细菌不饱和脂肪酸合成研究进展

脂肪酸不仅是细菌细胞膜组分，还是许多生物活性物质的合成原料。不饱和脂肪酸(unsaturated fatty acid, UFA)具有更低的相变温度，是细菌调节细胞膜流动性的重要分子，因此UFA合成途径是重要的抗菌药物筛选靶点。细菌可利用厌氧途径合成UFA，其中模式生物大肠杆菌利用经典的FabA-FabB途径合成UFA，但不同细菌中UFA合成的厌氧途径具有多样性，相关催化酶类也不尽相同；细菌还可以利用需氧途径合成UFA，利用脂肪酸脱饱和酶直接将饱和脂肪酸(saturated fatty acid, SFA)转化为不饱和脂肪酸，而不同脱饱和酶会生成不同结构的UFA，在逆境耐受、致病力等多方面发挥重要作用；细菌还可以利用单加氧酶，将脂肪酸合成途径中癸酰酰基载体蛋白(acyl carrier protein, ACP)转化为顺-3-癸烯酰ACP，并最终合成UFA。细菌脂肪酸合成相关的其他酶类在UFA合成或不同种类UFA调节中也发挥着重要作用。本文系统地总结了细菌UFA合成途径与相关酶类的多样性研究进展，旨在为进一步了解细菌UFA合成机制，并以此为靶点开发抗菌药物等方面提供理论支撑。     

Altered membrane free unsaturated fatty acid composition in human colorectal cancer tissue

Polyunsaturated free fatty acids (PUFAs) participate in normal functioning of the cell, particularly in control intracellular cell signalling. As nutritional components they compose a human diet with an indirect promoting influence on tumourogenesis. The PUFAs level depends on the functional state of the membrane. This work is focused on changes only of free unsaturated fatty acids amount (AA – arachidonic acid, LA – linoleic acid, ALA – α-linolenic acid, palmitoleic acid (PA) and oleic acid) in cell membranes of colorectal cancer of pT3 stage, G2 grade without metastasis. Qualitative and quantitative composition of free unsaturated fatty acids in the membrane was determined by high-performance liquid chromatography. It was shown that the malignant transformation was accompanied by a decrease in amount of LA and ALA while arachidonic and oleic acids increased. It is of interest that free AA levels are elevated in colon cancer, as AA is the precursor to biologically active eicosanoids.     

Peroxisome proliferator-activated receptor alpha is required for feedback regulation of highly unsaturated fatty acid synthesis

Delta6 desaturase (D6D), the rate-limiting enzyme for highly unsaturated fatty acid (HUFA) synthesis, is induced by essential fatty acid-deficient diets. Sterol regulatory element-binding protein-1c (SREBP-1c) in part mediates this induction. Paradoxically, D6D is also induced by ligands of peroxisome proliferator-activated receptor alpha (PPARalpha). Here, we report a novel physiological role of PPARalpha in the induction of genes specific for HUFA synthesis by essential fatty acid-deficient diets. D6D mRNA induction by essential fatty acid-deficient diets in wild-type mice was diminished in PPARalpha-null mice. This impaired D6D induction in PPARalpha-null mice was not attributable to feedback suppression by tissue HUFAs because PPARalpha-null mice had lower HUFAs in liver phospholipids than did wild-type mice. Furthermore, PPARalpha-responsive genes were induced in wild-type mice under essential fatty acid deficiency, suggesting the generation of endogenous PPARalpha ligand(s). Contrary to genes for HUFA synthesis, the induction of other lipogenic genes under essential fatty acid deficiency was higher in PPARalpha-null mice than in wild-type mice even though mature SREBP-1c protein did not differ between the genotypes. The expression of PPARgamma was markedly increased in PPARalpha-null mice and might have contributed to the induction of genes for de novo lipogenesis. Our study suggests that PPARalpha, together with SREBP-1c, senses HUFA status and confers pathway-specific induction of HUFA synthesis by essential fatty acid-deficient diets.     

