Expression of a novel 90-kDa protein, Lsd90, involved in the metabolism of very long-chain fatty acid-containing phospholipids in a mitosis-defective fission yeast mutant

The fission yeast lsd1/fas2 strain carries a temperature-sensitive mutation of the fatty-acid-synthase alpha-subunit, exhibiting an aberrant mitosis lsd phenotype, with accumulation of very-long-chain fatty-acid-containing phospholipid (VLCFA-PL). A novel 90-kDa protein, Lsd90 (SPBC16E9.16c), was found to be newly expressed in small particle-like structures in lsd1/fas2 cells under restrictive conditions. Two mismatches leading to a double frame shift were found between the sequences of the lsd90(+) gene registered in the genomic database and the sequences determined experimentally at the amino acid, cDNA and genomic DNA levels. Unexpectedly, overexpression and disruption of the lsd90(+) gene in either lsd1/fas2 or wild-type cells did not affect either cell growth or expression of the lsd phenotype. The amounts of VLCFA-PL that accumulated in lsd90-overexpressing lsd1/fas2 cells were significantly lower than those in lsd1/fas2 cells, suggesting the involvement of Lsd90 in the metabolism of VLCFA-PL.     

Aberrant mitosis in fission yeast mutants defective in fatty acid synthetase and acetyl CoA carboxylase

Two fission yeast temperature-sensitive mutants, cut6 and lsd1, show a defect in nuclear division. The daughter nuclei differ dramatically in size (the phenotype designated lsd, large and small daughter). Fluorescence in situ hybridization (FISH) revealed that sister chromatids were separated in the lsd cells, but appeared highly compact in one of the two daughter nuclei. EM showed asymmetric nuclear elongation followed by unequal separation of nonchromosomal nuclear structures in these mutant nuclei. The small nuclei lacked electron- dense nuclear materials and contained highly compacted chromatin. The cut6+ and lsd1+ genes are essential for viability and encode, respectively, acetyl CoA carboxylase and fatty acid synthetase, the key enzymes for fatty acid synthesis. Gene disruption of lsd1+ led to the lsd phenotype. Palmitate in medium fully suppressed the phenotypes of lsd1. Cerulenin, an inhibitor for fatty acid synthesis, produced the lsd phenotype in wild type. The drug caused cell inviability during mitosis but not during the G2-arrest induced by the cdc25 mutation. A reduced level of fatty acid thus led to impaired separation of non- chromosomal nuclear components. We propose that fatty acid is directly or indirectly required for separating the mother nucleus into two equal daughters.     

A role for unsaturated fatty acids in mitochondrial movement and inheritance

Yeast cells with the mdm2 mutation display temperature-sensitive growth and defective intracellular mitochondrial movement at the non-permissive temperature. The latter phenotype includes both an absence of mitochondrial transfer into daughter buds of mitotically growing cells and an aberrant mitochondrial distribution in cells exposed to mating pheromone. The wild-type MDM2 gene was cloned by complementation, and DNA sequence analysis revealed a large open reading frame encoding a putative protein of 58.4 kD. The predicted protein sequence is identical to that reported for the yeast OLE1 gene encoding fatty acid desaturase. Unsaturated fatty acid levels are substantially decreased in mdm2 cells after a prolonged incubation at the non-permissive temperature. The addition of oleic acid complements the temperature-sensitive growth and mitochondrial distribution defects of the mutant cells. These results indicate that mdm2 is a temperature-sensitive allele of OLE1 and demonstrate an essential role for unsaturated fatty acids in mitochondrial movement and inheritance.     

