Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.     

Alteration of low-temperature susceptibility of the cyanobacterium Synechococcus sp. PCC 7002 by genetic manipulation of membrane lipid unsaturation

Cyanobacteria acclimate to low temperature by desaturating their membrane lipids. Mutant strains of Synechococcus sp. PCC 7002 containing insertionally inactivated desA (Δ12 acyl-lipid desaturase) and desB (ω3 acyl-lipid desaturase) genes were produced, and their low-temperature susceptibility was characterized. The desA mutant synthesized no linoleic acid or α-linolenic acid, and the desB mutant did not produce α-linolenic acid. The desA mutant grew more slowly than the wild-type at 22° C and could not grow at 15° C. The desB mutant could not continuously grow at 15° C, although no observable phenotype appeared at higher temperatures. It has been shown that expression of the desA gene occurs at 38° C and is up-regulated at 22° C, and that the desB gene is only expressed at 22° C. These results indicate that the expression of the desA and desB genes occurs at higher temperatures than those at which a significant decline in physiological activities is caused by the absence of their products. The temperature dependency of photosynthesis was not affected by these mutations. Since chlorosis and inability to grow at 15° C with nitrate was suppressed by the substitution of urea as a nitrogen source, it is very likely that the chilling susceptibility of the desaturase mutants is attributable to nutrient limitation. Received: 24 April 1997 / Accepted: 5 August 1997     

植物脂肪酸的生物合成与基因工程

植物脂肪酸既具重要生理功能,又有巨大食用和工业价值。其生物合成途径较为复杂,涉及乙酰－ＣｏＡ羟化酶、脂肪酸合成酶、脂肪酸去饱和酶和脂肪酸延长酶等一系列酶。近年来,对脂肪酸生物合成途径进行了大量研究,克隆出许多相关基因,初步阐明了脂肪酸合成规律,并在此基础上开展了利用基因工程技术调控脂肪酸合成研究,取得可喜进展。本文详细介绍了植物饱和脂肪酸、不饱和脂肪酸和超长链脂肪酸的生物合成与基因工程研究的新结果     

The effects of temperature on the fatty acid and phospholipid composition of four obligately psychrophilicVibrio Spp.

The free fatty acid and phospholipid composition of 4 psychrophilic marineVibrio spp. have been determined in chemostat culture with glucose as the limiting substrate over a temperature range 0–20°C. All the isolates show maximum glucose and lactose uptake at 0°C and this correlates with maximum cell yield. None of the isolates contain fatty acids with a chain length exceeding 17 carbon atoms.Vibrio AF-1 andVibrio AM-1 respond to decreased growth temperatures by synthesizing increased proportions of unsaturated fatty acids (C15:1, C16:1 and C17:1) whereas inVibrio BM-2 the fatty acids undergo chain length shortening. The fourth isolate (Vibrio BM-4) contains high levels (60%) of hexadecenoic acid at all growth temperatures and the fatty acid composition changes little with decreasing temperature. The principal phospholipid components of the four psychrophilic vibrios were phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Lyso-phosphatidylethanolamine and 2 unknown phospholipids were additionally found inVibrio AF-1. The most profound effect of temperature on the phospholipid composition of these organisms was the marked increase in the total quantities synthesized at 0°C. At 15°C phosphatidylglycerol accumulated in the isolates as diphosphatidylglycerol levels decreased. Additionally inVibrio BM-2 andVibro BM-4 phosphatidylserine accumulates as phosphatidylethanolamine biosynthesis was similarly impaired. The observed changes in fatty acid and phospholipid composition in these organisms at 0°C may explain how solute transport is maintained at low temperature.Abbreviations PS Phosphatidylserine - PE phosphatidylethanolamine - PG phosphatidylglycerol - DPG diphosphatidylglycerol - lyso PE lysophosphatidylethanolamine     

Improvement of polyunsaturated fatty acids synthesis by the coexpression of CYB5 with desaturase genes in Saccharomyces cerevisiae