Microbial production of a novel trihydroxy unsaturated fatty acid from linoleic acid

A bacterium isolated from a dry soil sample collected from McCalla, AL, USA, converted linoleic acid to a novel compound, 12,13,17-trihydroxy-9 (Z)-octadecenoic acid (THOA). The organism is a Gram-positive, non-motile rod (0.5 μ m × 2 μ m). It was identified as a species of Clavibacter ALA2. The product was purified by high pressure liquid chromatography, and its structure was determined by ¹H and ¹³C nuclear magnetic resonance and Fourier transform infrared spectroscopies, and by mass spectrometer. Maximum production of THOA with 25% conversion of the substrate was reached after 5–6 days of reaction. THOA was not further metabolized by strain ALA2. This is the first report of a 12,13,17-trihydroxy unsaturated fatty acid and its production by microbial transformation. Some dihydroxy intermediates were also detected. THOA has a structure similar to those of known plant self-defense substances. Received 13 January 1997/ Accepted in revised form 05 May 1997     

Ethanol-induced decreases in membrane long-chain unsaturated fatty acids correlate with impaired chick brain development

Exposure to ethanol at 0 days of development induced changes in total membrane fatty acid composition at 18 days of development. When exposed to ethanol concentrations ranging from 0–743.27μm/kg egg wt, decreased levels of long-chain, unsaturated membrane fatty acids and increased levels of short-chain, saturated membrane fatty acids were observed in embryonic chick brains at 18 days of development. The ratios of unsaturated membrane/saturated membrane fatty acids correlated with an ethanol-induced reduction in neuron densities within the cerebral hemispheres and three different regions of the optic lobes with correlation coefficients (r) ranging from 0.44 [F = (1, 32) 7.84; P ≤ 0.009] to 0.59 [F = (1, 32) 17.38; P ≤ 0.0002]. The ratios of long-chain/short-chain membrane fatty acids also correlated with an ethanol-induced reduction in neuron densities within the cerebral hemispheres and three different regions of the optic lobes with correlation coefficients (r) ranging from 0.51 [F = (1, 32) 11.27; P≤ 0.002] to 0.66 [F = (1, 32) 24.40; P ≤ 0.0001]. Cell fractionation studies indicated that the ethanol-induced changes in brain membrane fatty acid composition were restricted to microsomal membranes.     

Lead-catalyzed peroxidation of essential unsaturated fatty acid

In the present study, the reaction mixtures (lead compounds with essential unsaturated fatty acids) were preincubated at 37°C for 24 h prior to the measurement of malondialdehyde (MDA) by HPLC. The metal-catalyzed reactions were also compared in the presence of butylated hydroxytoluene (BHT), a free radical scavenger. Our results showed that according to the difference in the number of double bonds of essential unsaturated fatty acids, the kinds of lead compounds, and the concentrations of lead compounds, the extent of lipid peroxidation was different. The addition of BHT to the reaction mixtures significantly reduced the production of MDA (P<0.01). These in vitro studies support prior in vivo reports that the important mechanism of the acute toxic effects of the lead compounds is owing at least in part, to metal-catalyzed peroxidation of polyun-saturated fatty acids.     

The synthesis and accumulation of stearidonic acid in transgenic plants: a novel source of 'heart-healthy' omega-3 fatty acids

