An FAO2a construct, with a TCG codon (codes for serine in E. coli) substituted for the CTG codon, was prepared and expressed in E. coli. Neither the substrate specificity nor the K_m values for the FAO2a variant with a serine at position 177 were radically different from those of the variant with a leucine at that position.     

Variation in Phospholipid Ester-Linked Fatty Acids and Carotenoids of Desiccated Nostoc commune (Cyanobacteria) from Different Geographic Locations

Profiles of phospholipid fatty acids and carotenoids in desiccated Nostoc commune (cyanobacteria) collected from China, Federal Republic of Germany, and Antarctica and in axenic cultures of the desiccation-tolerant strains N. commune UTEX 584 and Hydrocoleum strain GOEI were analyzed. The phospholipid fatty acid contents of the three samples of desiccated Nostoc species were all similar, and the dominant compounds were 16:1ω7c, 16:0, 18:2ω6, 18:3ω3, and 18:1ω7c. In comparison with the field materials, N. commune UTEX 584 had a much higher ratio of 18:2ω6 to 18:3ω3 (5.36) and a significantly lower ratio of 18:1ω7c to 18:1ω9c (1.86). Compound 18:3 was present in large amounts in the samples of desiccated Nostoc species which had been subject, in situ, to repeated cycles of drying and rewetting, but represented only a small fraction of the total fatty acids of the strains grown in liquid culture. This finding is in contrast to the data obtained from studies on the effects of drought and water stress on higher plants. Field materials of Nostoc species contained, in contrast to the axenic strains, significant amounts of apocarotenoids and a P384 pigment which, upon reduction with NaBH₄, yielded a mixture of a chlorophyll derivative and a compound with an absorption maximum of 451 nm. A clear distinction can be made between the carotenoid contents of the axenic cultures and the desiccated field materials. In the former, β-carotene and echinenone predominate; in the latter, canthaxanthin and the β-γ series of carotenoids are found.     

The Microbial Community Structure of Drinking Water Biofilms Can Be Affected by Phosphorus Availability

Microbial communities in biofilms grown for 4 and 11 weeks under the flow of drinking water supplemented with 0, 1, 2, and 5 μg of phosphorus liter⁻¹ and in drinking and warm waters were compared by using phospholipid fatty acids (PLFAs) and lipopolysaccharide 3-hydroxy fatty acids (LPS 3-OH-FAs). Phosphate increased the proportion of PLFAs 16:1ω7c and 18:1ω7c and affected LPS 3-OH-FAs after 11 weeks of growth, indicating an increase in gram-negative bacteria and changes in their community structure. Differences in community structures between biofilms and drinking and warm waters can be assumed from PLFAs and LPS 3-OH-FAs, concomitantly with adaptive changes in fatty acid chain length, cyclization, and unsaturation.     

Catabolism of (2E)-4-Hydroxy-2-nonenal via ω- and ω-1-Oxidation Stimulated by Ketogenic Diet

Oxidative stress triggers the peroxidation of ω-6-polyunsaturated fatty acids to reactive lipid fragments, including (2E)-4-hydroxy-2-nonenal (HNE). We previously reported two parallel catabolic pathways of HNE. In this study, we report a novel metabolite that accumulates in rat liver perfused with HNE or 4-hydroxynonanoic acid (HNA), identified as 3-(5-oxotetrahydro-2-furanyl)propanoyl-CoA. In experiments using a combination of isotopic analysis and metabolomics studies, three catabolic pathways of HNE were delineated following HNE conversion to HNA. (i) HNA is ω-hydroxylated to 4,9-dihydroxynonanoic acid, which is subsequently oxidized to 4-hydroxynonanedioic acid. This is followed by the degradation of 4-hydroxynonanedioic acid via β-oxidation originating from C-9 of HNA breaking down to 4-hydroxynonanedioyl-CoA, 4-hydroxyheptanedioyl-CoA, or its lactone, 2-hydroxyglutaryl-CoA, and 2-ketoglutaric acid entering the citric acid cycle. (ii) ω-1-hydroxylation of HNA leads to 4,8-dihydroxynonanoic acid (4,8-DHNA), which is subsequently catabolized via two parallel pathways we previously reported. In catabolic pathway A, 4,8-DHNA is catabolized to 4-phospho-8-hydroxynonanoyl-CoA, 3,8-dihydroxynonanoyl-CoA, 6-hydroxyheptanoyl-CoA, 4-hydroxypentanoyl-CoA, propionyl-CoA, and acetyl-CoA. (iii) The catabolic pathway B of 4,8-DHNA leads to 2,6-dihydroxyheptanoyl-CoA, 5-hydroxyhexanoyl-CoA, 3-hydroxybutyryl-CoA, and acetyl-CoA. Both in vivo and in vitro experiments showed that HNE can be catabolically disposed via ω- and ω-1-oxidation in rat liver and kidney, with little activity in brain and heart. Dietary experiments showed that ω- and ω-1-hydroxylation of HNA in rat liver were dramatically up-regulated by a ketogenic diet, which lowered HNE basal level. HET0016 inhibition and mRNA expression level suggested that the cytochrome P450 4A are main enzymes responsible for the NADPH-dependent ω- and ω-1-hydroxylation of HNA/HNE.     