Molecular cloning of fatty acid synthetase genes from Saccharomyces cerevisiae

Fatty acid synthetase from Saccharomyces cerevisiae is a multifunctional enzyme which catalyzes the synthesis of long chain fatty acids from acetyl- and malonyl-CoA. The enzyme is composed of two nonidentical subunits, alpha (Mr = 212,000) and beta (Mr = 203,000), which are coded for by two unlinked genes FAS2 and FAS1, respectively. Individual yeast strains containing mutations in either of the FAS genes were transformed with a bank of yeast DNA sequences in the vector YEp13. Plasmids YEpFAS1 and YEpFAS2 were selected by their ability to complement the fas1 or fas2 mutations, respectively. Additionally, we utilized an immunologic screening of a second yeast DNA bank and selected two clones 33F1 and 102B5 which produce antigenically reactive material to anti-yeast fatty acid synthetase antibodies. Through Southern hybridization experiments and restriction endonuclease mapping, a region of 5.3 kilobase pairs of 33F1 was shown to be homologous with YEpFAS1, and a span of 3.4 kilobase pairs of 102B5 was homologous with YEpFAS2. These experiments identify the yeast DNA sequences cloned into 33F1 as originating from the FAS1 gene and those DNA sequences in 102B5, from the FAS2 gene.     

Lipid droplet formation protects against gluco/lipotoxicity in Candida parapsilosis: An essential role of fatty acid desaturase Ole1

Elevated levels of glucose and lipids can result in cellular dysfunction in eukaryotic cells ranging from Saccharomyces cerevisiae yeasts to human cells. Moreover, glucotoxicity and lipotoxicity can cause cell death, although the mechanism(s) for lethality is unclear. In the present study, we utilized Candida parapsilosis fatty acid desaturase (OLE1) and fatty acid synthase (FAS2) gene deletion mutants and wild-type (WT) yeast cells to unravel the relationship to glucose and lipid induced cell death in eukaryotic cells. Incubation of WT yeast cells with glucose led to the rapid accumulation of lipid droplets, whereas lipid droplet formation was severely impaired in yeast cells with deletion of OLE1 (ole1Δ/Δ) or FAS2 (fas2Δ/Δ). Interestingly, ole1Δ/Δ yeast cells died within hours in a 1% glucose medium without fatty acid supplementation, whereas the WT or fas2Δ/Δ yeast cells did not. In glucose medium, ole1Δ/Δ yeast cells accumulated saturated fatty acids, while fas2Δ/Δ did not. Addition of saturated fatty acids (e.g., palmitic acid) enhanced ole1Δ/Δ yeast cell death, whereas the addition of unsaturated fatty acids (e.g., oleic or palmitoleic acid) rescued cell death. Furthermore, palmitic acid and glucose medium induced apopotic cell death in ole1Δ/Δ yeast cells, which was dependent on mitochondrial function. Thus, our results show that glucotoxicity is directly linked to lipotoxicity, which we demonstrate is mediated by mitochondrial function.     

解脂耶罗维亚酵母工程菌合成超长链脂肪酸及温度的影响

[背景]解脂耶罗维亚酵母属于产油微生物,大量研究表明该酵母能够高产长链脂肪酸和油脂,但是应用该酵母合成超长链脂肪酸仍待研究。[目的]工程化解脂耶罗维亚酵母合成高值超长链脂肪酸,并研究温度对脂肪酸合成的影响。[方法]合成密码子优化的拟南芥(Arabidopsis thaliana)延长酶基因AtFAE1、非洲芥菜(Brassica tournefortii)延长酶基因BtFAE1和碎米芥属植物Cardamine graeca的延长酶基因CgKCS,分别构建质粒pYLEX1-AtFAE1、pYLEX1-BtFAE1、pYLEX1-CgKCS和pYLEX1-AtFAE1-BtFAE1-CgKCS。以解脂耶罗维亚酵母菌株Po1g为宿主,通过化学法分别转化上述4个质粒,获得工程菌Po1g-AtFAE1、Po1g-BtFAE1、Po1g-CgKCS和Po1g-AtFAE1-BtFAE1-CgKCS,比较评价超长链脂肪酸的合成。在此基础上,过表达内源二酯酰甘油酰基转移酶基因DGAT1(diacylglycerol acyltransferase)提高产油量,并研究温度对生物量、产油、脂肪酸组成的影响。[结果]在解脂耶罗维亚酵母中3个延长酶的延长能力明显不同,AtFAE1主要催化C20:1脂肪酸的合成,BtFAE1更有利于芥酸(C22:1)的合成,而CgKCS能够催化合成神经酸(C24:1),但是三者共表达并未提高神经酸产量。在表达CgKCS基因的菌株中过表达DGAT1基因,细胞油脂含量提高50%。温度实验表明,低温有利于解脂耶罗维亚酵母合成不饱和脂肪酸,反之,高温利于其合成饱和脂肪酸。[结论]脂肪酸延长酶基因CgKCS可直接催化C18:1脂肪酸合成C24:1的超长链脂肪酸,并且通过优化培养温度可提高不饱和脂肪酸的合成。本研究为构建超长链脂肪酸细胞工厂以及发酵优化提供理论和技术参考。     

Only one of the five Ralstonia solanacearum long-chain 3-ketoacyl-acyl carrier protein synthase homologues functions in fatty acid synthesis

Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C(16:0)), palmitoleic (C(16:1)) and cis-vaccenic (C(18:1)) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I.     

Beta-hydroxydecanoyl thio ester dehydrase does not catalyze a rate-limiting step in Escherichia coli unsaturated fatty acid synthesis

The intracellular level of beta-hydroxydecanoyl thio ester dehydrase, the product of the fabA gene of Escherichia coli, was increased by isolation of a putative promotor mutant (termed fabAup) or by molecular cloning of the wild-type fabA gene into plasmid pBR322. The fabAup and plasmid-carrying strains overproduced dehydrase by about 15- and 10-fold, respectively. The phospholipids of all strains that overproduced the dehydrase contained significantly higher levels of saturated fatty acids than isogenic strains producing a normal level of dehydrase. No increased levels of unsaturated fatty acids were observed. This result indicates that, although the dehydrase is required for unsaturated fatty acid synthesis, the level of dehydrase activity in wild-type cells does not limit the rate of unsaturated fatty acid synthesis. The introduction of a plasmid carrying the structural gene for beta-ketoacyl acyl carrier protein synthase I into a fabAup strain overcame the effect of dehydrase overproduction on fatty acid composition.     

Identification and characterization of a very long-chain fatty acid elongase gene in the methylotrophic yeast, Hansenula polymorpha

To understand the biosynthetic network of fatty acids in the methylotrophic yeast Hansenula polymorpha, which is able to produce poly-unsaturated fatty acids, we have attempted to identify genes encoding fatty acid elongase. Here we have characterized HpELO1, a fatty acid elongase gene encoding a 319-amino-acid protein containing five predicted membrane-spanning regions that is conserved throughout the yeast Elo protein family. Phylogenetic analysis of the deduced amino acid sequence suggests that HpELO1 is an ortholog of the Saccharomyces cerevisiae ELO3 gene that is involved in the elongation of very long-chain fatty acids (VLCFAs). In the fatty acid profile of the Hpelo1Delta disruptant by gas chromatography/mass spectrometry, the amount of C24:0 and C26:0 decreased to undetectable levels, whereas there was a large accumulation of C22:0, suggesting that the HpELO1 is involved in the elongation of VLCFAs and is essential for the production of C24:0. Expression of HpELO1 suppressed the lethality of the S. cerevisiae elo2Delta elo3Delta double disruptant and recovered the synthesis of VLCFAs. Similar to the S. cerevisiae elo3Delta strain, the Hpelo1Delta disruptant exhibited the extraordinary growth sensitivity to fumonisin B(1), a ceramide synthase inhibitor. Furthermore, cells of the Hpelo1Delta disruptant were more sensitive to Zymolyase and more flocculent than the wild-type cells, clumping together and falling rapidly out of suspension, suggesting that the Hpelo1Delta mutation causes changes in cell wall composition and structure.     

Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.     

Coordinate genetic control of yeast fatty acid synthase genes FAS1 and FAS2 by an upstream activation site common to genes involved in membrane lipid biosynthesis.