Dietary omega-3 polyunsaturated fatty acids have a proven role in reducing the risk of cardiovascular disease and precursor disease states such as metabolic syndrome. Although most studies have focussed on the predominant omega-3 fatty acids found in fish oils (eicosapentaenoic acid and docosahexaenoic acid), recent evidence suggests similar health benefits from their common precursor, stearidonic acid. Stearidonic acid is a Δ6-unsaturated C18 omega-3 fatty acid present in a few plant species (mainly the Boraginaceae and Primulaceae ) reflecting the general absence of Δ6-desaturation from higher plants. Using a Δ6-desaturase from Primula vialii , we generated transgenic Arabidopsis and linseed lines accumulating stearidonic acid in their seed lipids. Significantly, the P. vialii Δ6-desaturase specifically only utilises α-linolenic acid as a substrate, resulting in the accumulation of stearidonic acid but not omega-6 γ-linolenic acid. Detailed lipid analysis revealed the accumulation of stearidonic acid in neutral lipids such as triacylglycerol but an absence from the acyl-CoA pool. In the case of linseed, the achieved levels of stearidonic acid (13.4% of triacylglycerols) are very similar to those found in the sole natural commercial plant source ( Echium spp.) or transgenic soybean oil. However, both those latter oils contain γ-linolenic acid, which is not normally present in fish oils and considered undesirable for heart-healthy applications. By contrast, the stearidonic acid-enriched linseed oil is essentially devoid of this fatty acid. Moreover, the overall omega-3/omega-6 ratio for this modified linseed oil is also significantly higher. Thus, this nutritionally enhanced linseed oil may have superior health-beneficial properties.     

Production of 8,11‐dihydroxy and 8‐hydroxy unsaturated fatty acids from unsaturated fatty acids by recombinant Escherichia coli expressing 8,11‐linoleate diol synthase from Penicillium chrysogenum

Hydroxy unsaturated fatty acids can be used as antimicrobial surfactants. 8,11‐Linoleate diol synthase (8,11‐LDS) catalyzes the conversion of unsaturated fatty acid to 8‐hydroperoxy unsaturated fatty acid, and it is subsequently isomerized to 8,11‐dihydroxy unsaturated fatty acid by the enzyme. The optimal reaction conditions of recombinant Escherichia coli expressing Penicillium chrysogenum 8,11‐LDS for the production of 8,11‐dihydroxy‐9,12(Z,Z)‐octadecadienoic acid (8,11‐DiHODE), 8,11‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid (8,11‐DiHOTrE), 8‐hydroxy‐9(Z)‐hexadecenoic acid (8‐HHME), and 8‐hydroxy‐9(Z)‐octadecenoic acid (8‐HOME) were pH 7.0, 25°C, 10 g/L linoleic acid, and 20 g/L cells; pH 6.0, 25°C, 6 g/L α‐linolenic acid, and 60 g/L cells; pH 7.0, 25°C, 8 g/L palmitoleic acid, and 25 g/L cells; and pH 8.5, 30°C, 6 g/L oleic acid, and 25 g/L cells, respectively. Under these optimized conditions, the recombinant cells produced 6.0 g/L 8,11‐DiHODE for 60 min, with a conversion of 60% (w/w) and a productivity of 6.0 g/L/h; 4.3 g/L 8,11‐DiHOTrE for 60 min, with a conversion of 72% (w/w) and a productivity of 4.3 g/L/h; 4.3 g/L 8‐HHME acid for 60 min, with a conversion of 54% (w/w) and a productivity of 4.3 g/L/h; and 0.9 g/L 8‐HOME for 30 min, with a conversion of 15% (w/w) and a productivity of 1.8 g/L/h. To best of our knowledge, this is the first report on the biotechnological production of 8,11‐DiHODE, 8,11‐DiHOTrE, 8‐HHME, and 8‐HOME. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:390–396, 2017     

Basal omega-3 fatty acid status affects fatty acid and oxylipin responses to high-dose n3-HUFA in healthy volunteers

A subject's baseline FA composition may influence the ability of dietary highly unsaturated omega-3 FAs (n3-HUFA) to change circulating profiles of esterified FAs and their oxygenated metabolites. This study evaluates the influence of basal n3-HUFA and n3-oxylipin status on the magnitude of response to n3-HUFA consumption. Blood was collected from fasting subjects (n = 30) before and after treatment (4 weeks; 11 ± 2 mg/kg/day n3-HUFA ethyl esters). Esterified FAs were quantified in erythrocytes, platelets, and plasma by GC-MS. Esterified oxylipins were quantified in plasma by LC-MS/MS. Treatment with n3-HUFAs increased n3-HUFAs and decreased n6-HUFAs in all reservoirs and increased plasma n3-oxylipins without significantly changing n6-oxylipin concentrations. As subject basal n3-HUFAs increased, treatment-associated changes decreased, and this behavior was reflected in the percentage of 20:5n3 + 22:6n3 in red blood cell membrane FAs (i.e., the omega-3 index). To maintain an omega-3 index of 8% and thus reduce cardiovascular disease risk, our analyses suggest a maintenance dose of 7 mg/kg/day n3-HUFA ethyl esters for a 70-kg individual. These results suggest that the basal n3 index may have clinical utility to establish efficacious therapeutic experimental feeding regimens and to evaluate the USDA Dietary Guidelines recommendations for n3-HUFA consumption.     

哺乳动物细胞多不饱和脂肪酸合成酶系的重建将亚油酸代谢转变为二十二碳六烯酸

二十二碳六烯酸(DHA,22:6n-3)是一种长度为22个碳原子且含有6个双键的ω-3系多不饱和脂肪酸,在人体中具有重要生物学功能。人体及其他哺乳动物体内只能合成少量的DHA,更多的需求必须从食物中获取。然而,DHA的天然资源(主要是深海鱼类等海洋产品)日趋枯竭,开发新型资源以满足不断扩大的市场需求势在必行。本研究利用转基因技术,在哺乳动物细胞中使Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶超表达,同时表达来源于秀丽隐杆线虫Caenorhabditis elegans的Δ15去饱和酶和小眼虫Euglena gracilis的Δ4去饱和酶,结果表明,这6种酶的表达或超表达能将ω-6系的亚油酸(LA,18:2n-6)有效地转化为DHA(22:6n-3),后者的含量从对照组的16.74%提高到实验组的25.3%。本研究的策略及技术路线为将来利用遗传改造的哺乳动物生产珍稀的DHA(22:6n-3)等长链多不饱和脂肪酸产品提供了重要的启示。     

Isolation and characterization of unsaturated fatty acids during bacteriorhodopsin preparation from Halobacterium halobium

AIMS: Isolation and characterization of unsaturated fatty acids during bacteriorhodopsin preparation from Halobacterium halobium. METHODS AND RESULTS: Halobacterium halobium was cultivated in a composite medium. Cells were collected by centrifugation followed by ultrasonic disruption, and the resulting suspension was subject to centrifugation for preparation of both pellet and supernatant. The pellet was saved in order to prepare bacteriorhodopsin, while the supernatant was used for the isolation of crude fatty acids by saponification and extraction. Crystallization then took place in acetone at -16 degrees C to remove fatty acids in which the carbon chain length was shorter than 13. The sample was obtained after purification and analysed by gas chromatography. The results demonstrated that Halobacterium halobium could synthesize multiple unsaturated fatty acids, particularly the three important polyunsaturated fatty acids arachidonic acid (1.12%), eicosapentaenoic acid (16.76%) and docosahexaenoic acid (9.38%). CONCLUSION: Important unsaturated fatty acids were isolated and characterized from the waste, which was produced during the preparation of bacteriorhodopsin from Halobacterium halobium. SIGNIFICANCE AND IMPACT OF THE STUDY: Halobacterium halobium has already been used for decades to prepare bacteriorhodopsin. We found that several important unsaturated fatty acids could be extracted from the bacterial waste, which extends its application scope and might bring additional benefits to humanity.     

Nutritional quality of fish faeces is enhanced by highly unsaturated fatty acid-producing heterotrophic protozoa

Highly unsaturated fatty acids such as 20:5n3 (EPA) are both hormone precursors and cell membrane components, making them important nutrients for aquatic animals. Many animals must obtain EPA from their diets because they cannot synthesize enough EPA to meet their requirements, and algae are the main source of EPA in aquatic ecosystems. In a previous study, we detected EPA in the faeces of Danio rerio, a freshwater fish, even though the fish consumed a green algae diet that did not contain EPA. The objective of this study was to determine why EPA was detected in fish faeces. A significant positive relationship was detected between the number of heterotrophic protozoa and the concentration of EPA in the faeces, which suggests that this EPA was of protozoan origin. In addition, another experiment showed that protozoa adhered to faeces far more than the green algal diet remnants, which indicates that protozoa preferred to swarm on faeces. Furthermore, we cultured protozoa in an EPA-free medium and fed them a bacterial diet also lacking EPA, and found that Cyclidium sp. synthesized EPA de novo. The results demonstrate that protozoa produce essential fatty acids and enhance the nutritional quality of animal faeces in detritus-based food webs in freshwater ecosystems.     

