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1.
In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Δ9Z-C16:1) and oleic acid (Δ9Z-C18:1), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C16:0) and stearic acid (C18:0), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Δ9 and Δ11) and substrate chain-length preferences (i.e., C16:0 and C18:0); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Δ11Z-C18:1), whereas neither Δ11Z-C16:1 nor palmitoleic acid (Δ9Z-C16:1) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.  相似文献   

2.
3.
In contrast to 16:3 plants like rapeseed (Brassica napus), which contain alpha-linolenic acid (18:3(Delta9,12,15)) and hexadecatrienoic acid (16:3(Delta7,10,13)) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5(Delta5,8,11,14,17)) and a different isomer of hexadecatrienoic acid (16:3(Delta6,9,12)). In this report, we describe the characterization of two cDNAs having sequence homology to Delta12-fatty acid desaturases from higher plants. These cDNAs were shown to code for a microsomal and a plastidial Delta12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively. Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6). The microsomal desaturases were similarly specific for oleic acid (18:1(Delta9)), suggesting that PtFAD2 is involved in the biosynthesis of EPA. In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed. Although the rapeseed plastidial desaturase showed high activity toward the omega9-fatty acids 18:1(Delta9) and 16:1(Delta7), in line with the fatty acid composition of rapeseed leaves, the enzyme of P. tricornutum was highly specific for 16:1(Delta9). Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P. tricornutum (16:3(Delta6,9,12)) is of plastidial origin.  相似文献   

4.
Transgenic tobacco plants O9 and T16 expressing the yeast acyl-CoA Delta9 desaturase and an insect acyl-CoA Delta11 desaturase, respectively, displayed altered profiles of fatty acids compared to wild-type tobacco plants and marked increases in cis-3-hexenal, a major leaf volatile derived from alpha-linolenic acid (18:3). As expected, O9 and T16 plants had increased levels of the major unsaturated fatty acid products formed by the transgenic desaturases they expressed, viz., palmitoleic acid (16:1(Delta9)) and palmitvaccenic acid (16:1(Delta11)), respectively. In addition, levels of 18:3 lipid declined slightly and the pool of free 18:3, which accounts for about 30% of free fatty acids in wild-type plants, disappeared completely in both transgenics. Both O9 and T16 plants were found to have a two-fold increase in 13-lipoxygenase (13-LOX) activity, which catalyzes the first of two steps leading to hexenal production from 18:3. In O9 and T16 plants, the activity of 9-lipoxygenase and hydroperoxide lyase, the latter catalyzing the formation of cis-3-hexenal from alpha-linolenic acid hydroperoxide, was significantly different from that of the wild-type plants. Although 16:1(Delta9) and 16:1(Delta11) had no direct effects on 13-LOX activity in vitro, cis-3-hexenal production increased in tobacco leaves treated with these fatty acids, suggesting that they may act in vivo by stimulating 13-LOX gene expression.  相似文献   

5.
When the cells of Saccharomyces cerevisiae are exposed to high concentration of ethanol, the content of oleic acid (C18:1n-9) increased as the initial concentration of ethanol increased. Based on this observation, we attempted to confer ethanol tolerance to S. cerevisiae by manipulating fatty acid composition of the cells. Rather than altering OLE1 expression [the desaturase making both C16:1n-7 (palmitoleic acid) and C18:1n-9], we introduced elongase genes. Introduction of rat elongase 1 gene (rELO1) into S. cerevisiae gave cis-vaccenic acid (cis-C18:1n-7) by conversion from C16:1n-7, and the increase in this C18:1 fatty acid did not confer ethanol tolerance to the cells. On the other hand, the introduction of rat elongase 2 gene (rELO2), which elongates C16:0 to C18:0, drastically increased C18:1n-9 content, and the cells acquired ethanol tolerance, emphasizing the specific role of C18:1n-9. Furthermore, the transformant of rELO2 also conferred tolerance to n-butanol, n-propanol, and 2-propanol.  相似文献   

6.
Brassica juncea plants transformed with the Arabidopsis ADS1 gene, which encodes a plant homologue of the mammalian and yeast acyl-CoA Delta9 desaturases and the cyanobateria acyl-lipid Delta9 desaturase, were found to have a statistically significant decrease in the level of saturated fatty acids in seeds. The decrease in the level of saturated fatty acids is largely attributable to decreases in palmitic acid (16:0) and stearic acid (18:0), although arachidic acid (20:0), behenic acid (22:0) and lignoceric acid (24:0) were also decreased in the transgenic seeds compared to the negative control lines. As a result, the level of oleic acid (18:1) was slightly increased in the transgenic seed lines compared to the non-transformed controls. However, a decrease in saturated fatty acid is not always accompanied by the corresponding increase in mono-unsaturated fatty acids. For example, palmitoleic acid (16:1), gondoic acid (20:1) and nervonic acid (24:1) were all found to be decreased in transgenic seeds. The levels of linoleic acid (18:2) and linolenic acid (18:3) were also notably changed in the transgenic lines compared to the controls. The present study provides preliminary experimental data suggesting that the Arabidopsis ADS1 encodes a fatty acid Delta9 desaturase and could be useful in genetic engineering for modifying the level of saturated fatty acids in oilseed crops. However, the effect of ADS1 gene expression on seed oil fatty acid composition is beyond the changes of total saturated and mono-unsaturated fatty acids, which suggests a complex mechanism is involved in the regulation of fatty acid metabolism.  相似文献   

