Biosynthesis of C(20) and C(22) Fatty Acids by Developing Seeds of Limnanthes alba: CHAIN ELONGATION AND Delta5 DESATURATION

The storage triacylglycerols of meadowfoam (Limnanthes alba) seeds are composed essentially of C₂₀ and C₂₂ fatty acids, which contain an unusual Δ5 double bond. When [1-¹⁴C]acetate was incubated with developing seed slices, ¹⁴C-labeled fatty acids were synthesized with a distribution similar to the endogenous fatty acid profile. The major labeled product was cis-5-eicosenoate, with smaller amounts of palmitate, stearate, oleate, cis-5-octadecenoate, eicosanoate, cis-11-eicosenoate, docosanoate, cis-5-docosenoate, cis-13-docosenoate, and cis-5,cis-13-docosadienoate. The label from [¹⁴C]acetate and [¹⁴C]malonate was used preferentially for the elongation of endogenous oleate to produce cis-[¹⁴C]11-eicosenoate, cis-13-[¹⁴C]docosenoate, and cis-5,cis-13-[¹⁴C]docosadienoate and for the elongation of endogenous palmitate to produce the remaining C₂₀ and C₂₂ acyl species. The Δ5 desaturation of the preformed acyl chain and chain elongation of oleate and palmitate were demonstrated in vivo by incubation of the appropriate 1-¹⁴C-labeled free fatty acids. Using [1-¹⁴C]acyl-CoA thioesters as substrates, these enzyme activities were also demonstrated in vitro with a cell-free homogenate.     

The effect of cold, acid and ethanol shocks on synthesis of membrane fatty acid, freeze-drying survival and malolactic activity of Oenococcus oeni

The effects of stress shocks on the freeze-drying viability, malolactic activity and membrane fatty acid composition of the Oenococcus oeni SD-2a cells were studied. O. oeni SD-2a cells after 2 h of stress exposure exhibited better freeze-drying viability and malolactic fermentation ability. A decrease in unsaturated fatty acids/saturated fatty acids (UFA/SFA) ratio and in the C18:1 relative concentration, and an increase in cyclopropane fatty acids (CFA) content mainly due to the increase in C19cyc11 relative concentration were observed in all stress shocked cells. There was a significant negative correlation between C19cyc11 and C18:lcis11, C16:0 in all stress shocks. The freeze-drying viability exhibited a significant positive correlation with the levels of C19cyc11 in cold and acid shocks. The only significant positive correlation between the ability of O. oeni SD-2a to conduct malic acid degradation and membrane composition existed with C14:0 in ethanol shocks. In general, freeze-drying viabilities were maximum for cells with low UFA/SFA ratio and high CFA levels, and, consequently, with low membrane fluidity. Moreover, CFA formation played a major role in protecting stress shocked cells from lyophilization. However, changes observed in membrane fatty acid composition are not enough to explain the greater freeze-drying viability of cells shocked at 8% ethanol. Thus, other mechanisms could be responsible for this increase in the bacterial resistance to lyophilization.     

Metabolic diversity in biohydrogenation of polyunsaturated fatty acids by lactic acid bacteria involving conjugated fatty acid production

Lactobacillus plantarum AKU 1009a effectively transforms linoleic acid to conjugated linoleic acids of cis-9,trans-11-octadecadienoic acid (18:2) and trans-9,trans-11–18:2. The transformation of various polyunsaturated fatty acids by washed cells of L. plantarum AKU 1009a was investigated. Besides linoleic acid, α-linolenic acid [cis-9,cis-12,cis-15-octadecatrienoic acid (18:3)], γ-linolenic acid (cis-6,cis-9,cis-12–18:3), columbinic acid (trans-5,cis-9,cis-12–18:3), and stearidonic acid [cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (18:4)] were found to be transformed. The fatty acids transformed by the strain had the common structure of a C18 fatty acid with the cis-9,cis-12 diene system. Three major fatty acids were produced from α-linolenic acid, which were identified as cis-9,trans-11,cis-15–18:3, trans-9,trans-11,cis-15–18:3, and trans-10,cis-15–18:2. Four major fatty acids were produced from γ-linolenic acid, which were identified as cis-6,cis-9,trans-11–18:3, cis-6,trans-9,trans-11–18:3, cis-6,trans-10–18:2, and trans-10-octadecenoic acid. The strain transformed the cis-9,cis-12 diene system of C18 fatty acids into conjugated diene systems of cis-9,trans-11 and trans-9,trans-11. These conjugated dienes were further saturated into the trans-10 monoene system by the strain. The results provide valuable information for understanding the pathway of biohydrogenation by anaerobic bacteria and for establishing microbial processes for the practical production of conjugated fatty acids, especially those produced from α-linolenic acid and γ-linolenic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterisation of two self-sufficient CYP102 family monooxygenases from Ktedonobacter racemifer DSM44963 which have new fatty acid alcohol product profiles

