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.

Identification and characterization of two fatty acid elongases in Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi is a potential cost-effective source for the production of microbial lipids. Fatty acid elongases have vital roles in the syntheses of long-chain fatty acids. In this study, two genes encoding fatty acid elongases of L. starkeyi, LsELO1, and LsELO2 were identified and characterized. Heterologous expression of these genes in Saccharomyces cerevisiae revealed that LsElo1 is involved in the production of saturated long-chain fatty acids with 24 carbon atoms (C24:0) and that LsElo2 is involved in the conversion of C16 fatty acids to C18 fatty acids. In addition, both LsElo1 and LsElo2 were able to elongate polyunsaturated fatty acids. LsElo1 elongated linoleic acid (C18:2) to eicosadienoic acid (C20:2), and LsElo2 elongated α-linolenic acid (C18:3) to eicosatrienoic acid (C20:3). Overexpression of LsElo2 in L. starkeyi caused a reduction in C16 fatty acids, such as palmitic and palmitoleic acids, and an accumulation of C18 fatty acids such as oleic and linoleic acids. Our findings have the potential to contribute to the remodeling of fatty acid composition and the production of polyunsaturated long-chain fatty acids in oleaginous yeasts.

Identification and characterization of Δ12 and Δ12/Δ15 bifunctional fatty acid desaturases in the oleaginous yeast Lipomyces starkeyi.

Fatty acid desaturases play vital roles in the synthesis of unsaturated fatty acids. In this study, Δ12 and Δ12/Δ15 fatty acid desaturases of the oleaginous yeast Lipomyces starkeyi, termed LsFad2 and LsFad3, respectively, were identified and characterized. Saccharomyces cerevisiae expressing LsFAD2 converted oleic acid (C18:1) to linoleic acid (C18:2), while a strain of LsFAD3-expressing S. cerevisiae converted oleic acid to linoleic acid, and linoleic acid to α-linolenic acid (C18:3), indicating that LsFad2 and LsFad3 were Δ12 and bifunctional Δ12/Δ15 fatty acid desaturases, respectively. The overexpression of LsFAD2 in L. starkeyi caused an accumulation of linoleic acid and a reduction in oleic acid levels. In contrast, overexpression of LsFAD3 induced the production of α-linolenic acid. Deletion of LsFAD2 and LsFAD3 induced the accumulation of oleic acid and linoleic acid, respectively. Our findings are significant for the commercial production of polyunsaturated fatty acids, such as ω-3 polyunsaturated fatty acids, in L. starkeyi.

High-efficiency extracellular release of free fatty acids from Aspergillus oryzae using non-ionic surfactants.

Free fatty acids (FFAs) are useful for generating biofuel compounds and functional lipids. Microbes are increasingly exploited to produce FFAs via metabolic engineering. However, in many microorganisms, FFAs accumulate in the cytosol, and disrupting cells to extract them is energy intensive. Thus, a simple cost-effective extraction technique must be developed to remove this drawback. We found that FFAs were released from cells of the filamentous fungus Aspergillus oryzae with high efficiency when they were cultured or incubated with non-ionic surfactants such as Triton X-100. The surfactants did not reduce hyphal growth, even at 5% (w/v). When the faaA disruptant was cultured with 1% Triton X-100, more than 80% of the FFAs synthesized de novo were released. When the disruptant cells grown without surfactants were incubated for 1h in 1% Triton X-100 solution, more than 50% of the FFAs synthesized de novo were also released. Other non-ionic surfactants in the same ether series, such as Brij 58, IGEPAL CA-630, and Tergitol NP-40, elicited a similar FFA release. The dry cell weight of total hyphae decreased when grown with 1% Triton X-100. The decrement was 4.9-fold greater than the weight of the released FFAs, implying release of other intracellular compounds. Analysis of the culture supernatant showed that intracellular lactate dehydrogenase was also released, suggesting that FFAs are not released by a specific transporter. Therefore, ether-type non-ionic surfactants probably cause non-specific release of FFAs and other intracellular compounds by increasing cell membrane permeability.

Modulation of gluconeogenesis and lipid production in an engineered oleaginous Saccharomyces cerevisiae transformant.