Since Saccharomyces cerevisiae contains Δ9 fatty acid desaturase (OLE1) as a sole fatty acid desaturase, it produces saturated and monounsaturated fatty acids of 16- and 18-carbon compounds. We showed earlier that Kluyveromyces lactis Δ12 (KlFAD2) and ω3 (KlFAD3) fatty acid desaturase genes enabled S. cerevisiae to make also polyunsaturated fatty acids (PUFAs), linoleic (18:2n-6), and α-linolenic (18:3n-3) acids. Unlike Δ9 fatty acid desaturase Ole1p, the two added fatty acid desaturases (KlFAD2and KlFAD3) do not contain a cytochrome b5 domain, and we now report on effects of the overexpression of K. lactis and S. cerevisiae cytochrome b5 (CYB5) genes as well as temperature effects on PUFA synthesis. Without extra cytochrome b5, while PUFA synthesis is significant at low temperature (20 °C), it was marginal at 30 °C. Overexpression of cytochrome b5 at 20 °C did not affect the fatty acid synthesis so much, but it significantly enhanced the synthesis of PUFA at 30 °C.     

The effect of low temperatures on fatty acid composition of crops with different cold resistance

The study was focused on fatty acid (FA) composition of lipids from the seedlings and roots of crops having different cold resistance and grown at 27°C or 4°C. Biosynthesis of FA in the lipids of seedlings and roots of cold-susceptible maize (Zea mays L.) at both growth temperatures was controlled by chloroplast ω6 desaturase and microsomal ω6 desaturase, respectively. The content of linoleic acid was 56.2% and 43.3% in the coleoptiles of maize seedlings grown at 4 and 27°C, respectively, and in the roots it was 52.2% and 38.5%, respectively. The content of α-linolenoic acid in the coleoptiles was 6.7–6.8% at both temperatures, while in the root lipids it was higher at low temperature (3.15% at 27°C vs. 4.7% at 4°C). FA biosynthesis in the seedling coleoptiles of wheat (Triticum aestivum L.) and Siberian wild rye (Elymus sibiricus L.) grown at low temperature was controlled by the chloroplast ω3 desaturase. A minor increase in the content of α-linolenoic acid was observed at low temperature: 29.7% to 30.2% in wheat and 22.8% to 25.8% in wild rye. In the root tissues of these species, the biosynthesis of α-linolenoic acid was controlled by the microsomal ω3 desaturase. The content of α-linolenoic acid was higher at low temperature: in wheat it was 6.1% at 27°C and 17.1%, and 4°C, while in Siberian wild rye, 7.1% and 12.0% at 4 and 27°C, respectively.     

Synergistic effect of high-light and low temperature on cell growth of the Δ12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Δ12 fatty acid desaturase mutant strains. Under a light intensity of 250 μmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 20–38 °C, but growth was non-exponential below 20 °C and ceased at 12 °C. The Δ12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25–38 °C but grew slowly at 22 °C, and no cell growth took place below 18 °C. Under a very high-light intensity of 2.5 mmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 30–38 °C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Δ12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 °C, but were killed at 30 °C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Δ12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 °C under the normal light intensity of 250 μmol m⁻² s⁻¹. These results suggest that growth of the Δ12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of QTLs controlling acylsugar fatty acid composition in an intraspecific population of Lycopersicon pennellii (Corr.) D’Arcy

 Acylsugars exuded by type IV glandular trichomes are responsible for insect resistances found in many Lycopersicon pennellii accessions. Acylsugars are complex mixtures composed of polyacylated sugars (glucose or sucrose) esterified to branched and straight-chain 4 : 0 to 12 : 0 fatty acids. The biogeneses of these unusual fatty acid constituents have their origins in branched-chain amino acid pathways. However, the mechanism of fatty acid elongation in these systems and the genetic control of carbon flux from amino acid to fatty acid pathways remain unclear. In this study, we used an intraspecific F₂ population derived from the cross between L. pennellii LA716 and L. pennellii LA1912 to examine the genetic basis of acylsugar fatty acid composition. Six QTLs were detected which, combined, explain 23–60% of the variance observed for each of the nine segregating fatty acid constituents. Both correlation data and QTL analysis data indicate that branched medium-chain fatty acids are synthesized through elongation of short-chain precursors in two-carbon increments. The proportion of iso-branched acylsugar fatty acids that have an even-carbon chain length was found to be primarily determined by a single locus that maps to a location 5.5 cM above TG117 on chromosome 8. QTL function in several cases can be inferred from discrete patterns of fatty acid composition; in other cases, control of acylsugar fatty acid composition appears to be complex. Received: 7 April 1998 / Accepted: 28 December 1998     

Effect of growth temperature on lipid fatty acids of four fungi (Aspergillus niger,Neurospora crassa,Penicillium chrysogenum,andTrichoderma reesei)

The effect of growth temperature on the lipid fatty acid composition was studied over a temperature range from 35 to 10° C with 5° C intervals in four exponentially growing fungi: Aspergillus niger, Neurospora crassa, Penicillium chrysogenum, and Trichoderma reesei. Fatty acid unsaturation increased in A. niger, P. chrysogenum, and T. reesei when the temperature was lowered to 20–15, 20, and 26–20° C, respectively. In A. niger and T. reesei, this was due to the increase in linolenic acid content. In P. chrysogenum, the linolenic acid content increased concomitantly with a more pronounced decrease in the less-unsaturated fatty acid, oleic acid, and in palmitic and linoleic acids; consequently, the fatty acid content decreased as the temperature was lowered to 20° C. In T. reesei, when the growth temperature was reduced below 26–20° C, fatty acid unsaturation decreased since the mycelial linolenic acid content decreased. In A. niger and P. chrysogenum, the mycelial fatty acid content increased greatly at temperatures below 20–15° C. In contrast, in N. crassa, fatty acid unsaturation was nearly temperature-independent, although palmitic and linoleic acid contents clearly decreased when the temperature was lowered between 26 and 20° C; concomitantly, the growth rate decreased. Therefore, large differences in the effects of growth temperature on mycelial fatty acids were observed among various fungal species. However, the similarities found may indicate common regulatory mechanisms causing the responses. Received: 1 March 1995 / Accepted: 8 May 1995     

Effect of environmental factors on the limpid composition membrane structure and permeability ofMicrosporum gypseum

Supplementation with unsaturated fatty acids, substitution of glucose by glycerol as carbon source and lowered growth temperature (20°C) increased the total phospholipid content ofMicrosporum gypseum spheroplasts. Levels of sterols increased with glycerol substitution and decreased in other growth conditions. Substantial changes were seen in the ratios of unsaturated to saturated fatty acids and phosphatidylcholine to phosphatidylethanolafne under all the experimental conditions. Changed lipid composition resulted in altered uptake of amino acids (L-lysine, L-aspartic acid and L-glycine) and increased number of binding sites for a fluorescent probe, 1-anilinonaphthalene-8-sulfonate.     

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.     

Enzymatic characterisation of high-palmitic acid sunflower (Helianthus annuus L.) mutants

Two high-palmitic acid sunflower (Helianthus annuus L.) mutants, CAS-5 and CAS-12, have been biochemically characterised. The enzymatic activities found to be responsible for the mutant characteristics are β-keto-acyl-acyl carrier protein synthetase II (KASII; EC 2.3.1.41) and acyl-acyl carrier protein thioesterase (EC 3.1.2.14). Our data suggest that the high-palmitic acid phenotype observed in both mutant lines is due to the combined effect of a lower KASII activity and a higher thioesterase activity with respect to palmitoyl-acyl carrier protein (16:0-ACP). The level of the latter enzyme appeared to be insufficient to hydrolyse the produced 16:0-ACP completely. As a consequence of this, three new fatty acids appear: palmitoleic acid (16:1 Δ9), asclepic acid (18:1 Δ11), and palmitolinoleic acid (16:2 Δ9 Δ12). These fatty acids should be synthesised from palmitoyl-ACP or a derivative by the action of the stearoyl-ACP desaturase, fatty acid synthetase II and oleoyl-phosphatidylcholine desaturase, respectively. Received: 11 July 1998 / Accepted: 10 October 1998     