BackgroundTwo self-sufficient CYP102 family encoding genes (Krac_0936 and Krac_9955) from the bacterium Ktedonobacter racemifer DSM44963, which possesses one of the largest bacterial genomes, have been identified.MethodsPhylogenetic analysis of both the encoded cytochrome P450 enzymes, Krac0936 and Krac9955. Both enzymes were produced and their turnovers with fatty acid substrates assessed in vitro and using a whole-cell oxidation system.ResultsKrac0936 hydroxylated straight chain, saturated fatty acids predominantly at the ω-1 and ω-2 positions using NADPH as the cofactor. Krac0936 was less active towards shorter unsaturated fatty acids but longer unsaturated acids were efficiently oxidised. cis,cis-9,12-Octadecadienoic and pentadecanoic acids were the most active substrates tested with Krac0936. Unusually Krac9955 showed very low levels of NAD(P)H oxidation activity though coupling of the reducing equivalents to product formation was high. The product distribution of tridecanoic, tetradecanoic and pentadecanoic acid oxidation by Krac9955 favoured oxidation at the ω-4, ω-5 and ω-6 positions, respectively.ConclusionKrac0936 and Krac9955 are self-sufficient P450 monooxygenases. Krac0936 has a preference for pentadecanoic acid over other straight chain fatty acids and showed little or no activity with dodecanoic or octadecanoic acids. Krac9955 preferably oxidised shorter fatty acids compared to Krac0936 with tridecanoic having the highest levels of product formation. Unlike Krac0936 and P450Bm3, Krac9995 showed lower activities with unsaturated fatty acids.General significanceIn this study of two of the CYP enzymes from K. racemifer we have shown that this bacterium from the Chloroflexi phylum contains genes which encode new proteins with novel activity.     

Flavour biogenesis. Partial purification and properties of a fatty acid hydroperoxide cleaving enzyme from fruits of cucumber

A membrane-bound enzyme, which catalyses the cleavage of fatty acid hydroperoxides to carbonyl fragments, has been partially purified from cucumber fruit. The isomeric 9- and 13-hydroperoxydienes (but not the hydroxydienes) derived from both linoleic and linolenic acids are cleaved by the enzyme but a mixture of 9- and 10-hydroperoxymonoenoic derivatives of oleic acid was not attacked. No evidence was obtained for free intermediates between fatty acid hydroperoxides and the cleavage products. Major volatile products were: cis-3-nonenal and hexanal (from 9- and 13-hydroperoxides of linoleic acid respectively) or cis-3,cis-6-nonadienal and cis-3-hexenal (from 9- and 13-hydroperoxides of linolenic acid). The increase in the ratio of cis-3- to trans-2-enal products with enzyme purification indicated that cis-3-enals are the immediate cleavage products and that the trans-2- forms are produced by subsequent isomerization.     

Fatty acid changes during maturation of Momordica charantia and Trichosanthes Anguina Seeds

Changes in fatty acids were studied during maturation of Momordica charantia and Trichosanthes anguina seeds, which contain cis-9, trans-11, trans-13-octadecatrienoic acid (α-eleostearic) and cis-9, trans-11, cis-13-octadecatrienoic acid (punicic), respectively. The two seeds matured 30 and 35 days after flowering, respectively. Total lipids as well as α-eleostearic acid accumulated rapidly from 10 to 20 days in M. charantia. In T. anguina the active period of lipid synthesis was from 15 to 30 days but punicic acid continued to be synthesized until maturity. In both species, the disappearance of linolenic acid and the reduction in concentration of linoleic acid were concomitant with the formation of conjugated fatty acids. The conjugated fatty acids were absent from monoacylglycerols and phospholipids of both species, and also from the diacylglycerols of M. charantia, throughout maturation     