Purification and Biochemical Analysis of the Cytoplasmic Membrane from the Desiccation-Tolerant Cyanobacterium Nostoc commune UTEX 584

The cytoplasmic membrane of the heterocystous cyanobacterium Nostoc commune UTEX 584 was isolated free of thylakoids and phycobiliprotein-membrane complexes by flotation centrifugation. Purified membranes had a buoyant density of 1.07 g cm⁻³ and were bright orange. Twelve major proteins were detected in the membrane, and of these, the most abundant had molecular masses of 83, 71, 68, 51, and 46 kilodaltons. The ester-linked fatty acids of the methanol fraction contained 16:0, 18:0, 18:1ω9c, 20:0, and 20:3ω3 with no traces of hydroxy fatty acids. Compound 20:3ω3 represented 56.8% of the total fatty acid methyl esters, a feature which distinguishes the cell membrane of N. commune UTEX 584 from those of all other cyanobacteria which have been characterized to date. Fatty acid 18:3 was not detected. Carotenoids were analyzed by highperformance liquid chromatography. The cytoplasmic membrane contained β-carotene and echinenone as the dominant carotenoids and lacked chlorophyll a and pheophytin a. Whole cells contained β-carotene and echinenone, and lesser amounts of zeaxanthin and (3R)-cryptoxanthin.     

Human Cytochrome P450 2E1 Structures with Fatty Acid Analogs Reveal a Previously Unobserved Binding Mode

Human microsomal cytochrome P450 (CYP) 2E1 is widely known for its ability to oxidize >70 different, mostly compact, low molecular weight drugs and other xenobiotic compounds. In addition CYP2E1 oxidizes much larger C9–C20 fatty acids that can serve as endogenous signaling molecules. Previously structures of CYP2E1 with small molecules revealed a small, compact CYP2E1 active site, which would be insufficient to accommodate medium and long chain fatty acids without conformational changes in the protein. In the current work we have determined how CYP2E1 can accommodate a series of fatty acid analogs by cocrystallizing CYP2E1 with ω-imidazolyl-octanoic fatty acid, ω-imidazolyl-decanoic fatty acid, and ω-imidazolyl-dodecanoic fatty acid. In each structure direct coordination of the imidazole nitrogen to the heme iron mimics the position required for native fatty acid substrates to yield the ω-1 hydroxylated metabolites that predominate experimentally. In each case rotation of a single Phe²⁹⁸ side chain merges the active site with an adjacent void, significantly altering the active site size and topology to accommodate fatty acids. The binding of these fatty acid ligands is directly opposite the channel to the protein surface and the binding observed for fatty acids in the bacterial cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium. Instead of the BM3-like binding mode in the CYP2E1 channel, these structures reveal interactions between the fatty acid carboxylates and several residues in the F, G, and B′ helices at successive distances from the active site.     

The PNPLA3 Ile148Met interacts with overweight and dietary intakes on fasting triglyceride levels

The Ile148Met (rs738409, G-allele) in the patatin-like phospholipase domain-containing protein 3 gene (PNPLA3) associates with liver fat content and may lead to loss-of-function (hydrolysis) or gain-of-function (CoA-dependent lysophosphatidic acid acyltransferase) defects. PNPLA3 is up-regulated by dietary carbohydrates, and interactions between rs738409 and carbohydrates, and sugar and ω6:ω3-polyunsaturated fatty acid (PUFA) ratio on hepatic fat accumulation have been reported. We examined interaction between rs738409 and overweight, and between rs738409 and dietary intakes (carbohydrates, sucrose and ω6:ω3-PUFA ratio), on fasting triglyceride levels. From the Malmo Diet and Cancer Study-Cardiovascular Cohort, 4,827 individuals without diabetes aged 58 ± 6 years, 2,346 with BMI ≤ 25 kg/m² and 2,478 with BMI > 25 kg/m², were included in cross-sectional analyses. Dietary data were collected by a modified diet history method. Overweight modified the association between rs738409 and fasting triglyceride levels (P_interaction = 0.003). G-allele associated with lower triglycerides only among overweight individuals (P = 0.01). Nominally, significant interaction on triglyceride levels was observed between rs738409 and sucrose among normal-weight individuals (P_interaction = 0.03). G-allele associated with lower triglycerides among overweight individuals in the lowest tertiles of carbohydrate and ω6:ω3-PUFA ratio (P = 0.04 and P = 0.001) and with higher triglycerides among normal-weight individuals in the highest tertile of sucrose (P = 0.001). We conclude that overweight and dietary sucrose may modify the association between rs738409 and fasting triglyceride levels.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0388-4) contains supplementary material, which is available to authorized users.     