The effect of bio-converted polyunsaturated fatty acids on the oxidation of TAG containing highly unsaturated fatty acids

Microbial modification of vegetable fatty acids can often lead to special changes in their structure and in biological function. A bacterial strain, Pseudomonas aeruginosa PR3, is known to carry out multiple hydroxylations on polyunsaturated fatty acids containing 1,4-cis, cis diene structural units, resulting in antibacterial activity. In this paper, in an effort to understand the overall mechanism involved in the varied biological functions of the complicated metabolites of bio-converted polyunsaturated fatty acids, we performed bioconversion of several polyunsaturated fatty acids using PR3, and determined their oxidative activities against fish oil. Bio-converted linoleic acid, eicosapentanoic acid, and docosahexanoic acid promoted effectively oxidation of fish oil. It is assumed that this oxidative effect could plausibly play an important role in the antimicrobial function of these bio-converted fatty acids.     

不饱和脂肪酸在逆境胁迫中的作用

生物膜是将细胞与环境分开的第一道屏障,是环境胁迫造成损伤的主要位点.脂肪酸是生物膜的主要组成成分,不饱和脂肪酸在决定生物膜的生理特性中具有重要作用,增加脂肪酸的不饱和程度能增加膜脂的流动性.近年来,很多研究发现,生物通过脂肪酸脱饱和维持膜的流动性来适应外界环境变化.本文主要从不饱和脂肪酸在环境温度胁迫、盐胁迫、氧化胁迫、酸碱胁迫、干旱胁迫、乙醇胁迫及铝胁迫中的作用研究进展进行了综述.     

Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio–product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.     

Influence of unsaturated fatty acids on the production of tumour necrosis factor and interleukin-6 by rat peritoneal macrophages

The effect of individual unsaturated fatty acids on the release of tumour necrosis factor (TNF) and interleukin 6 (IL6) was investigated in thioglycollate — induced rat peritoneal macrophages. The intracellular mechanisms associated with the changes of cytokine production in response to fatty acids were also studied. Incubation of macrophages with 100 M docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) increased TNF (21% and 15% respectively) and IL6 (69% and 40% respectively) production. Linoleic acid (LA) diminished TNF production by 16%. At 100 M oleic acid (OA), LA and EPA concentration an increase in macrophage adenylate cyclase activity (110%, 72% and 39% respectively) and a decrease (14%) in the presence of DHA was observed. PGE₂ production in the presence of 100 M DHA was reduced by 36%, whereas in the presence of 100 M LA an increase (75%) was observed. Phospholipase A₂ (PLA₂) activity was also found to be modified in the presence of EPA and DHA at 50 M (20% and 60% respectively) and 100 M (34% and 62% respectively) concentrations. The activities of both protein kinase A (PKA) and protein kinase C (PKC) were effected by the different fatty acids. At 50 M all fatty acids suppressed PKA activity except OA which enhanced PKA activity by 14%. At 100 M fatty acid concentration, EPA suppressed PKA activity by 40%. PKC activity was enhanced by LA and OA, by 18% and 21% respectively. However, at 100 M EPA and DHA, PKC activity was suppressed by 37% and 17% respectively, whereas PKC activity was enhanced by 146% in the presence of 100 M LA. These results show for the first time that unsaturated fatty acids have an effect on macrophage PLA₂ activity and that PGE₂ may be a potent modulator of IL6 production. From these studies it is tempting to speculate that macrophage TNF and IL6 release may, in part, occur via a PKC and PKA independent pathway and that PLA₂ activity and PGE₂ concentration are inversely related to production of TNF and IL6.