毕赤酵母中外源表达脂肪酰-CoA还原酶生产脂肪醇

【目的】通过在毕赤酵母Komagataella pastoris GS115中外源表达来源于霍霍巴[Simmondsia chinensis(Jojoba)]的脂肪酰-Co A还原酶Jojoba FAR,利用微生物发酵生产脂肪醇。【方法】以质粒p RL105为模板PCR扩增获得霍霍巴脂肪酰-Co A还原酶的编码基因,以p GAPZαA为载体构建重组表达质粒p GAP-far,并通过电转化法转入K.pastoris GS115,筛选转化子并发酵,气相色谱-质谱联用检测发酵产物。【结果】构建了毕赤酵母重组菌株p GAPZ-far-GS115,通过摇瓶发酵检测到脂肪醇的合成。随后在7 L规模的发酵罐上发酵验证,得到脂肪醇产量为134.74 mg/L,产率为1.22 mg/(L·h)。【结论】实现了脂肪醇在毕赤酵母中的生物合成,为工业上利用毕赤酵母生产脂肪醇奠定了一定基础。     

茄科雷尔氏菌脂酰-CoA合成酶的功能鉴定

【目的】茄科雷尔氏菌是一种常见的农作物致病菌,引起植物青枯病。研究其脂肪酸代谢途径将有助于寻找新的抗菌药物靶点,为防治青枯病害提供新的思路。【方法】利用大肠杆菌FadD序列,进行同源比对发现茄科雷尔氏菌GMI1000中RSc2857(RsFadD)具有较高的相似性,推测其具有脂酰-CoA合成酶活性。采用PCR扩增方法获得RsfadD基因,连入表达载体pBAD24M后互补大肠杆菌fadD突变株,并检测转化子的生长情况。RsfadD与pET-28b连接后,在大肠杆菌BL(DE3)中表达,并利用Ni-NTA纯化获得带有组氨酸标签的RsFadD,体外测定RsFadD的活性。利用同源重组方法,获得RsfadD敲除突变株,分析突变株的生长性状。【结果】RsfadD异体互补大肠杆菌fadD突变株,恢复突变株在以脂肪酸为碳源的基础培养基上生长。体外活性测定RsFadD具有脂酰-CoA合成酶活性,对不同链长的脂肪酸都具有活性,但活性低于大肠杆菌FadD。RsfadD突变株在添加不同链长脂肪酸的基础培养上仅能微弱生长,而在丰富培养基上生长无差异。【结论】茄科雷尔氏菌中RsfadD编码脂酰-CoA合成酶,在脂肪酸利用过程中发挥重要作用。但RsfadD突变株在基础培养基上微弱生长,说明茄科雷尔氏菌基因组中还有其他的脂酰-CoA合成酶基因。以上研究结果为进一步研究茄科雷尔氏菌中脂酰-CoA合成酶以及脂肪酸利用机制奠定了基础。     

The fatty acyl-CoA reductase Waterproof mediates airway clearance in Drosophila

The transition from a liquid to a gas filled tubular network is the prerequisite for normal function of vertebrate lungs and invertebrate tracheal systems. However, the mechanisms underlying the process of gas filling remain obscure. Here we show that waterproof, encoding a fatty acyl-CoA reductase (FAR), is essential for the gas filling of the tracheal tubes during Drosophila embryogenesis, and does not affect branch network formation or key tracheal maturation processes. However, electron microscopic analysis reveals that in waterproof mutant embryos the formation of the outermost tracheal cuticle sublayer, the envelope, is disrupted and the hydrophobic tracheal coating is damaged. Genetic and gain-of-function experiments indicate a non-cell-autonomous waterproof function for the beginning of the tracheal gas filling process. Interestingly, Waterproof reduces very long chain fatty acids of 24 and 26 carbon atoms to fatty alcohols. Thus, we propose that Waterproof plays a key role in tracheal gas filling by providing very long chain fatty alcohols that serve as potential substrates for wax ester synthesis or related hydrophobic substances that ultimately coat the inner lining of the trachea. The hydrophobicity in turn reduces the tensile strength of the liquid inside the trachea, leading to the formation of a gas bubble, the focal point for subsequent gas filling. Waterproof represents the first enzyme described to date that is necessary for tracheal gas filling without affecting branch morphology. Considering its conservation throughout evolution, Waterproof orthologues may play a similar role in the vertebrate lung.     