Biocatalyst Engineering by Assembly of Fatty Acid Transport and Oxidation Activities for In Vivo Application of Cytochrome P-450BM-3 Monooxygenase

The application of whole cells containing cytochrome P-450_BM-3 monooxygenase [EC 1.14.14.1] for the bioconversion of long-chain saturated fatty acids to ω-1, ω-2, and ω-3 hydroxy fatty acids was investigated. We utilized pentadecanoic acid and studied its conversion to a mixture of 12-, 13-, and 14-hydroxypentadecanoic acids by this monooxygenase. For this purpose, Escherichia coli recombinants containing plasmid pCYP102 producing the fatty acid monooxygenase cytochrome P-450_BM-3 were used. To overcome inefficient uptake of pentadecanoic acid by intact E. coli cells, we made use of a cloned fatty acid uptake system from Pseudomonas oleovorans which, in contrast to the common FadL fatty acid uptake system of E. coli, does not require coupling by FadD (acyl-coenzyme A synthetase) of the imported fatty acid to coenzyme A. This system from P. oleovorans is encoded by a gene carried by plasmid pGEc47, which has been shown to effect facilitated uptake of oleic acid in E. coli W3110 (M. Nieboer, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1996). By using a double recombinant of E. coli K27, which is a fadD mutant and therefore unable to consume substrates or products via the β-oxidation cycle, a twofold increase in productivity was achieved. Applying cytochrome P-450_BM-3 monooxygenase as a biocatalyst in whole cells does not require the exogenous addition of the costly cofactor NADPH. In combination with the coenzyme A-independent fatty acid uptake system from P. oleovorans, cytochrome P-450_BM-3 recombinants appear to be useful alternatives to the enzymatic approach for the bioconversion of long-chain fatty acids to subterminal hydroxylated fatty acids.Cytochrome P-450_BM-3 monooxygenase (CytP450_BM-3) is a soluble NADPH-dependent monooxygenase from Bacillus megaterium ATCC 14581 (13). It is a class II P-450 enzyme that contains flavin adenine dinucleotide, flavin mononucleotide, and a heme moiety (17). Unlike most CytP450 monooxygenases, which consist of a distinct monooxygenase and a reductase, CytP450_BM-3 contains these functionalities in a single polypeptide (3, 15, 18).The enzyme hydroxylates a variety of long-chain aliphatic substrates, such as fatty acids, alkanols, and alkylamides at the ω-1, ω-2, and ω-3 positions (4, 17), and oxidizes unsaturated fatty acids to epoxides in vitro (17, 23) with high enantioselectivity. Oxidation of eicosapentenoic acid (C_20:5) and arachidonic acid (C_20:4) yielded 17(S),18(R)-epoxyeicosatetraenoic acid (94% enantiomeric excess [e.e.]) for the former and a mixture of 18-(R)-hydroxyarachidonic acid (92% e.e.) and 14(S),15(R)-epoxyeicosatrienoic acid at 98% e.e. for the latter substrate (8). Recently, it has been demonstrated that the enzyme also produces α,ω diacids from ω-oxo fatty acids by oxidation of the terminal aldehyde functionality (9). The catalytic constant (k_cat) of CytP450_BM-3 is among the highest found for P-450 monooxygenases, ranging from 15 s⁻¹ for laureate to 75 s⁻¹ for pentadecanoic acid (11). For comparison, a typical microsomal P-450 monooxygenase from human liver (CYP2J2) had a k_cat of 10⁻³ s⁻¹ for arachidonic acid (32), compared to a k_cat of 55 s⁻¹ for CytP450_BM-3 for the same substrate (8).This high catalytic efficiency prompted us to investigate the applicability of CytP450_BM-3 as a biocatalyst for the subterminal hydroxylation of long-chain fatty acids (LCFAs). Since these subterminal hydroxy LCFAs are chiral molecules, their application in the production of enantiopure synthetic building blocks, especially for pharmaceutical agents, could be envisioned. Further, long-chain hydroxy acids find applications as precursors for polymers or cyclic lactones, which are used as components of fragrances and as antibiotics. Although chemical syntheses have been developed for ω-1 hydroxy fatty acids (from C₁₂ to C₁₈) (26, 28, 29) and for ω-2 and ω-3 hydroxyoctadecanoic acids (2), they require expensive functionalized substrates and are in general complicated, multistep processes (26, 28, 29) which cannot be carried out with unmodified fatty acids as inexpensive starting material. In principle, such inexpensive substrates can be oxidized to hydroxy fatty acids by biocatalysts, either in vitro or in vivo. The latter is preferred, since whole cells actively regenerate the NADPH required for fatty acid oxidation with monooxygenases such as CytP450_BM-3. In designing a suitable whole-cell biocatalyst, several additional points had to be considered.First, uptake must be efficient. Second, degradation of substrate or product must be avoided. In fact, biotransformations of fatty acids with whole cells are usually inefficient due to limited uptake of these compounds at neutral pH, and when taken up, they are degraded via β-oxidation. The transport of LCFAs in Escherichia coli is mediated via the fadL and fadD gene products. FadL is the transporter that carries LCFAs across the outer membrane and is absolutely required for LCFA transport (20). FadD, the acyl coenzyme A (CoA) synthetase, is located at the inner side of the cytoplasmic membrane and is required for formation of the acyl coenzyme A thioester, after which the activated fatty acids are channeled into the β-oxidation cycle for fatty acid degradation (21, 22). Thus, we used a FadD mutant, E. coli K27, as a suitable host for the production of subterminal hydroxyalkanoic acids (20). E. coli K27 cannot couple free fatty acids to coenzyme A, thus preventing substrate or product degradation by the host. Such fadD mutants are, however, also impaired in efficient uptake of fatty acids (20). We circumvented this by introducing a fatty acid uptake system from Pseudomonas oleovorans encoded on pGEc47. Finally, we introduced the P-450_BM-3 monooxygenase on pCYP102 into the fadD mutant E. coli. The resulting recombinant, E. coli K27(pCYP102, pGEc47), is a promising tailored biocatalyst for the oxidation of saturated LCFAs to ω-1, ω-2, and ω-3 hydroxy fatty acids.     