High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase

Analysis of fatty acids from the cyanobacterium Cyanothece sp. PCC 8801 revealed that this species contained high levels of myristic acid (14:0) and linoleic acid in its glycerolipids, with minor contributions from palmitic acid (16:0), stearic acid, and oleic acid. The level of 14:0 relative to total fatty acids reached nearly 50%. This 14:0 fatty acid was esterified primarily to the sn-2 position of the glycerol moiety of glycerolipids. This characteristic is unique because, in most of the cyanobacterial strains, the sn-2 position is esterified exclusively with C16 fatty acids, generally 16:0. Transformation of Synechocystis sp. PCC 6803 with the PCC8801_1274 gene for lysophosphatidic acid acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase) from Cyanothece sp. PCC 8801 increased the level of 14:0 from 2% to 17% in total lipids and the increase in the 14:0 content was observed in all lipid classes. These findings suggest that the high content of 14:0 in Cyanothece sp. PCC 8801 might be a result of the high specificity of this acyltransferase toward the 14:0-acyl-carrier protein.     

Fatty acids and breast cancer: The role of stem cells

Studies with animal models in vivo as well as with animal and human tumor cells in vitro suggest that specific fatty acids could reduce breast tumorigenesis. The most striking dietary fatty acid studies in animal models that show promise for reduction of breast cancer risk in humans are with conjugated linoleic acids (CLA) and n-3 fatty acids. Although a number of mechanisms have been proposed, the specific target of those fatty acids is not yet known. We sought to determine whether the effects of those fatty acids on terminally differentiated tumor cell seen could be due to alteration of breast cancer stem cells. The isomers, cis9, trans11-CLA and trans10, cis12-CLA, and the n-3 fatty acids, docosahexaenoic and eicosapentaenoic, reduced the proliferation of, and had increased toxicity towards, mammary tumor initiating cells. One mechanism involved in the effect of n-3 fatty acids may be due to alteration of the profile of prostaglandins. These results indicate that select fatty acids may be useful for preventing or reducing the risk of breast cancer as they may target the tumor initiating cell.     

Conjugated Linoleic Acid Accumulation via 10-Hydroxy-12-Octadecaenoic Acid during Microaerobic Transformation of Linoleic Acid by Lactobacillus acidophilus

Specific isomers of conjugated linoleic acid (CLA), a fatty acid with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from linoleic acid by washed cells of Lactobacillus acidophilus AKU 1137 under microaerobic conditions, and the metabolic pathway of CLA production from linoleic acid is explained for the first time. The CLA isomers produced were identified as cis-9, trans-11- or trans-9, cis-11-octadecadienoic acid and trans-9, trans-11-octadecadienoic acid. Preceding the production of CLA, hydroxy fatty acids identified as 10-hydroxy-cis-12-octadecaenoic acid and 10-hydroxy-trans-12-octadecaenoic acid had accumulated. The isolated 10-hydroxy-cis-12-octadecaenoic acid was transformed into CLA during incubation with washed cells of L. acidophilus, suggesting that this hydroxy fatty acid is one of the intermediates of CLA production from linoleic acid. The washed cells of L. acidophilus producing high levels of CLA were obtained by cultivation in a medium containing linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After 4 days of reaction with these washed cells, more than 95% of the added linoleic acid (5 mg/ml) was transformed into CLA, and the CLA content in total fatty acids recovered exceeded 80% (wt/wt). Almost all of the CLA produced was in the cells or was associated with the cells as free fatty acid.     

The biohydrogenation of α-linoleic acid and oleic acid by rumen micro-organisms

1. α-[U-¹⁴C]Linolenic acid was incubated with the rumen contents of sheep and the metabolic products were characterized by thin-layer chromatography, gas–liquid chromatography and absorption spectroscopy in the ultraviolet and infrared. 2. A tentative scheme for the biohydrogenation route to stearic acid is presented. The main pathway is through diconjugated cis–cis–cis-octadecatrienoic acid, non-conjugated trans–cis (cis–trans)-octadecadienoic acid and trans-octadecenoic acid, but other pathways are apparent. 3. Washed rumen micro-organisms possessed only a limited capacity to hydrogenate α-linolenic acid and oleic acid but the rate was greatly stimulated by a factor(s) present in the supernatant rumen liquor. 4. Pure cultures of Clostridium perfringens, Streptococcus faecalis, Escherichia coli and a coliform organism isolated from sheep faeces possessed negligible ability to hydrogenate unsaturated fatty acids compared with a mixed population of rumen micro-organisms. Butyrivibrio fibrisolvens slowly converted linoleic acid into octadecenoic acid.     