Modulation by dexamethasone of the fatty acyl-CoA desaturase system in Tetrahymena microsomes

Dexamethasone produced an increased activity of stearoyl-CoA desaturase through the enhancement of delta 9-terminal component activity, and a corresponding decrease of oleoyl-CoA desaturase activity via the reduced activity of delta 12-terminal component in Tetrahymena microsomes. However, the content of cytochrome b5 as well as the activities of NAD(P)H-cytochrome c and NADH-ferricyanide reductases showed no significant changes by dexamethasone. Additionally, dexamethasone evoked a 3.5-fold increase of intracellular cyclic AMP content 2 hr after administration. These results suggest that dexamethasone may modulate microsomal fatty acyl-CoA desaturase system in Tetrahymena by increasing intracellular cyclic AMP content.     

Microsomal fatty acid desaturase activities in vitamin A-deficient rat liver

The activity of microsomal fatty acid delta 9-desaturase was significantly higher in liver microsomes of vitamin A-deficient rats as compared with their controls. Feeding of vitamin A-supplemented control diet to the deficient rats restored the delta 9-desaturase activity to the control values. The activity of delta 6-desaturase was not affected by vitamin A deficiency.     

In vivo cholesterol removal from liver microsomes induces changes in fatty acid desaturase activities

Rats fed a 1% cholesterol and 0.5% cholate diet for 21 days were transferred to a sterol-free diet after variable periods of time. The effect of cholesterol removal on liver microsomal composition and fatty acid desaturases was studied. Some changes were already observed after 1 day. However, after 21 days of a sterol-free diet, the cholesterol content of liver microsomes decreased as well as that of phosphatidylcholine. So did the cholesterol/phospholipid ratio. Phosphatidylinositol, phosphatidylserine and sphingomyelin slightly increased along with time. The total fatty acid composition was altered by a decrease in monounsaturated acids and an increase in the saturated acids, palmitic and stearic acids. The arachidonic acid content rose. A similar pattern of change was found in the fatty acid composition of the main phospholipids: phosphatidylcholine and phosphatidylethanolamine. delta 9-Desaturase activity steadily decreased along with cholesterol removal, whereas delta 5- and delta 6-desaturase activities were enhanced towards the end of the removal period. The microsomal membrane became more 'fluid', according to the decrease of fluorescence anisotropy of the 1,6-diphenyl-1,3,5-hexatriene incorporated into the membrane.     

Improved ethanol tolerance of Saccharomyces cerevisiae strains by increases in fatty acid unsaturation via metabolic engineering

To enhance the ethanol tolerance of Saccharomyces cerevisiae, the Arabidopsis thaliana FAD2 gene and/or the S. cerevisiae OLE1 gene were over-expressed in this yeast. The transformant over-expressing both these genes could not only synthesize dienoic fatty acids but also increased the unsaturated fatty acid content of membrane lipid and then showed the highest viability in the presence of 15% (v/v) ethanol.     

Chronic ethanol administration depresses fatty acid synthesis in rat adipose tissue.

The temperature-dependence of both the lipid order parameter (SDPH) and acetylcholinesterase (AChE) activity from native and cholesterol-enriched human erythrocyte membranes was investigated. Cholesterol enrichment abolishes an inflection observed around 30 degrees C in the temperature-dependence of native membrane lipid order parameter, whereas the Arrhenius plot of the enzymic activity is substantially unaffected. These results support the view that the breaks in the Arrhenius plot of the enzyme activity are not related to sudden changes of bulk membrane physical state, but arise from a direct effect of temperature on enzyme conformation.     

Chronic leptin administration decreases fatty acid uptake and fatty acid transporters in rat skeletal muscle.

Chronic leptin administration reduces triacylglycerol content in skeletal muscle. We hypothesized that chronic leptin treatment, within physiologic limits, would reduce the fatty acid uptake capacity of red and white skeletal muscle due to a reduction in transport protein expression (fatty acid translocase (FAT/CD36) and plasma membrane-associated fatty acid-binding protein (FABPpm)) at the plasma membrane. Female Sprague-Dawley rats were infused for 2 weeks with leptin (0.5 mg/kg/day) using subcutaneously implanted miniosmotic pumps. Control and pair-fed animals received saline-filled implants. Leptin levels were significantly elevated (approximately 4-fold; p < 0.001) in treated animals, whereas pair-fed treated animals had reduced serum leptin levels (approximately -2-fold; p < 0.01) relative to controls. Palmitate transport rates into giant sarcolemmal vesicles were reduced following leptin treatment in both red (-45%) and white (-84%) skeletal muscle compared with control and pair-fed animals (p < 0.05). Leptin treatment reduced FAT mRNA (red, -70%, p < 0.001; white, -48%, p < 0.01) and FAT/CD36 protein expression (red, -32%; p < 0.05) in whole muscle homogenates, whereas FABPpm mRNA and protein expression were unaltered. However, in leptin-treated animals plasma membrane fractions of both FAT/CD36 and FABPpm protein expression were significantly reduced in red (-28 and -34%, respectively) and white (-44 and -56%, respectively) muscles (p < 0.05). Across all experimental treatments and muscles, palmitate uptake by giant sarcolemmal vesicles was highly correlated with the plasma membrane FAT/CD36 protein (r = 0.88, p < 0.01) and plasma membrane FABPpm protein (r = 0.94, p < 0.01). These studies provide the first evidence that protein-mediated long chain fatty acid transport is subject to long term regulation by leptin.     

Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002

Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C₁₈ fatty acids increased in cells grown at lower temperature for all lipid classes, and ω3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the ω3 position of C₁₈ fatty acids increased gradually during a 12-h period after a temperature shift-down to 22°C. However, the mRNA levels of the desA (Δ12 desaturase), desB (ω3 desaturase) and desC (Δ9 desaturase) genes increased within 15 min after a temperature shift-down to 22°C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38°C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22°C and 38°C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium.     

Binge ethanol exposure decreases neurogenesis in adult rat hippocampus

Alcoholism is associated with cognitive deficits and loss of brain mass. Recent studies have indicated that neural progenitor cells proliferate throughout life forming neurons, astrocytes, and oligodendrocytes. The dentate gyrus is one neurogenic region of the adult brain containing neural progenitor cells. To determine if binge ethanol (EtOH) exposure alters neural progenitor cell proliferation and survival, bromodeoxyuridine was administered to adult male rats following an acute or chronic binge exposure paradigm. For an acute binge, rats were gavaged with a 5 g/kg dose of EtOH or vehicle, administered bromodeoxyuridine, and killed either 5 h or 28 days after EtOH treatment. In a 4-day, chronic-binge paradigm, rats were infused with EtOH three times per day (mean dose 9.3 g/kg/day) or isocaloric control diet. Rats were given bromodeoxyuridine once a day for the 4 days of chronic binge treatment, then perfused either immediately following the last dose of EtOH or 28 days later. In both EtOH treatment groups, binge EtOH decreased neural progenitor cell proliferation. Following the chronic four-day binge, neural progenitor cell survival was decreased. These studies are the first to show EtOH inhibition of neural progenitor cell proliferation and survival in the adult, a possible new mechanism underlying alcoholic cognitive dysfunction.     

Chronic ethanol consumption alters cardiovascular functions in conscious rats

Chronic ethanol intake and hypertension are related. In the present work, we investigated the effect of chronic ethanol (20% v/v) intake for 2, 6 and 10 weeks on basal arterial blood pressure, baroreflex and heart rate levels, as well as on the cardiovascular responses to the infusion of vasoactive agents in unanesthetized rats. Mild hypertension was observed after 2 weeks, 6 weeks or 10 weeks of treatment. On the other hand, no changes were observed in heart rate after long-term ethanol intake. Similar baroreflex changes were observed in 2- or 6-week ethanol-treated rats, and affected all parameters of baroreflex sigmoid curves, when compared to the control group. These changes were characterized by an enhanced baroreflex sympathetic component and a reduction in the baroreflex parasympathetic component. No differences in baroreflex parameters were observed in 10-week ethanol-treated animals. The pressor effects of i.v. phenylephrine were enhanced in 2-week ethanol-treated rats; not affected in 6-week treated animals and reduced in 10-week ethanol-treated rats, when compared to respective control and isocaloric groups. The hypotensive response to i.v. sodium nitroprusside (SNP) was enhanced at all different times of treatment, when compared to respective control and isocaloric groups. In conclusion, the present findings showed increased arterial pressure in the early phase of chronic ethanol consumption, which was consequent of rise in both systolic and diastolic pressures. Ethanol intake affected both the sympathetic and the parasympathetic components of the baroreflex. Vascular responsiveness to the pressor agent phenylephrine was initially enhanced and later on decreased during chronic ethanol intake. Vascular responsiveness to the depressor agent SNP was enhanced during chronic ethanol intake.     