Organic Production Enhances Milk Nutritional Quality by Shifting Fatty Acid Composition: A United States–Wide, 18-Month Study

Over the last century, intakes of omega-6 (ω-6) fatty acids in Western diets have dramatically increased, while omega-3 (ω-3) intakes have fallen. Resulting ω-6/ω-3 intake ratios have risen to nutritionally undesirable levels, generally 10 to 15, compared to a possible optimal ratio near 2.3. We report results of the first large-scale, nationwide study of fatty acids in U.S. organic and conventional milk. Averaged over 12 months, organic milk contained 25% less ω-6 fatty acids and 62% more ω-3 fatty acids than conventional milk, yielding a 2.5-fold higher ω-6/ω-3 ratio in conventional compared to organic milk (5.77 vs. 2.28). All individual ω-3 fatty acid concentrations were higher in organic milk—α-linolenic acid (by 60%), eicosapentaenoic acid (32%), and docosapentaenoic acid (19%)—as was the concentration of conjugated linoleic acid (18%). We report mostly moderate regional and seasonal variability in milk fatty acid profiles. Hypothetical diets of adult women were modeled to assess milk fatty-acid-driven differences in overall dietary ω-6/ω-3 ratios. Diets varied according to three choices: high instead of moderate dairy consumption; organic vs. conventional dairy products; and reduced vs. typical consumption of ω-6 fatty acids. The three choices together would decrease the ω-6/ω-3 ratio among adult women by ∼80% of the total decrease needed to reach a target ratio of 2.3, with relative impact “switch to low ω-6 foods” > “switch to organic dairy products” ≈ “increase consumption of conventional dairy products.” Based on recommended servings of dairy products and seafoods, dairy products supply far more α-linolenic acid than seafoods, about one-third as much eicosapentaenoic acid, and slightly more docosapentaenoic acid, but negligible docosahexaenoic acid. We conclude that consumers have viable options to reduce average ω-6/ω-3 intake ratios, thereby reducing or eliminating probable risk factors for a wide range of developmental and chronic health problems.