7.
絮凝特性对自絮凝颗粒酵母耐酒精能力的影响及作用机制   总被引:7,自引:2,他引:5  
首次报道絮凝特性提高酵母菌耐酒精能力的现象及其机制。融合株SPSC与其两亲本粟酒裂殖酵母变异株和酿酒酵母变异株于 30℃经 18% (V/V)酒精冲击 7h的存活率分别为 52%、37%和 9%。细胞膜磷脂脂肪酸组成分析表明 ,两絮凝酵母 (融合株SPSC和粟酒裂殖酵母变异株 )的棕榈酸含量均约为非絮凝酵母 (酿酒酵母变异株 )的两倍 ,而棕榈油酸和油酸的含量明显低于后者。研究表明 ,当两絮凝酵母在培养中由于柠檬酸钠的作用 (抑制絮凝体的形成 )而以游离细胞生长存在时 ,其细胞膜磷脂棕榈酸含量显著下降 ,而棕榈油酸和油酸的含量明显增加 ,结果细胞膜磷脂脂肪酸组成特点与酿酒酵母变异株相似 ;而且实验表明 ,絮凝特性的消失伴随菌体耐酒精能力的急剧下降 ,变得与酿酒酵母变异株的水平相当。这些结果提示两絮凝酵母具有较强的耐酒精能力与其细胞膜磷脂脂肪酸组成中含有更高比例的棕榈酸有关。  相似文献   

8.
9.
The methylotrophic yeast Pichia pastoris GS115, a widely used strain in production of various heterologous proteins, especially membrane-bound enzymes, can also produce linoleic and linolenic acids, which indicates the existence of membrane-bound Delta12 and Delta15-fatty acid desaturases. This paper describes the cloning and functional characterization of a novel Delta12-fatty acid desaturase gene from this methylotrophic yeast. The open reading frame of the gene (named Pp-FAD12) is 1263 bp in size and encodes a 420-amino-acid peptide. The deduced Pp-FAD12 protein shows high identity (50-67%) with Delta12-fatty acid desaturases from other fungi. It also shows a high identity (57%) with Delta15-fatty acid desaturase (named Sk-FAD15) from Saccharomyces kluyveri. Expression of Pp-FAD12 in polyunsaturated fatty acids non-producing yeast Saccharomyces cerevisiae demonstrated that its product converted oleic acid (18 : 1) to linoleic acid (18 : 2). This result suggests that Pp-FAD12 encodes a novel Delta12-fatty acid desaturase in P. pastoris GS115. This is the first report about the cloning and functional characterization of Delta12-fatty acid desaturase gene in methylotrophic yeast.  相似文献   

10.
11.
Based on the sequence information for delta 9-desaturase genes (from rat, mouse and yeast), which are involved in the desaturation of palmitic acid and stearic acid to palmitoleic acid and oleic acid, respectively, the corresponding cDNA and genomic gene were cloned from the fungal strain, Mortierella alpina 1S-4, which industrially produces arachidonic acid. There was a cytochrome b5-like domain linked to the carboxyl terminus of this Mortierella desaturase, as also seen in the yeast delta 9-desaturase. The Mortierella delta 9-desaturase genomic gene had only one intron, in which a novel phenomenon was observed: there was a GC-end at the 5'-terminus instead of a GT-end that is, in general, found in introns of eukaryotic genes. The full-length cDNA clone was expressed under the control of an amyB promoter in a filamentous fungus, Aspergillus oryzae, resulting in drastic changes in the fatty acid composition in the transformant cells; the contents of palmitoleic acid (16:1) and oleic acid (18:1) increased significantly, with accompanying decreases in palmitic acid (16:0) and stearic acid (18:0). These changes were controlled by the addition of maltose as a carbon source to the medium. Also, the expression of the gene caused a significant change in the lipid composition in the Aspergillus transformant. Genomic Southern blot analysis of the transformant with the Mortierella delta 9-desaturase gene as a probe confirmed the integration of this gene into the genome of A. oryzae.  相似文献   

12.
Meesapyodsuk D  Qiu X 《Plant physiology》2008,147(3):1325-1333
Claviceps purpurea, a fungal pathogen responsible for ergot diseases in many agriculturally important cereal crops, produces high levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. It has been believed for many years that the biosynthesis of this fatty acid in C. purpurea involves a hydration process with linoleic acid as the substrate. Using degenerate polymerase chain reaction, we cloned a gene from the sclerotia encoding an enzyme (CpFAH) that has high sequence similarity to the C. purpurea oleate desaturase, but only low similarity to plant oleate hydroxylases. Functional analysis of CpFAH in yeast (Saccharomyces cerevisiae) indicated it acted predominantly as a hydroxylase, introducing hydroxyl groups at the 12-position of oleic acid and palmitoleic acid. As well, it showed Delta(12) desaturase activities on 16C and 18C monounsaturated fatty acids and, to a much lesser extent, omega(3) desaturase activities on ricinoleic acid. Heterologous expression of CpFAH under the guidance of a seed-specific promoter in Arabidopsis (Arabidopsis thaliana) wild-type and mutant (fad2/fae1) plants resulted in the accumulation of relatively higher levels of hydroxyl fatty acids in seeds. These data indicate that the biosynthesis of ricinoleic acid in C. purpurea is catalyzed by the fungal desaturase-like hydroxylase, and CpFAH, the first Delta(12) oleate hydroxylase of nonplant origin, is a good candidate for the transgenic production of hydroxyl fatty acids in oilseed crops.  相似文献   

13.
The effect of change in unsaturated fatty acid composition on ethanol tolerance in Saccharomyces cerevisiae overexpressing ScOLE1 (?9 fatty acid desaturase gene of S. cerevisiae), CaFAD2 (?12 fatty acid desaturase gene of Candida albicans), or CaFAD3 (ω3 fatty acid desaturase gene of C. albicans) was examined. ScOLE1 over-expression increased the total unsaturated fatty acid content and enhanced ethanol tolerance, compared with a control strain. In contrast, overexpression of CaFAD2 and CaFAD3, which led to production of linoleic acid (18:2) and α-linolenic acid (18:3), respectively, neither changed total unsaturated fatty acids nor enhanced ethanol tolerance. The total unsaturated fatty acid content rather than the degree of unsaturation is thus an important factor for ethanol tolerance.  相似文献   

14.
15.
Dimorphecolic acid (9-OH-18:2Delta(10)(trans)(,12)(trans)) is the major fatty acid of seeds of Dimorphotheca species. This fatty acid contains structural features that are not typically found in plant fatty acids, including a C-9 hydroxyl group, Delta(10),Delta(12)-conjugated double bonds, and trans-Delta(12) unsaturation. Expressed sequence tag analysis was conducted to determine the biosynthetic origin of dimorphecolic acid. cDNAs for two divergent forms of Delta(12)-oleic acid desaturase, designated DsFAD2-1 and Ds-FAD2-2, were identified among expressed sequence tags generated from developing Dimorphotheca sinuata seeds. Expression of DsFAD2-1 in Saccharomyces cerevisiae and soybean somatic embryos resulted in the accumulation of the trans-Delta(12) isomer of linoleic acid (18: 2Delta(9)(cis)(,12)(trans)) rather than the more typical cis-Delta(12) isomer. When co-expressed with DsFAD2-1 in soybean embryos or yeast, DsFAD2-2 converted 18:2Delta(9)(cis)(,12)(trans) into dimorphecolic acid. When DsFAD2-2 was expressed alone in soybean embryos or together with a typical cis-Delta(12)-oleic acid desaturase in yeast, trace amounts of the cis-Delta(12) isomer of dimorphecolic acid (9-OH-18:2Delta(10)(trans,)(12)(cis)) were formed from DsFAD2-2 activity with cis-Delta(12)-linoleic acid [corrected]. These results indicate that DsFAD2-2 catalyzes the conversion of the Delta(9) double bond of linoleic acid into a C-9 hydroxyl group and Delta(10)(trans) double bond and displays a substrate preference for the trans-Delta(12), rather than the cis-Delta(12), isomer of linoleic acid. Overall these data are consistent with a biosynthetic pathway of dimorphecolic acid involving the concerted activities of DsFAD2-1 and DsFAD2-2. The evolution of two divergent Delta(12)-oleic acid desaturases for the biosynthesis of an unusual fatty acid is unprecedented in plants.  相似文献   

16.
ABSTRACT A cDNA encoding pheromone Δ9 acyl-CoA desaturase, Slit KPSE was isolated from sex pheromone gland of the tobacco cutworm, Spodoptera litura which uses a diene unsaturated fatty acid (UFA) derivative, Z9E11-14 : 2 as a major pheromone component. The fulllength open reading frame coding region of Slit KPSE was inserted in a yeast shuttle vector, YEpOLEX, and two kinds of yeast ( Saccharomyces cerevisiae ) mutant strains were transformed with the recombinant vector. In the desaturase-deficient ole 1 strain, Slit KPSE expressed a complementary enzyme producing two kinds of diene UFAs, more 9–16 : 1 and less 9–18 : 1 at a ratio of 1 : 0.74 exhibiting a typical functional characteristics as one of the pheromone Δ9 acyl-CoA desaturase lineage group, KPSE, but no Δ9 14C monoene was detectable because of too small amount of 14C saturated fatty acid precursor to be reliably used by Slit KPSE in the transformed cells. However, the another transformed yeast strain elo 1 which is deficient of elongase 1, an enzyme converting 14C to 16C hydrocarbon substrate, was supplemented with some myristic acid (14 : 0) in the medium, and produced a significant amount of 9–14 : 1 in due to a much enhanced level of the 14C substrate suggesting that Slit KPSE may be responsible for making the Δ9 double bond on the diene pheromone component.  相似文献   

17.
The free-living soil protozoon Acanthamoeba castellanii synthesizes a range of polyunsaturated fatty acids, the balance of which can be altered by environmental changes. We have isolated and functionally characterized in yeast a microsomal desaturase from A. castellanii, which catalyzes the sequential conversion of C(16) and C(18) Delta9-monounsaturated fatty acids to di- and tri-unsaturated forms. In the case of C(16) substrates, this bifunctional A. castellanii Delta12,Delta15-desaturase generated a highly unusual fatty acid, hexadecatrienoic acid (16:3Delta(9,12,15)(n-1)). The identification of a desaturase, which can catalyze the insertion of a double bond between the terminal two carbons of a fatty acid represents a new addition to desaturase functionality and plasticity. We have also co-expressed in yeast the A. castellanii bifunctional Delta12,Delta15-desaturase with a microsomal Delta6-desaturase, resulting in the synthesis of the highly unsaturated C(16) fatty acid hexadecatetraenoic acid (16:4Delta(6,9,12,15)(n-1)), previously only reported in marine microorganisms. Our work therefore demonstrates the feasibility of the heterologous synthesis of polyunsaturated fatty acids of the n-1 series. The presence of a bifunctional Delta12,Delta15-desaturase in A. castellanii is also considered with reference to the evolution of desaturases and the lineage of this protist.  相似文献   

18.
We have cloned a Caenorhabditis elegans cDNA encoding a Delta12 fatty acid desaturase and demonstrated its activity by heterologous expression in Saccharomyces cerevisiae. The predicted protein is highly homologous both to the cloned plant genes with similar function and to the published sequence of the C. elegans omega-3 fatty acid desaturase. In addition, it conforms to the structural constraints expected of a membrane-bound fatty acid desaturase including the canonical histidine-rich regions. This is the first report of a cloned animal Delta(12) desaturase gene. Expression of this cDNA in yeast resulted in the accumulation of 16:2 and 18:2 (linoleic) acids. The increase of membrane fluidity brought about by this change in unsaturation was measured. The production of polyunsaturated fatty acids in yeast cells and the concomitant increase in membrane fluidity was correlated with a modest increase in growth rate at low temperature and with increased resistance to ethanol and oxidative stress.  相似文献   

19.
20.
Two cDNAs encoding acyl-CoA Z9-desaturase from the fat body and Z10-desaturase from the pheromone gland of the greenhead leafroller moth, Planotortrix octo, were obtained by RACE PCR. The Z9-desaturase (Pocto-Z9) cDNA spans 2291 nt with an ORF encoding a 352 amino-acid protein, which has 65% identity to Trichoplusia ni Delta 9 desaturase (Tni-Z9). The Z10-desaturase (Pocto-Z10) cDNA spans 2777 nt with an ORF encoding a protein with 356 amino acids. Pocto-Z10 shows lower identity to Pocto-Z9 and Tni-Z9 (48 and 46%, respectively) and relatively higher identity to the Delta 11 desaturases of T. ni and Helicoverpa zea (57 and 56%, respectively). The ORFs of these two P. octo cDNAs were constructed into an expression vector, YEpOLEX, that complemented the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient ole1 strain of Saccharomyces cerevisiae. Expression of Pocto-Z9 produced a 5:2 ratio of Z9-16 and Z9-18 acids, with minor amounts (<4%) of Z9-14, Z9-15, and Z9-17 acids. Pocto-Z10 was successfully expressed in the YEpOLEX system when complemented with Z11-18:Me, and the major desaturase product proved to be Z10-16:Acid. The results confirm the regio- and stereo-selectivity of this unusual Delta 10 desaturase.  相似文献   

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