Association study between copy number variation and beef fatty acid profile of Nellore cattle

The aim of this study was to analyze the association between the copy number variation regions (CNVRs) and fatty acid profile phenotypes for saturated (SFA), monosaturated (MUFA), polyunsaturated (PUFA), ω6 and ω3 fatty acids, PUFA/SFA and ω6/ω3 ratios, as well as for their sums, in Nellore cattle (Bos primigenius indicus). A total of 963 males were finished in feedlot and slaughtered with approximately 2 years of age. Animals were genotyped with the BovineHD BeadChip (Illumina Inc., San Diego, CA, USA). The copy number variation (CNV) detection was performed using the PennCNV algorithm. Log R ratio (LRR) and allele B frequency (BAF) were used to estimate the CNVs. The association analyses were done using the CNVRuler software and applying a logistic regression model. The phenotype was adjusted using a linear model considering the fixed effects of contemporary group and the animal age at slaughter. The fatty acid profile was analyzed on samples of longissimus thoracis muscle using gas chromatography with a 100-m capillary column. For the association analysis, the adjusted phenotypic values were considered for the traits, while the data was adjusted for the effects of the farm and year of birth, management groups at birth, weaning, and superannuation. A total of 186 CNVRs were significant for SFA (43), MUFA (42), PUFA (66), and omega fatty acid (35) groups, totaling 278 known genes. On the basis of the results, several genes were associated with several fatty acids of different saturations. Olfactory receptor genes were associated with C12:0, C14:0, and C18:0 fatty acids. The SAMD8 and BSCL2 genes, both related to lipid metabolic process, were associated with C12:0. The RAPGEF6 gene was found to be associated with C18:2 cis-9 cis-12 n-6, and its function is related to regulation of GTPase activity. Among the results, we highlighted the olfactory receptor activity (GO:0004984), G-protein-coupled receptor activity (GO:0004930), potassium:proton antiporter activity (GO:0015386), sodium:proton antiporter activity (GO:0015385), and odorant-binding (GO:0005549) molecular functions. A large number of genes associated with fatty acid profile within the CNVRs were identified in this study. These findings must contribute to better elucidate the genetic mechanism underlying the fatty acid profile of intramuscular fat in Nellore cattle.     

Antifungal 3-Hydroxy Fatty Acids from Lactobacillus plantarum MiLAB 14

We report the identification and chemical characterization of four antifungal substances, 3-(R)-hydroxydecanoic acid, 3-hydroxy-5-cis-dodecenoic acid, 3-(R)-hydroxydodecanoic acid and 3-(R)-hydroxytetradecanoic acid, from Lactobacillus plantarum MiLAB 14. The concentrations of the 3-hydroxy fatty acids in the supernatant followed the bacterial growth. Racemic mixtures of the saturated 3-hydroxy fatty acids showed antifungal activity against different molds and yeasts with MICs between 10 and 100 μg ml⁻¹.     

Peculiarities of fatty acid composition of the genusCaulobacter

The fatty acid composition of 35 strains of stalked bacteria belonging to 17 of the hitherto described 19 species and 10 unidentified strains of the genusCaulobacter was studied. ll-Methyl-cis-octadec-11-enoic acid presumably synthesized fromcis-vaccenic acid was detected in all the strains in amounts of 0.4 – 34.7 % and was considered as a chemotaxonomic marker of the genus. During growth on a peptone-yeast medium, the caulobacters synthesized, along with the fatty acids which are typical of gram-negative bacteria, some normal and branched fatty acids with 15 and 17 carbon atoms (1–49 %). The synthesis of these acids was inhibited by glucose. The cell shape of stalked bacteria (fusiform, vibrioid or bacteroid) is not obviously associated with the contents of individual fatty acids.     

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. ¹³C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.     