A systematic search for upstream controlling elements necessary for efficient expression of the yeast fatty acid synthase genes FAS1 and FAS2 revealed identical activation sites, UASFAS, in front of both FAS genes. The individual element confers, in a heterologous yeast test system, an approximately 40-fold stimulation of basal gene expression. The UASFAS motifs identified have the consensus sequence TYTTCACATGY and function in either orientation. The same sequence motif is found in the upstream regions of all so far characterized yeast genes encoding enzymes of phospholipid biosynthesis. In gel retardation assays, a protein factor, Fbf1 (FAS binding factor), was identified which interacted with UASFAS. The UASFAS motif proved to be an inositol/choline responsive element (ICRE) conferring strict repression by exogenous inositol and choline on a heterologous reporter gene. Its core sequence perfectly matches the CANNTG motif typical of basic helix-loop-helix DNA-binding proteins. In contrast to the individual UASFAS element, the intact yeast FAS promoters are not significantly influenced by inositol and choline, and thus allow nearly constitutive fatty acid synthase production. Available evidence suggests that additional cis- and trans-acting elements, other than UASFAS and Fbf1, are involved in this constitutive FAS gene expression.     

Dietary fatty acids modulate liver mitochondrial cardiolipin content and its fatty acid composition in rats with non alcoholic fatty liver disease

No data are reported on changes in mitochondrial membrane phospholipids in non-alcoholic fatty liver disease. We determined the content of mitochondrial membrane phospholipids from rats with non alcoholic liver steatosis, with a particular attention for cardiolipin (CL) content and its fatty acid composition, and their relation with the activity of the mitochondrial respiratory chain complexes. Different dietary fatty acid patterns leading to steatosis were explored. With high-fat diet, moderate macrosteatosis was observed and the liver mitochondrial phospholipid class distribution and CL fatty acids composition were modified. Indeed, both CL content and its C18:2n-6 content were increased with liver steatosis. Moreover, mitochondrial ATP synthase activity was positively correlated to the total CL content in liver phospholipid and to CL C18:2n-6 content while other complexes activity were negatively correlated to total CL content and/or CL C18:2n-6 content of liver mitochondria. The lard-rich diet increased liver CL synthase gene expression while the fish oil-rich diet increased the (n-3) polyunsaturated fatty acids content in CL. Thus, the diet may be a significant determinant of both the phospholipid class content and the fatty acid composition of liver mitochondrial membrane, and the activities of some of the respiratory chain complex enzymes may be influenced by dietary lipid amount in particular via modification of the CL content and fatty acid composition in phospholipid.     

Mapping of a complex gene locus coding for part of the Saccharomyces cerevisiae fatty acid synthetase multienzyme complex

Summary Using two different experimental approaches—UV induced mitotic recombination and meiotic segregation—a fatty acid synthetase gene locus has been mapped on chromosome Fragment V of the Saccharomyces cerevisiae genetic map. This locus has been tentatively designated as fas1AB since it is a complex locus coding for at least two different fatty acid synthetase component enzymes, namely the -hydroxyacid dehydratase and the enoyl reductase. According to the meiotic segregation patterns obtained, fas1AB is 41.6 centimorgans from ura1 and 35.7 centimorgans from trp3. Furthermore, the same criteria of mitotic sectoring and meiotic segregation indicate that the second known fatty acid synthetase gene cluster in yeast is genetically unlinked to fas1AB or to any other of the known genetic loci on Fragment V.     

Elevated levels of glyoxylate shunt enzymes in Escherichia coli strains constitutive for fatty acid degradation.

Mutants of Escherichia coli K-12 constitutive for the synthesis of the enzymes of fatty acid degradation (fadR) have elevated levels of the glyoxylate shunt enzymes, isocitrate lyase and malate synthase. A temperature-sensitive fadR strain has high levels of glyoxylate shunt enzymes when grown at elevated temperatures but has low, inducible levels of glyoxylate shunt enzymes when grown at low temperatures. The increased activity of glyoxylate shunt enzymes did not appear to be due to the degradation of intracellular fatty acids in fadR strains or differences in allosteric effectors in fadR versus fadR+ strains. These studies suggest that the fadR gene product may be involved in the regulation of the glyoxylate operon.