Molecular cloning and ontogenic mRNA expression of fatty acid desaturase in the carnivorous striped snakehead fish (Channa striata)

There is very little information on the capacity of freshwater carnivorous fish to biosynthesize highly unsaturated fatty acids (HUFA). The striped snakehead fish (Channa striata) is a carnivorous species cultured inland of several Southeast Asian countries due to its pharmaceutical properties in wound healing enhancement. We described here the full-length cDNA cloning of a striped snakehead fatty acid desaturase (fads), which is responsible for desaturation of unsaturated fatty acids in the HUFA biosynthesis. Bioinformatics analysis reveals a protein coding region with length of 445 amino acids containing all characteristic features of desaturase enzyme, including a cytochrome b5-domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. The striped snakehead fads amino acid sequence shares high similarity with known fads of other teleosts. The mRNA expression of striped snakehead fads also showed an ontogenic-related increase in expression in 0-20 days after hatch larva. Using ISH, we localized the presence of fads in larva brain, liver and intestinal tissues.     

Cholesterol binding reserve of hyperlipemic rat serum lipoproteins in chronic ethanol administration

Chronic administration of ethanol in rats caused the reduction of serum cholesterol binding reserve. The very low density and high density lipoproteins, main serum cholesterol binding reserves, were slightly increased with corresponding increases in their lipid and protein components during initial stage of alcohol consumption. However, these capacities get deminished during reversal of hyperlipemia induced by prolonged action of ethanol. This situation may be an early indicator for the initiation of hepatic damage and a variety of secondary effects of ethanol.     

Fatty acid composition of phospholipids in mitochondria and microsomes during diethylnitrosamine carcinogenesis in rat liver

Changes in lipid composition and function of subcellular organelles have been described in transplanted and primary tumours. We examine here the fatty acid composition of individual phospholipids (PL) in hyperplastic nodules and primary hepatoma induced by diethylnitrosamine (DEN), compared to that of normal liver and of transplantable Yoshida AH-130 hepatoma. Phosphatidylcholine and phosphatidylethanolamine fatty acid composition in mitochondria and microsomes from primary hepatoma were markedly different from normal liver; C18:0/C18:1 ratio was lower and the ratio between monosaturated and polyunsaturated fatty acids was higher. Linoleic acid content of mitochondrial cardiolipin, usually very high in normal rat liver, was notably lower in primary hepatoma. Cholesterol/phospholipid ratio in both microsomes and mitochondria from DEN-induced hepatoma was higher than in normal liver. Hyperplastic nodules showed no changes in cholesterol content whereas modifications in fatty acid composition were already observable. These modifications of membrane structure may be related to the functional changes found in nodular cells. Changes in fatty acid composition of membrane phospholipids, occurring in both primary hepatoma and preneoplastic nodules, might be one of the causes for decreased rate of lipid peroxidation peculiar to these tissues.     

Effect of the non-steroidal anti-inflammatory drugs on the acyl-CoA synthetase activity toward medium-chain,long-chain and polyunsaturated fatty acids in mitochondria of mouse liver and brain

Effect of eleven non-steroidal anti-inflammatory drugs on the acyl-CoA synthetase activities toward octanoic, palmitic, arachidonic and docosahexaenoic acids was evaluated in mouse liver and brain mitochondria. The drugs tested were aspirin, salicylic acid, diflunisal, mefenamic acid, indomethacin, etodolac, ibuprofen, ketoprofen, naproxen, loxoprofen, flurbiprofen. In mouse liver mitochondria, diflunisal and mefenamic acid exhibited the inhibitory activities not only for octanoic acid (IC₅₀?=?78.7 and 64.7 µM) and but also for palmitic acid (IC₅₀?=?236.5 and 284.4 µM), respectively. Aspirin was an inhibitor for the activation of octanoic acid only (IC₅₀?=?411.0 µM). In the brain, mefenamic acid and diflunisal inhibited strongly palmitoyl-CoA formation (IC₅₀?=?57.3 and 114.0 µM), respectively. The activation of docosahexaenoic acid in brain was sensitive to inhibition by diflunisal and mefenamic acid compared with liver.     

Long-chain fatty acids and their acyl-CoA esters cause the translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction of rat liver

A translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction was promoted in cell-free extracts of rat liver by oleate and palmitate and their CoA esters. Oleate was more potent in this respect than palmitate and the CoA esters were more effective than the unesterified acids. Octanoate, octanoyl-CoA and CoA did not cause the translocation. It is proposed that the interaction of phosphatidate phosphohydrolase with the membranes that synthesize glycerolipids causes it to become metabolically active. This enables the liver to increase its capacity for triacylglycerol synthesis in response to an increased supply of fatty acids.     

Changes in activities of some ammonia-metabolizing enzymes in the rat liver and the brain after chronic ethanol administration

The changes in the activities of ammonia-metabolizing enzymes in liver and brain after ethanol intoxication has been investigated in rats. After administration of ethanol 30% (w/v) 6g kg-1 for 4 weeks we found an increase in liver glutamate dehydrogenase and glutaminase activity. In brain tissue the glutaminase activity was significantly higher and glutamate dehydrogenase was significantly lower. Glutamine synthetase activity in liver and brain was practically unchanged. The reasons for these changes in the activities of some ammonia-metabolizing enzymes in liver and brain after ethanol ingestion have been discussed.     

Resveratrol, a red wine polyphenol, attenuates ethanol-induced oxidative stress in rat liver

The involvement of oxidative stress in the pathogenesis of alcoholic diseases in the liver has been repeatedly confirmed. Resveratrol, a natural phytoalexin present in grape skin and red wine possesses a variety of biological activities including antioxidant. This study was conducted to evaluate whether resveratrol has a preventive effect on the main indicators of hepatic oxidative status as an expression of the cellular damage caused by free radicals, and on antioxidant defence mechanism during chronic ethanol treatment. Wistar rats were treated daily with 35% ethanol solution (3 g/kg/day i.p.) during 6 weeks and fed basal diet or basal diet containing 5 g/kg resveratrol. Control rats were treated with i.p. saline and fed basal diet. Experimentally, chronic ethanol administration leads to hepatotoxicity as monitored by the increase in the level of hepatic marker enzymes and the appearance of fatty change, necrosis, fibrosis and inflammation in liver sections. Ethanol also enhanced the formation of MDA in the liver indicating an increase in lipid peroxidation, a major end-point of oxidative damage, and caused drastic alterations in antioxidant defence systems. Particularly the activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were found reduced by ethanol treatment while glutathione reductase (GR) activity was unchanged. Dietary supplementation with resveratrol during ethanol treatment inhibited hepatic lipid peroxidation and ameliorated SOD, GPx and CAT activities in the liver. Conclusively, we can suggest that resveratrol could have a beneficial effect in inhibiting the oxidative damage induced by chronic ethanol administration, which was proved by the experiments that we conducted on rats.     

Age-dependent changes in rat liver lipid peroxidation and glutathione content induced by acute ethanol ingestion

The study of the influence of the age of animals (13 to 53 weeks) on total liver thiobarbituric acid reactive substances (TBAR) content showed an increase which is maximal in rats of 39 weeks of age compared to young animals (13 weeks), followed by a dimunition in the 53 weeks old group. In this situation, the content of hepatic GSH and total GSH equivalents as well as the GSH/GSSG ratio were decreased with ageing, while GSSG levels were enhanced in the oldest group studied. Acute ethanol intoxication resulted in a marked increase in liver TBAR content in young animals, together with a decline in GSH, total GSH equivalents and GSH/GSSG ratio, and an enhancement in GSSG. These changes elicited by ethanol intake were reduced with ageing. It is concluded that ethanol-induced oxidative stress in the liver is diminished during ageing, despite the progressive decrease in the glutathione content of the tissue observed in control animals.