Fatty Acids In Yeast Research Articles

Reconstruction of a fatty acid synthesis cycle from acyl carrier protein and cofactor structural snapshots

Fatty acids (FAs) play a central metabolic role in living cells as constituents of membranes, cellular energy reserves, and second messenger precursors. A 2.6 MDa FA synthase (FAS), where the enzymatic reactions and structures are known, is responsible for FA biosynthesis in yeast. Essential in the yeast FAS catalytic cycle is the acyl carrier protein (ACP) that actively shuttles substrates, biosynthetic intermediates, and products from one active site to another. We resolve the S.cerevisiae FAS structure at 1.9Å, elucidating cofactors and water networks involved in their recognition. Structural snapshots of ACP domains bound to various enzymatic domains allow the reconstruction of a full yeast FA biosynthesis cycle. The structural information suggeststhat each FAS functional unit could accommodate exogenous proteins to incorporate various enzymaticactivities, and we show proof-of-concept experiments where ectopic proteins are used to modulate FAS product profiles.

Acyl CoA oxidase: from its expression, structure, folding, and import to its role in human health and disease.

β-oxidation of fatty acids is an important metabolic pathway and is a shared function between mitochondria and peroxisomes in mammalian cells. On the other hand, peroxisomes are the sole site for the degradation of fatty acids in yeast. The first reaction of this pathway is catalyzed by the enzyme acyl CoA oxidase housed in the matrix of peroxisomes. Studies in various model organisms have reported the conserved function of the protein in fatty acid oxidation. The importance of this enzyme is highlighted by the lethal conditions caused in humans due to its altered function. In this review, we discuss various aspects ranging from gene expression, structure, folding, and import of the protein in both yeast and human cells. Further, we highlight recent findings on the role of the protein in human health and aging, and discuss the identified mutations in the protein associated with debilitating conditions in patients.

The Role of Metabolic Engineering Technologies for the Production of Fatty Acids in Yeast.

Simple SummaryMetabolic engineering involves the sustainable production of high-value products. E. coli and yeast, in particular, are used for such processes. Using metabolic engineering, the biosynthetic pathways of these cells are altered to obtain a high production of desired products. Fatty acids (FAs) and their derivatives are products produced using metabolic engineering. However, classical methods used for engineering yeast metabolic pathways for the production of fatty acids and their derivatives face problems such as the low supply of key precursors and product tolerance. This review introduces the different ways FAs are being produced in E. coli and yeast and the genetic manipulations for enhanced production of FAs. The review also summarizes the latest techniques (i.e., CRISPR–Cas and synthetic biology) for developing FA-producing yeast cell factories.Metabolic engineering is a cutting-edge field that aims to produce simple, readily available, and inexpensive biomolecules by applying different genetic engineering and molecular biology techniques. Fatty acids (FAs) play an important role in determining the physicochemical properties of membrane lipids and are precursors of biofuels. Microbial production of FAs and FA-derived biofuels has several advantages in terms of sustainability and cost. Conventional yeast Saccharomyces cerevisiae is one of the models used for FA synthesis. Several genetic manipulations have been performed to enhance the citrate accumulation and its conversation into acetyl-CoA, a precursor for FA synthesis. Success has been achieved in producing different chemicals, including FAs and their derivatives, through metabolic engineering. However, several hurdles such as slow growth rate, low oleaginicity, and cytotoxicity are still need to be resolved. More robust research needs to be conducted on developing microbes capable of resisting diverse environments, chemicals, and cost-effective feed requirements. Redesigning microbes to produce FAs with cutting-edge synthetic biology and CRISPR techniques can solve these problems. Here, we reviewed the technological progression of metabolic engineering techniques and genetic studies conducted on S. cerevisiae, making it suitable as a model organism and a great candidate for the production of biomolecules, especially FAs.

Understanding the Role of Populus ECERIFERUM2-Likes in the Biosynthesis of Very-Long-Chain Fatty Acids for Cuticular Waxes.

Cuticular waxes are derived from very-long-chain fatty acid (VLCFA) precursors made by the concerted action of four enzymes that form the fatty acid (FA) elongation complex. The condensing enzyme of the complex confers specificity to substrates of different chain lengths, yet on its own cannot account for the biosynthesis of VLCFAs longer than 28 carbons (C28). Recent evidence from Arabidopsis thaliana points to a synergistic role of clade II BAHD acyltransferases and condensing enzymes in the elongation of VLCFAs beyond C28. In Populus trichocarpa, clade II is composed of seven uncharacterized paralogous genes (PtCER2-like1-7). In the present study, five of these genes were heterologously expressed in yeast and their respective FA profiles were determined. PtCER2-likes differentially altered the accumulation of C28 and C30 FAs when expressed in the presence of the condensing enzyme AtCER6. Among these, PtCER2-like5 produced the highest levels of C28 FAs in yeast and its expression was localized to the epidermis in β-glucuronidase-reporter poplar lines, consistent with a role in cuticular wax biosynthesis. Complementation of the A. thaliana cer2-5 mutant with PtCER2-like5 increased the levels of C28-derived cuticular waxes at the expense of C30-derived components. Together, these results demonstrate that the role of CER2-likes in cuticular wax biosynthesis is conserved in Populus clade II BAHD acyltransferases.

Production of 10-methyl branched fatty acids in yeast

BackgroundDespite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation.ResultsWe cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids.ConclusionsWe report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications.

Identification and characterization of Pseudozyma antarctica Δ12 fatty acid desaturase and its utilization for the production of polyunsaturated fatty acids

Fatty acid desaturases, especially Δ12 fatty acid desaturases, are key enzymes for the production of unsaturated fatty acids in oleaginous yeasts. In this study, we identified and characterized a gene encoding Δ12 fatty acid desaturase of Pseudozyma antarctica named PaFAD2. Almost all oleic acid (C18:1) was converted to linoleic acid by the heterologous expression of the PaFAD2 gene in Saccharomyces cerevisiae and Lipomyces starkeyi oleaginous yeast. Notably, PaFad2 converted not only oleic acid to linoleic acid, but also palmitoleic acid (C16:1) to 9,12-hexadecadienoic acid (C16:2). These results indicated that the PaFAD2 gene was very useful for the production of polyunsaturated fatty acids in yeast, including oleaginous yeast.

PEROXIREDOXIN Q stimulates the activity of the chloroplast 16:1Δ3trans FATTY ACID DESATURASE4.

Thylakoid membrane lipids, comprised of glycolipids and the phospholipid phosphatidylglycerol (PG), are essential for normal plant growth and development. Unlike other lipid classes, chloroplast PG in nearly all plants contains a substantial fraction of the unusual trans fatty acid 16:1Δ3trans or 16:1t. We determined that, in Arabidopsis thaliana, 16:1t biosynthesis requires both FATTY ACID DESATURASE4 (FAD4) and a thylakoid-associated redox protein, PEROXIREDOXIN Q (PRXQ), to produce wild-type levels of 16:1t. The FAD4-PRXQ biochemical relationship appears to be very specific in planta, as other fatty acids (FA) desaturases do not require peroxiredoxins for their activity, nor does FAD4 require other chloroplast peroxiredoxins under standard growth conditions. Although most of chloroplast PG assembly occurs at the inner envelope membrane, FAD4 was primarily associated with the thylakoid membranes facing the stroma. Furthermore, co-production of PRXQ with FAD4 was required to produce Δ3-desaturated FAs in yeast. Alteration of the redox state of FAD4 or PRXQ through site-directed mutagenesis of conserved cysteine residues impaired Δ3 FA production. However, these mutations did not appear to directly alter disulfide status of FAD4. These results collectively demonstrate that the production of 16:1t is linked to the redox status of the chloroplast through PRXQ associated with the thylakoids.

Enhanced Production of High-Value Cyclopropane Fatty Acid in Yeast Engineered for Increased Lipid Synthesis and Accumulation.

The unique strained ring structure in cyclopropane fatty acids (CFA) conveys oxidative stability and lubricity to lipids. These attributes are highly valuable for industrial applications such as cosmetics and specialist lubrication but there is currently no commercial source of the lipid. Here, built on recently engineered strains of Saccharomyces cerevisiae, the authors have developed an efficient strategy for CFA production. Expression of the Escherichia coli cyclopropane fatty acid synthetase (Ec.CFAS) in the engineered yeast resulted in formation of cis-9,10-methylene-hexadecanoic and octadecanoic acids in both the phospholipid (PL) and triacylglycerol (TAG) fractions. CFA concentration in TAG of engineered yeast is 12 mg CFA g-1 DCW (fourfold above the strain expressing CFAS only). The yield of CFA increases from 13.2 to 68.3 mg L-1 , the highest reported in yeast, using a two-stage bioprocess strategy that separated cell growth from the lipid modification stage. Strategies for further improvement of this valuable lipid are proposed.

Mutation in MON2, Which Encodes a Protein Implicated in Vesicular Transport, Affects Response to Exogenous Fatty Acids in Yeast

Metabolism of exogenous fatty acids requires cells to take up, traffic, and molecularly respond to fed fatty acids. Our lab has determined that a mutation in mon2 results in defective growth on the fatty acid 10‐undecenoic acid. Like wild type cells, mon2Δ mutants are capable of normal response to the feeding of long chain unsaturated fatty acids (UFAs) by reducing transcription of OLE1, (which encodes the Δ9 desaturase), and by reducing the accumulation of endogenously produced UFAs. However, wild type cells when fed 10‐undecenoic acid will continue to express OLE1 and increase their content of endogenously produced UFAs. In contrast, mon2Δ mutants do not differentially respond to the two fatty acids. Staining with a BODIPY‐labeled fluorescent fatty acid analog indicates that there is a difference in localization/amount of the supplied fatty acid in mon2Δ cells compared to wild type cells. Thus, it appears that the mon2 mutation decreases the cell's ability to differentiate between fatty acids, perhaps due to mislocalization of the fed fatty acid or regulatory proteins.Support or Funding InformationThis work was supported by a Jacob Nyenhuis Faculty Award (VM), NSF‐REU (AG, MS, BG) and a Summer Scholar award from the Dean for Natural and Applied Science (RD).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elongation of very Long-Chain (>C24) Fatty Acids in Clarias gariepinus: Cloning, Functional Characterization and Tissue Expression of elovl4 Elongases

Elongation of very long-chain fatty acid 4 (Elovl4) proteins participate in the biosynthesis of very long-chain (>C24) saturated and polyunsaturated fatty acids (FA). Previous studies have shown that fish possess two different forms of Elovl4, termed Elovl4a and Elovl4b. The present study aimed to characterize both molecularly and functionally two elovl4 cDNA from the African catfish Clarias gariepinus. The results confirmed that C. gariepinus possessed two elovl4-like elongases with high homology to two previously characterized Elovl4 from Danio rerio, and thus they were termed accordingly as Elovl4a and Elovl4b. The C. gariepinus Elovl4a and Elovl4b have open reading frames (ORF) of 945 and 915 base pairs, respectively, encoding putative proteins of 314 and 304 amino acids, respectively. Functional characterization in yeast showed both Elovl4 enzymes have activity towards all the PUFA substrates assayed (18:4n-3, 18:3n-6, 20:5n-3, 20:4n-6, 22:5n-3, 22:4n-6 and 22:6n-3), producing elongated products of up to C36. Moreover, the C. gariepinus Elovl4a and Elovl4b were able to elongate very long-chain saturated FA (VLC-SFA) as denoted by increased levels of 28:0 and longer FA in yeast transformed with elovl4 ORF compared to control yeast. These results confirmed that C. gariepinus Elovl4 play important roles in the biosynthesis of very long-chain FA. Tissue distribution analysis of elovl4 mRNAs showed both genes were widely expressed in all tissues analyzed, with high expression of elovl4a in pituitary and brain, whereas female gonad and pituitary had the highest expression levels for elovl4b.

Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin-Fatty Acid Biosynthetic Pathway.

Dicarboxylic acids are commodity chemicals used in the production of plastics, polyesters, nylons, fragrances, and medications. Bio-based routes to dicarboxylic acids are gaining attention due to environmental concerns about petroleum-based production of these compounds. Some industrial applications require dicarboxylic acids with specific carbon chain lengths, including odd-carbon species. Biosynthetic pathways involving cytochrome P450-catalyzed oxidation of fatty acids in yeast and bacteria have been reported, but these systems produce almost exclusively even-carbon species. Here we report a novel pathway to odd-carbon dicarboxylic acids directly from glucose in Escherichia coli by employing an engineered pathway combining enzymes from biotin and fatty acid synthesis. Optimization of the pathway will lead to industrial strains for the production of valuable odd-carbon diacids.

Trans 18-carbon monoenoic fatty acid has distinct effects from its isomeric cis fatty acid on lipotoxicity and gene expression in Saccharomyces cerevisiae

Epidemiological studies have suggested that an excess intake of trans-unsaturated fatty acids increases the risk of coronary heart disease. However, the mechanisms of action of trans-unsaturated fatty acids in eukaryotic cells remain unclear. Since the budding yeast Saccharomyces cerevisiae can grow using fatty acids as the sole carbon source, it is a simple and suitable model organism for understanding the effects of trans-unsaturated fatty acids at the molecular and cellular levels. In this study, we compared the physiological effects of Δ9 cis and trans 18-carbon monoenoic fatty acids (oleic acid and elaidic acid) in yeast cells. The results obtained revealed that the two types have distinct effects on the expression of OLE1, which encodes Δ9 desaturase, and lipotoxicity in are1Δare2Δdga1Δlro1Δ and gat1Δ cells. Our results suggest that cis and trans 18-carbon monoenoic fatty acids exert different physiological effects in the regulation of gene expression and processing of excess fatty acids in yeast.

The Yeast ATF1 Acetyltransferase Efficiently Acetylates Insect Pheromone Alcohols: Implications for the Biological Production of Moth Pheromones.

Many moth pheromones are composed of mixtures of acetates of long-chain (≥10 carbon) fatty alcohols. Moth pheromone precursors such as fatty acids and fatty alcohols can be produced in yeast by the heterologous expression of genes involved in insect pheromone production. Acetyltransferases that subsequently catalyze the formation of acetates by transfer of the acetate unit from acetyl-CoA to a fatty alcohol have been postulated in pheromone biosynthesis. However, so far no fatty alcohol acetyltransferases responsible for the production of straight chain alkyl acetate pheromone components in insects have been identified. In search for a non-insect acetyltransferase alternative, we expressed a plant-derived diacylglycerol acetyltransferase (EaDAcT) (EC 2.3.1.20) cloned from the seed of the burning bush (Euonymus alatus) in a yeast system. EaDAcT transformed various fatty alcohol insect pheromone precursors into acetates but we also found high background acetylation activities. Only one enzyme in yeast was shown to be responsible for the majority of that background activity, the acetyltransferase ATF1 (EC 2.3.1.84). We further investigated the usefulness of ATF1 for the conversion of moth pheromone alcohols into acetates in comparison with EaDAcT. Overexpression of ATF1 revealed that it was capable of acetylating these fatty alcohols with chain lengths from 10 to 18 carbons with up to 27- and 10-fold higher in vivo and in vitro efficiency, respectively, compared to EaDAcT. The ATF1 enzyme thus has the potential to serve as the missing enzyme in the reconstruction of the biosynthetic pathway of insect acetate pheromones from precursor fatty acids in yeast.

“Malonate Uptake and Metabolism in Saccharomyces cerevisiae”

Malonyl-CoA plays an important role in the synthesis and elongation of fatty acids in yeast Saccharomyces cerevisiae. Malonyl-CoA is at a low concentration inside the cell and is produced mainly from acetyl-CoA through the enzyme acetyl-CoA carboxylase. It would be beneficial to find an alternative source of malonyl-CoA to increase its intracellular concentration and overall synthesis of the fatty acids. MatB gene from the bacteria Rhizobium leguminosarium bv. trifolii encodes for a malonyl-CoA synthetase which catalyzes the formation of the malonyl-CoA directly from malonate and CoA. However, results from high-performance liquid chromatography (HPLC) proved that Saccharomyces cerevisiae itself does not contain enough cytoplasmic malonate within them and is unable to uptake exogenously supplied malonate in the form of malonic acid. A dicarboxylic acid plasma membrane transporter with the ability to uptake exogenous malonic acid was identified from another species of yeast known as Schizosaccharomyces pombe and the gene encoding this transporter is identified as the mae1 gene. From the experiments thus far, the mae1 gene had been successfully cloned and transformed into Saccharomyces cerevisiae. The expression and functional ability of the encoded plasma membrane dicarboxylic acid transporter were also demonstrated and verified using specialized technologies such as RT-PCR, yeast immunofluorescence, HPLC, and LC-MS.

Transcriptional repression by glycerol of genes involved in the assimilation ofn-alkanes and fatty acids in yeastYarrowia lipolytica

The yeast Yarrowia lipolytica assimilates n-alkanes or fatty acids as carbon sources. Transcriptional activation by n-alkanes of ALK1 encoding a cytochrome P450 responsible for the terminal hydroxylation has been well studied so far, but its regulation by other carbon sources is poorly understood. Here, we analyzed the transcriptional regulation of ALK1 by glycerol. Glycerol is a preferable carbon source compared to glucose for Y.lipolytica. The n-decane-induced transcript levels of ALK1 as well as the reporter gene under the control of ALK1 promoter were significantly decreased in the simultaneous presence of glycerol, but not of glucose. Similarly, the expression of PAT1 encoding acetoacetyl-CoA thiolase involved in β-oxidation was induced by n-decane or oleic acid, but its transcript level was decreased when glycerol was supplemented. These results indicate that glycerol represses the transcription of the genes involved in the metabolism of hydrophobic carbon sources in Y.lipolytica. Repression of ALK1 transcription by glycerol was not observed in the deletion mutant of GUT1 encoding glycerol kinase, implying that the phosphorylation of glycerol is required for the glycerol repression.

A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory

BackgroundMoths (Lepidoptera) are highly dependent on chemical communication to find a mate. Compared to conventional unselective insecticides, synthetic pheromones have successfully served to lure male moths as a specific and environmentally friendly way to control important pest species. However, the chemical synthesis and purification of the sex pheromone components in large amounts is a difficult and costly task. The repertoire of enzymes involved in moth pheromone biosynthesis in insecta can be seen as a library of specific catalysts that can be used to facilitate the synthesis of a particular chemical component. In this study, we present a novel approach to effectively aid in the preparation of semi-synthetic pheromone components using an engineered vector co-expressing two key biosynthetic enzymes in a simple yeast cell factory.ResultsWe first identified and functionally characterized a ∆11 Fatty-Acyl Desaturase and a Fatty-Acyl Reductase from the Turnip moth, Agrotis segetum. The ∆11-desaturase produced predominantly Z11-16:acyl, a common pheromone component precursor, from the abundant yeast palmitic acid and the FAR transformed a series of saturated and unsaturated fatty acids into their corresponding alcohols which may serve as pheromone components in many moth species. Secondly, when we co-expressed the genes in the Brewer’s yeast Saccharomyces cerevisiae, a set of long-chain fatty acids and alcohols that are not naturally occurring in yeast were produced from inherent yeast fatty acids, and the presence of (Z)-11-hexadecenol (Z11-16:OH), demonstrated that both heterologous enzymes were active in concert. A 100 ml batch yeast culture produced on average 19.5 μg Z11-16:OH. Finally, we demonstrated that oxidized extracts from the yeast cells containing (Z)-11-hexadecenal and other aldehyde pheromone compounds elicited specific electrophysiological activity from male antennae of the Tobacco budworm, Heliothis virescens, supporting the idea that genes from different species can be used as a molecular toolbox to produce pheromone components or pheromone component precursors of potential use for control of a variety of moths.ConclusionsThis study is a first proof-of-principle that it is possible to “brew” biologically active moth pheromone components through in vitro co-expression of pheromone biosynthetic enzymes, without having to provide supplementary precursors. Substrates present in the yeast alone appear to be sufficient.

Synthesis and production of unsaturated and polyunsaturated fatty acids in yeast: current state and perspectives

Recently, many genes involved in the formation of unsaturated and polyunsaturated fatty acids (PUFAs) were isolated. In most cases, their activities were confirmed by expressing them in the well-studied model organism Saccharomyces cerevisiae because its fatty acid compositions are very simple and it does not contain PUFAs. Taking advantage of its genetic tractability and increasing wealth of accessible data, many groups are attempting to produce various useful fatty acids in the model yeasts, mainly in S. cerevisiae. This review describes typical such examples including a very recent study on the expression of a fatty acid hydroxylase gene in fission yeast Schizosaccharomyces pombe. Furthermore, the impact of the genetically engineered alteration of fatty acid composition on the stress tolerance is presented because unsaturated fatty acids have crucial roles in membrane fluidity and signaling processes. Lastly, recent attempts at increasing lipid content in S. cerevisiae are discussed.

Dynamic Changes in the Subcellular Distribution of Gpd1p in Response to Cell Stress

Gpd1p is a cytosolic NAD(+)-dependent glycerol 3-phosphate dehydrogenase that also localizes to peroxisomes and plays an essential role in the cellular response to osmotic stress and a role in redox balance. Here, we show that Gpd1p is directed to peroxisomes by virtue of an N-terminal type 2 peroxisomal targeting signal (PTS2) in a Pex7p-dependent manner. Significantly, localization of Gpd1p to peroxisomes is dependent on the metabolic status of cells and the phosphorylation of aminoacyl residues adjacent to the targeting signal. Exposure of cells to osmotic stress induces changes in the subcellular distribution of Gpd1p to the cytosol and nucleus. This behavior is similar to Pnc1p, which is coordinately expressed with Gpd1p, and under conditions of cell stress changes its subcellular distribution from peroxisomes to the nucleus where it mediates chromatin silencing. Although peroxisomes are necessary for the beta-oxidation of fatty acids in yeast, the localization of Gpd1p to peroxisomes is not. Rather, shifts in the distribution of Gpd1p to different cellular compartments in response to changing cellular status suggests a role for Gpd1p in the spatial regulation of redox potential, a process critical to cell survival, especially under the complex stress conditions expected to occur in the wild.

Production of polyunsaturated fatty acids in yeast Saccharomyces cerevisiae and its relation to alkaline pH tolerance

Saccharomyces cerevisiae produces saturated and monounsaturated fatty acids of 16- and 18-carbon atoms and no polyunsaturated fatty acids (PUFAs) with more than two double bonds. To study the biological significance of PUFAs in yeast, we introduced Kluyveromyces lactis Delta12 fatty acid desaturase (KlFAD2) and omega3 fatty acid desaturase (KlFAD3) genes into S. cerevisiae to produce linoleic and alpha-linolenic acids in S. cerevisiae. The strain producing linoleic and alpha-linolenic acids showed an alkaline pH-tolerant phenotype. DNA microarray analyses showed that the transcription of a set of genes whose expressions are under the repression of Rim101p were downregulated in this strain, suggesting that Rim101p, a transcriptional repressor which governs the ion tolerance, was activated. In line with this activation, the strain also showed elevated resistance to Li(+) and Na(+) ions and to zymolyase, a yeast lytic enzyme preparation containing mainly beta-1,3-glucanase, indicating that the cell wall integrity was also strengthened in this strain. Our findings demonstrate a novel influence of PUFA production on transcriptional control that is likely to play an important role in the early stage of alkaline stress response. The Accession No. for microarray data in the Center for Information Biology Gene Expression database is CBX68.

Incorporation and remodeling of extracellular phosphatidylcholine with short acyl residues in Saccharomyces cerevisiae

The pem1/cho2 pem2/opi3 double mutant of Saccharomyces cerevisiae, which is auxotrophic for choline because of the deficiency in methylation activities of phosphatidylethanolamine, grew in the presence of 0.1 mM dioctanoyl-phosphatidylcholine (diC 8PC). Analysis of the metabolism of methyl- 13C-labeled diC 8PC (( methyl- 13C) 3-diC 8PC) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) revealed that it was rapidly converted to ( methyl- 13C) 3-PCs containing C16 or C18 acyl chains. ( Methyl- 13C) 3-8:0-lyso-PC, ( methyl- 13C) 3-8:0-16:0-PC and ( methyl- 13C) 3-8:0-16:1-PC, which are the probable intermediate molecular species of acyl chain remodeling, appeared immediately after 5 min of pulse-labeling and decreased during the subsequent chase period. These results indicate that diC 8PC was taken up by the pem1 pem2 double mutant and that the acyl chains of diC 8PC were exchanged with longer yeast fatty acids. The temporary appearance of ( methyl- 13C) 3-8:0-lyso-PC suggests that the remodeling reaction may consist of deacylation and reacylation by phospholipase activities and acyltransferase activities, respectively. The detailed analyses of the structures of ( methyl- 13C) 3-8:0-16:0-PC and ( methyl- 13C) 3-8:0-16:1-PC by MS/MS and MS 3 strongly suggest that most ( methyl- 13C) 3-8:0-16:0-PCs have a C16:0 acyl chain at sn-1 position, whereas ( methyl- 13C) 3-8:0-16:1-PCs have a C16:1 acyl chain at either sn-1 or sn-2 position in a similar frequency, implying that the initial C16:0 acyl chain substitution prefers the sn-1 position; however, the C16:1 acyl chain substitution starts at both sn-1 and sn-2 positions. The current study provides a pivotal insight into the acyl chain remodeling of phospholipids in yeast.