Combined effects of oleic,linoleic and linolenic acids on lactation performance and the milk fatty acid profile in lactating dairy cows

The potential combined effects of oleic, linoleic and linolenic acids supplementation on lactation performance and the milk fatty acid (FA) profile in dairy cows have not been well investigated. Our objective was to examine the effects of supplementation with a combination of these FA as well as the effects of removing each from the combination on lactation performance and the milk FA profile in dairy cows. Eight Holstein cows (101±11 days in milk) received four intravenously infused treatments in a 4×4 Latin square design, and each period lasted for 12 days which consisted of 5 days of infusion and 7 days of recovery. The control treatment (CTL) contained 58.30, 58.17 and 39.96 g/day of C18: 1 cis-9; C18: 2 cis-9, cis-12; and C18: 3 cis-9, cis-12, cis-15, respectively. The other three treatments were designated −−C18: 1 (20.68, 61.17 and 41.72 g/day of C18: 1 cis-9; C18: 2 cis-9, cis-12; and C18: 3 cis-9, cis-12, cis-15, respectively), −C18: 2 (61.49, 19.55 and 42.13 g/day of C18: 1 cis-9; C18: 2 cis-9, cis-12; and C18: 3 cis-9, cis-12, cis-15, respectively) and −C18: 3 (60.89, 60.16 and 1.53 g/day of C18: 1 cis-9; C18: 2 cis-9, cis-12; and C18: 3 cis-9, cis-12, cis-15, respectively). Dry matter intake and lactose content were not affected by the treatments, but the milk protein content was lower in cows treated with −C18: 2 than that in CTL-treated cows. Milk yield as well as milk fat, protein and lactose yields were higher in cows treated with −C18: 3 than the yields in CTL-treated cows, and these yields increased linearly as the unsaturation degree of the supplemental FA decreased. Compared with the CTL treatment, the −C18: 2 treatment decreased milk C18: 2 cis-9 content (by 2.80%) and yield (by 22.12 g/day), and the −C18: 3 treatment decreased milk C18: 3 cis-9, cis-12, cis-15 content (by 2.72%) and yield (by 22.33 g/day). In contrast, removing C18: 1 cis-9 did not affect the milk content or yield of C18: 1 cis-9. The −C18: 2-treated cows had a higher C18: 1 cis-9 content and tended to have a higher C18: 1 cis-9 yield than CTL-treated cows. The yields of C8: 0, C14: 0 and C16: 0 as well as <C16: 0 tended to increase linearly as the unsaturation degree of the supplemental FA decreased (P=0.06, 0.07, 0.07 and 0.09, respectively). These results indicated that supplementation with C18 unsaturated FA might not independently affect the lactation performance and the milk FA profile of dairy cows.     

Hexadecenoic Fatty Acid Isomers in Human Blood Lipids and Their Relevance for the Interpretation of Lipidomic Profiles

Monounsaturated fatty acids (MUFA) are emerging health biomarkers, and in particular the ratio between palmitoleic acid (9cis-16:1) and palmitic acid (16:0) affords the delta-9 desaturase index that is increased in obesity. Recently, other positional and geometrical MUFA isomers belonging to the hexadecenoic family (C16 MUFA) were found in circulating lipids, such as sapienic acid (6cis-16:1), palmitelaidic acid (9trans-16:1) and 6trans-16:1. In this work we report: i) the identification of sapienic acid as component of human erythrocyte membrane phospholipids with significant increase in morbidly obese patients (n = 50) compared with age-matched lean controls (n = 50); and ii) the first comparison of erythrocyte membrane phospholipids (PL) and plasma cholesteryl esters (CE) in morbidly obese patients highlighting that some of their fatty acid levels have opposite trends: increases of both palmitic and sapienic acids with the decrease of linoleic acid (9cis,12cis-18:2, omega-6) in red blood cell (RBC) membrane PL were reversed in plasma CE, whereas the increase of palmitoleic acid was similar in both lipid species. Consequentially, desaturase enzymatic indexes gave different results, depending on the lipid class used for the fatty acid content. The fatty acid profile of morbidly obese subjects also showed significant increases of stearic acid (C18:0) and C20 omega-6, as well as decreases of oleic acid (9cis-18:1) and docosahexaenoic acid (C22:6 omega-3) as compared with lean healthy controls. Trans monounsaturated and polyunsaturated fatty acids were also measured and found significantly increased in both lipid classes of morbidly obese subjects. These results highlight the C16 MUFA isomers as emerging metabolic marker provided that the assignment of the double bond position and geometry is correctly performed, thus identifying the corresponding lipidomic pathway. Since RBC membrane PL and plasma CE have different fatty acid trends, caution must also be used in the choice of lipid species for the interpretation of lipidomic profiles.     

Effect of exogenous fatty acids on growth,membrane fluidity,and phospholipid fatty acid composition in yeast

The growth response of a double-mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis-Δ¹¹- eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity. There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 1 5:0, and 1 6:0. We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism.