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 functional characterization of two cDNAs encoding NADPH-dependent 3-ketoacyl-CoA reductased from developing cotton fibers

Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulated during early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA reductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCRI and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed during the cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2 showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAs were cloned and expressed in yeast haploid ybr159wA mutant that was deficient in 3-ketoacyl-CoA reductase activity.Wild-type growth rate was restored in vbr159wA cells that expressed either GhKCRI or 2. Further analysis showed that GhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to the yeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductase activity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest that GhKCR1 and 2 are functional orthologues of ScYbr159p.     

Co-transcribed genes for long chain polyunsaturated fatty acid biosynthesis in the protozoon Perkinsus marinus include a plant-like FAE1 3-ketoacyl coenzyme A synthase

The marine parasitic protozoon Perkinus marinus synthesizes the polyunsaturated fatty acid arachidonic acid via the unusual alternative Delta8 pathway in which elongation of C18 fatty acids generates substrate for two sequential desaturations. Here we have shown that genes encoding the three P. marinus activities responsible for arachidonic acid biosynthesis (C18 Delta9-elongating activity, C20 Delta8 desaturase, C20 Delta5 desaturase) are genomically clustered and co-transcribed as an operon. The acyl elongation reaction, which underpins this pathway, is catalyzed by a FAE1 (fatty acid elongation 1)-like 3-ketoacyl-CoA synthase class of condensing enzyme previously only reported in higher plants and algae. This is the first example of an elongating activity involved in the biosynthesis of a polyunsaturated fatty acid that is not a member of the ELO/SUR4 family. The P. marinus FAE1-like elongating activity is sensitive to the herbicide flufenacet, similar to some higher plant 3-ketoacyl-CoA synthases, but unable to rescue the yeast elo2Delta/elo3Delta mutant consistent with a role in the elongation of polyunsaturated fatty acids. P. marinus represents a key organism in the taxonomic separation of the single-celled eukaryotes collectively known as the alveolates, and our data imply a lineage in which ancestral acquisition of plant-like genes, such as FAE1-like 3-ketoacyl-CoA synthases, occurred via endosymbiosis. The P. marinus FAE1-like elongating activity is also indicative of the independent evolution of the alternative Delta8 pathway, distinct from ELO/SUR4-dependent examples.     

Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the fox2 gene and gene product.

The gene encoding the multifunctional protein (MFP) of peroxisomal beta-oxidation in Saccharomyces cerevisiae was isolated from a genomic library via functional complementation of a fox2 mutant strain. The open reading frame consists of 2700 base pairs encoding a protein of 900 amino acids. The predicted molecular weight (98,759) is in close agreement with that of the isolated polypeptide (96,000). Analysis of the deduced amino acid sequence revealed similarity to the MFPs of two other fungi but not to that of rat peroxisomes or the multifunctional subunit of the Escherichia coli beta-oxidation complex. The FOX2 gene was overexpressed from a multicopy vector (YEp352) in S. cerevisiae and the gene product purified to apparent homogeneity. A truncated version of MFP lacking 271 carboxyl-terminal amino acids was also overexpressed and purified. Experiments to study the enzymatic properties of the wild-type MFP demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA). The truncated form contained only the D-3-hydroxyacyl-CoA dehydrogenase activity. These results clearly demonstrate that the beta-oxidation of fatty acids in S. cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate.     

A study of the role of the hexose monophosphate pathway with respect to fatty acid biosynthesis in sporulation of Saccharomyces cerevisiae

13C NMR was used to study the pattern of label incorporation from [2-13C]acetate into trehalose during sporulation in Saccharomyces cerevisiae. A wild-type strain and a strain homozygous for the zwf1 mutation (which affects glucose-6-phosphate dehydrogenase) were used. In the wild-type it was possible to deduce the cycling of glucose 6-phosphate around the hexose monophosphate pathway whilst in the mutant strain this did not occur. The requirement of the hexose monophosphate pathway for providing NADPH for fatty acid biosynthesis was examined using 13C NMR and GC/MS. The wild-type strain produced a typical profile of fatty acids with palmitoleic acid being the most abundant whereas the mutant contained only one-quarter the amount of total fatty acid. As zwf1 homozygous diploids are able to sporulate this indicates that the large amount of fatty acid biosynthesis observed in sporulation of wild-type strains is not essential to the process.     

极长链脂肪酸的合成缺陷对酵母细胞膜的稳定性和多烯类药物敏感性的影响

【背景】脂肪酸延长酶家族参与脂肪酸代谢具有真核生物的高度保守性,且与膜脂的代谢密切相关。但细胞极长链脂肪酸(Very long-chain fatty acid,VLCFA)的合成缺陷对膜的稳定性及多烯类药物的敏感性影响并不完全明晰。【目的】探究细胞VLCFA延长酶ELO1、ELO2和ELO3的作用及功能。【方法】研究脂肪酸延长酶缺陷型elo1?、elo2?和elo3?对多烯类药物两性霉素B (Amphotericin B,AmB)、制霉菌素(Nystatin,Ny)及唑类硝酸益康唑(Econazolenitrate,Eco)的响应,检测不同酵母细胞的麦角固醇,检测其对Na+的响应及胞内钠钾离子水平。【结果】发现细胞VLCFA延长酶ELO2和ELO3缺陷后对AmB高度敏感;VLCFA延长酶缺陷突变株elo2?和elo3?对其它多烯类药物Ny及唑类药物Eco也十分敏感;细胞膜不饱和脂肪酸增加也会改变膜的稳定性,实验结果表明外源油酸(Oleic acid,OLA)增加了elo2?和elo3?突变体的AmB敏感性;相对野生型BY4741和elo1?,缺陷菌株elo2?和elo3?中麦角固醇的含量有显著下降;钠钾离子平衡是维护细胞正常生理的必要条件,也是检测细胞膜稳定性的重要参数,发现VLCFA的合成缺陷菌株对高浓度的NaCl比野生型菌株更敏感,使用ICP-AES检测不同浓度AmB胁迫下细胞内钠钾离子水平,也显示VLCFA延长酶缺陷菌株中,钠水平表现出上升趋势,并且细胞内钾含量明显降低。【结论】细胞VLCFA的合成缺陷会导致细胞膜更脆弱、稳定性下降,从而提高真菌对多烯类药物的敏感性,也表明脂肪酸延长酶是潜在的抗真菌治疗靶点。     

A specific structural requirement for ergosterol in long-chain fatty acid synthesis mutants important for maintaining raft domains in yeast

Fungal sphingolipids contain ceramide with a very-long-chain fatty acid (C26). To investigate the physiological significance of the C26-substitution on this lipid, we performed a screen for mutants that are synthetically lethal with ELO3. Elo3p is a component of the ER-associated fatty acid elongase and is required for the final elongation cycle to produce C26 from C22/C24 fatty acids. elo3delta mutant cells thus contain C22/C24- instead of the natural C26-substituted ceramide. We now report that under these conditions, an otherwise nonessential, but also fungal-specific, structural modification of the major sterol of yeast, ergosterol, becomes essential, because mutations in ELO3 are synthetically lethal with mutations in ERG6. Erg6p catalyzes the methylation of carbon atom 24 in the aliphatic side chain of sterol. The lethality of an elo3delta erg6delta double mutant is rescued by supplementation with ergosterol but not with cholesterol, indicating a vital structural requirement for the ergosterol-specific methyl group. To characterize this structural requirement in more detail, we generated a strain that is temperature sensitive for the function of Erg6p in an elo3delta mutant background. Examination of raft association of the GPI-anchored Gas1p and plasma membrane ATPase, Pma1p, in the conditional elo3delta erg6(ts) double mutant, revealed a specific defect of the mutant to maintain raft association of preexisting Pma1p. Interestingly, in an elo3delta mutant at 37 degrees C, newly synthesized Pma1p failed to enter raft domains early in the biosynthetic pathway, and upon arrival at the plasma membrane was rerouted to the vacuole for degradation. These observations indicate that the C26 fatty acid substitution on lipids is important for establishing raft association of Pma1p and stabilizing the protein at the cell surface. Analysis of raft lipids in the conditional mutant strain revealed a selective enrichment of ergosterol in detergent-resistant membrane domains, indicating that specific structural determinants on both sterols and sphingolipids are required for their association into raft domains.     

Functional differentiation and selective inactivation of multiple <Emphasis Type="Italic"> Saccharomyces cerevisiae </Emphasis>genes involved in very-long-chain fatty acid synthesis

While de novo fatty acid synthesis uses acetyl-CoA, fatty acid elongation uses longer-chain acyl-CoAs as primers. Several mutations that interfere with fatty acid elongation in yeast have already been described, suggesting that there may be different elongases for medium- and long-chain acyl-CoA primers. In the present study, an experimental approach is described that allows differential characterization of the various yeast elongases in vitro. Based on their characteristic primer specificities and product patterns, at least three different yeast elongases are defined. Elongase I extends C12-C16 fatty acyl-CoAs to C16-C18 fatty acids. Elongase II elongates palmitoyl-CoA and stearoyl-CoA up to C22 fatty acids, and elongase III synthesizes 20-26-carbon fatty acids from C18-CoA primers. Elongases I, II and III are specifically inactivated in, respectively, elo1, elo2 and elo3 mutants. Elongases II and III share the same 3-ketoacyl reductase, which is encoded by the YBR159w gene. Inactivation of YBR159w inhibits in vitro fatty acid elongation after the first condensation reaction. Although in vitro elongase activity is absent, the mutant nevertheless contains 10-30% of normal VLCFA levels. On the basis of this finding, an additional elongating activity is inferred to be present in vivo. ybr159Delta cells show synthetic lethality in the presence of cerulenin, which inactivates fatty acid synthase. An involvement of FAS in VLCFA synthesis may account for these findings, but remains to be demonstrated directly. Alternatively, a vital role for C18 and C20 hydroxyacids, which are dramatically overproduced in ybr159Delta cells, may be postulated.     

Multiple regulatory roles of a novel Saccharomyces cerevisiae protein, encoded by YOL002c, in lipid and phosphate metabolism

The yeast open reading frame YOL002c encodes a putative membrane protein. This protein is evolutionarily conserved across species, including humans, although the function of each of these proteins remains unknown. YOL002c is highly expressed in yeast cells that are grown in the presence of saturated fatty acids such as myristate. Furthermore, cells in which the YOL002c gene is disrupted grow poorly on this carbon source. These mutant cells are also resistant to the polyene antibiotic, nystatin. Gene chip analysis on yol002cDelta cells revealed that a variety of genes encoding proteins involved in fatty acid metabolism and in the phosphate signaling pathway are induced in this mutant strain. In addition, our studies demonstrated that in the disruption strain acid phosphatase activity is expressed constitutively, and the cells accumulate polyphosphate to much higher levels than wild-type cells. A homologous human protein is able to partially rescue these defects in phosphate metabolism. We propose that YOL002c encodes a Saccharomyces cerevisiae protein that plays a key role in metabolic pathways that regulate lipid and phosphate metabolism.     

Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids

Whereas the physiological significance of microsomal fatty acid elongation is generally appreciated, its molecular nature is poorly understood. Here, we describe tissue-specific regulation of a novel mouse gene family encoding components implicated in the synthesis of very long chain fatty acids. The Ssc1 gene appears to be ubiquitously expressed, whereas Ssc2 and Cig30 show a restricted expression pattern. Their translation products are all integral membrane proteins with five putative transmembrane domains. By complementing the homologous yeast mutants, we found that Ssc1 could rescue normal sphingolipid synthesis in the sur4/elo3 mutant lacking the ability to synthesize cerotic acid (C(26:0)). Similarly, Cig30 reverted the phenotype of the fen1/elo2 mutant that has reduced levels of fatty acids in the C(20)-C(24) range. Further, we show that Ssc1 mRNA levels were markedly decreased in the brains of myelin-deficient mouse mutants known to have very low fatty acid chain elongation activity. Conversely, the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity. Our results strongly implicate this new mammalian gene family in tissue-specific synthesis of very long chain fatty acids and sphingolipids.     

Enhanced levels of Pis1p (phosphatidylinositol synthase) improve the growth of Saccharomyces cerevisiae cells deficient in Rsp5 ubiquitin ligase

The Rsp5 ubiquitin ligase plays a role in many cellular processes including the biosynthesis of unsaturated fatty acids. The PIS1 (phosphatidylinositol synthase gene) encoding the enzyme Pis1p which catalyses the synthesis of phosphatidylinositol from CDP-diacyglycerol and inositol, was isolated in a screen for multicopy suppressors of the rsp5 temperature sensitivity phenotype. Suppression was allele non-specific. Interestingly, expression of PIS1 was 2-fold higher in the rsp5 mutant than in wild-type yeast, whereas the introduction of PIS1 in a multicopy plasmid increased the level of Pis1p 6-fold in both backgrounds. We demonstrate concomitantly that the expression of INO1 (inositol phosphate synthase gene) was also elevated approx. 2-fold in the rsp5 mutant as compared with the wild-type, and that inositol added to the medium improved growth of rsp5 mutants at a restrictive temperature. These results suggest that enhanced phosphatidylinositol synthesis may account for PIS1 suppression of rsp5 defects. Analysis of lipid extracts revealed the accumulation of saturated fatty acids in the rsp5 mutant, as a consequence of the prevention of unsaturated fatty acid synthesis. Overexpression of PIS1 did not correct the cellular fatty acid content; however, saturated fatty acids (C(16:0)) accumulated preferentially in phosphatidylinositol, and (wild-type)-like fatty acid composition in phosphatidylethanolamine was restored.     

Defects in very long chain fatty acid synthesis enhance alpha-synuclein toxicity in a yeast model of Parkinson's disease

We identified three S. cerevisiae lipid elongase null mutants (elo1Δ, elo2Δ, and elo3Δ) that enhance the toxicity of alpha-synuclein (α-syn). These elongases function in the endoplasmic reticulum (ER) to catalyze the elongation of medium chain fatty acids to very long chain fatty acids, which is a component of sphingolipids. Without α-syn expression, the various elo mutants showed no growth defects, no reactive oxygen species (ROS) accumulation, and a modest decrease in survival of aged cells compared to wild-type cells. With (WT, A53T or E46K) α-syn expression, the various elo mutants exhibited severe growth defects (although A30P had a negligible effect on growth), ROS accumulation, aberrant protein trafficking, and a dramatic decrease in survival of aged cells compared to wild-type cells. Inhibitors of ceramide synthesis, myriocin and FB1, were extremely toxic to wild-type yeast cells expressing (WT, A53T, or E46K) α-syn but much less toxic to cells expressing A30P. The elongase mutants and ceramide synthesis inhibitors enhance the toxicity of WT α-syn, A53T and E46K, which transit through the ER, but have a negligible effect on A30P, which does not transit through the ER. Disruption of ceramide-sphingolipid homeostasis in the ER dramatically enhances the toxicity of α-syn (WT, A53T, and E46K).     

Excretion of anthranilate and 3-hydroxyanthranilate by Saccharomyces cerevisiae: relationship to iron metabolism.

Resting suspensions of cells of Saccharomyces cerevisiae grown in iron-rich or iron-deficient conditions were studied by following the fluorescence emission changes (lambda em. 400-460 nm, lambda exc. 300-340 nm) occurring in these suspensions upon addition of glucose and ferric iron. The results show that, in addition to NAD(P)H, metabolites of the aromatic amino acid pathway interfere with the fluorescence measurements, and that they could be involved in ferric iron reduction. Wild-type strains of S. cerevisiae are known to excreted anthranilic acid and 3-hydroxyanthranilic acid in response to glucose. The major fluorescing compound excreted by a chorismate-mutase-deficient mutant strain of S. cerevisiae was identified as anthranilic acid. The excretion of anthranilic and 3-hydroxyanthranilic acids was correlated with the ferric-reducing capacity of the extracellular medium. Excretion during growth was much greater by cells cultured in iron-rich medium than by cells grown in iron-deficient medium. The possibility was examined that a link could exist between the biosynthesis of aromatics and the ferri-reductase activity of the cells, via chorismate synthase and its putative diaphorase-associated activity. Two ferri-reductase-deficient mutants excreted much less 3-hydroxyanthranilate than did the parental wild-type strains. However, the ferri-reductase activity of a chorismate-synthase-deficient mutant was comparable to that of the parental strain.     

Mutants of Saccharomyces cerevisiae deficient in acyl-CoA synthetases secrete fatty acids due to interrupted fatty acid recycling

In the present study, acyl-CoA synthetase mutants of Saccharomyces cerevisiae were employed to investigate the impact of this activity on certain pools of fatty acids. We identified a genotype responsible for the secretion of free fatty acids into the culture medium. The combined deletion of Faa1p and Faa4p encoding two out of five acyl-CoA synthetases was necessary and sufficient to establish mutant cells that secreted fatty acids in a growth-phase dependent manner. The mutants accomplished fatty acid export during exponential growth-phase followed by fatty acid re-import into the cells during the stationary phase. The data presented suggest that the secretion is driven by an active component. The fatty acid re-import resulted in a severely altered ultrastructure of the mutant cells. Additional strains deficient of any cellular acyl-CoA synthetase activity revealed an almost identical phenotype, thereby proving transfer of fatty acids across the plasma membrane independent of their activation with CoA. Further experiments identified membrane lipids as the origin of the observed free fatty acids. Therefore, we propose the recycling of endogenous fatty acids generated in the course of lipid remodelling as a major task of both acyl-CoA synthetases Faa1p and Faa4p.