The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast

Candida lipolytica yeast was grown batchwise on two different carbon sources, glucose and n-hexadecane. Free ceramides were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography. Their composition, after acid methanolysis, was analysed by gas-liquid chromatography. The ceramide content accounted for 2.6% of the total cell lipids in hexadecane-grown cells, which was 1.5 times higher than in glucose-grown cells. The fatty acid composition of ceramides was characterized by the predominance of fatty acids shorter than 20 carbon atoms and by high concentrations of fatty acids with 16 carbon atoms after growth on both carbon sources. The dominant fatty acid was hydroxylated 16:0 in the glucose-grown cells and 16:0 in the hexadecane-grown cells. The striking finding was the low degree of fatty acid hydroxylation and relatively high proportion of odd-numbered fatty acids in ceramide of the n-hexadecane-grown cells. The ceramides contained an unusual long-chain base composition. In hexadecane-grown cells more than 60% of the long-chain bases were C₁₉ phytosphingosine. In glucose-grown cells more than one-half of the total long-chain bases were tetrahydroxy bases, 4,5-dihydroxysphinganine and 4,5-dihydroxyeicosasphinganine. Received: 20 April 1998 / Received revision: 10 July 1998 / Accepted: 29 July 1998     

The glucose dehydrogenase-mediated energization of Acinetobacter calcoaceticus as a tool for evaluating its susceptibility to,and defence against,hazardous chemicals

Cells of Acinetobacter calcoaceticus 69-V could be energized by glucose oxidation after the growth on acetate, ethanol, hexanol and benzoate. The velocities of glucose oxidation-driven ATP syntheses were relatively constant in the range from pH 5.4 to 7.5. With decreasing pH values (7.0, 6.0, 5.4) ATP synthesis was inhibited more strongly by the action of 2,4-dinitrophenol and at the same pH value glucose oxidation was nearly unimpaired or inhibited more weakly. This finding is expressed by a decrease of the P/O ratios, indicating the uncoupling of the electron-transport phosphorylation by 2,4-dinitrophenol. The sensitivity towards this uncoupling effect was higher in ethanol-grown cells of Acinetobacter calcoaceticus 69-V than in hexanol- or acetate-grown cells. This increase in sensitivity was accompanied by a decrease of the ratio of saturated (mainly C16:0) to unsaturated (C16:1, C18:1) fatty acids in ethanol-grown cells compared with hexanol-grown ones. The knowledge of such differences in the susceptibility and its molecular background, e.g. possible substrate-induced changes of the fatty acid composition of the cytoplasmic membranes, should help elucidate mechanisms of poisoning by membrane-active hazardous chemicals and develop defence strategies.     

Co-expression of the borage Δ6 desaturase and the Arabidopsis Δ15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean

Two relatively rare fatty acids, γ-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, α-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Δ⁶ desaturase and an Arabidopsis Δ¹⁵ desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Δ⁶ desaturase event with the Δ¹⁵ desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean β-conglycinin promoter. Soybean events that carried only the Δ¹⁵ desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids.     

植物脂肪酸的生物合成与基因工程

植物脂肪酸既具重要生理功能,又有巨大食用和工业价值。其生物合成途径较为复杂,涉及乙酰_CoA羧化酶、脂肪酸合成酶、脂肪酸去饱和酶和脂肪酸延长酶等一系列酶。近年来,对脂肪酸生物合成途径进行了大量研究,克隆出许多相关基因,初步阐明了脂肪酸合成规律,并在此基础上开展了利用基因工程技术调控脂肪酸合成研究,取得可喜进展。本文详细介绍了植物饱和脂肪酸、不饱和脂肪酸和超长链脂肪酸的生物合成与基因工程研究的新结果。     

A transketolase mutant of Corynebacterium glutamicum

A transketolase mutant was first isolated from Corynebacterium glutamicum, an organism of industrial importance. The mutant strain exhibited an absolute requirement for shikimic acid or the aromatic amino acids and vitamins for growth, and also failed to grow on ribose or gluconic acid as sole carbon source, even with the aromatic supplement. All of these defective properties were fully restored in spontaneous revertants, indicating the existence of a single transketolase in C. glutamicum that was indispensable both for aromatic biosynthesis and for utilization of these carbohydrates in vivo. The transketolase mutant accumulated ribulose extracellularly when cultivated in glucose medium with shikimic acid, but no ribose was detected. Received: 10 April 1998 / Received revision: 26 May 1998 / Accepted: 14 June 1998