We previously created an oleaginous Saccharomyces cerevisiae transformant as a dga1 mutant overexpressing Dga1p lacking 29 amino acids at the N-terminal (Dga1∆Np). Because we have already shown that dga1 disruption decreases the expression of ESA1, which encodes histone acetyltransferase, the present study was aimed at exploring how Esa1p was involved in lipid accumulation. We based our work on the previous observation that Esa1p acetylates and activates phosphoenolpyruvate carboxykinase (PEPCK) encoded by PCK1, a rate-limiting enzyme in gluconeogenesis, and subsequently evaluated the activation of Pck1p by yeast growth with non-fermentable carbon sources, thus dependent on gluconeogenesis. This assay revealed that the ∆dga1 mutant overexpressing Dga1∆Np had much lower growth in a glycerol-lactate (GL) medium than the wild-type strain overexpressing Dga1∆Np. Moreover, overexpression of Esa1p or Pck1p in mutants improved the growth, indicating that the ∆dga1 mutant overexpressing Dga1∆Np had lower activities of Pck1p and gluconeogenesis due to lower expression of ESA1. In vitro PEPCK assay showed the same trend in the culture of the ∆dga1 mutant overexpressing Dga1∆Np with 10 % glucose medium, indicating that Pck1p-mediated gluconeogenesis decreased in this oleaginous transformant under the lipid-accumulating conditions introduced by the glucose medium. The growth of the ∆dga1 mutant overexpressing Dga1∆Np in the GL medium was also improved by overexpression of acetyl-CoA synthetase, Acs1p or Acs2p, indicating that supply of acetyl-CoA was crucial for Pck1p acetylation by Esa1p. In addition, the ∆dga1 mutant without Dga1∆Np also showed better growth in the GL medium, indicating that decreased lipid accumulation was enhancing Pck1p-mediated gluconeogenesis. Finally, we found that overexpression of Ole1p, a fatty acid ∆9-desaturase, in the ∆dga1 mutant overexpressing Dga1∆Np improved its growth in the GL medium. Although the exact mechanisms leading to the effects of Ole1p were not clearly defined, changes of palmitoleic and oleic acid contents appeared to be critical. This observation was supported by experiments using exogenous palmitoleic and oleic acids or overexpression of elongases. Our findings provide new insights on lipid accumulation mechanisms and metabolic engineering approaches for lipid production.

Addition of methionine and low cultivation temperatures increase palmitoleic acid production by engineered Saccharomyces cerevisiae.

Palmitoleic acid (POA) has recently gained attention for its health benefits and as a potential resource for industrial feedstock. This study focused on the use of Saccharomyces cerevisiae, which has a high POA content but low lipid content, for POA production. We created an oleaginous S. cerevisiae as a dga1 mutant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1∆Np). This was performed to further increase POA content in the oleaginous S. cerevisiae through optimization of culture conditions and genetic modifications. We found that high concentrations of methionine (2.0 g/l) increased POA production in a concentration-dependent way, while other amino acids such as cysteine, glycine, and glutamine showed no effect. It was not clear if the effect of methionine was mediated through S-adenosylmethionine, mainly because its addition did not increase POA content as did the addition of methionine. We increased POA content up to 55% by incubation of the dga1 transformant in a medium containing 2 g/l methionine at lower than normal temperatures ranging from 20 to 25 °C. Cultivation at such temperatures increased dry cell weight, but did not affect the lipid content, thereby increasing total POA production. The effects of methionine and low temperatures (20-25 °C) on POA content were more apparent in the strains overexpressing Dga1∆Np than those harboring empty vectors, which was consistent with the observation that POA was enriched in triacylglycerol. Overexpression of Ole1p, the enzyme responsible for POA production, did not increase POA content of the dga1 mutant overexpressing Dga1∆Np, but increased that of the wild-type strain overexpressing Dga1∆Np. The results suggested that genomic Ole1p in the dga1 mutant was active enough to achieve the optimal POA production under these conditions. Finally, the POA production by the S. cerevisiae transformant was increased 2.5-fold, which demonstrates that oleaginous S. cerevisiae is a potential source of POA.

Increase in stearidonic acid by increasing the supply of histidine to oleaginous Saccharomyces cerevisiae.

Increasing concentration of histidine significantly increased stearidonic acid production and cell growth in oleaginous Saccharomyces cerevisiae that has been genetically modified by Δsnf2 disruption, DGA1 and Δ6 desaturase gene overexpression, and LEU2 expression. High concentration of histidine in wild-type transformant and HIS3 expression in Δsnf2 transformant also increased stearidonic acid.

Overexpression of the active diacylglycerol acyltransferase variant transforms Saccharomyces cerevisiae into an oleaginous yeast.

Lipid production by Saccharomyces cerevisiae was improved by overexpression of the yeast diacylglycerol acyltransferase Dga1p lacking the N-terminal 29 amino acids (Dga1∆Np), which was previously found to be an active form in the ∆snf2 mutant. Overexpression of Dga1∆Np in the ∆snf2 mutant, however, did not increase lipid content as expected, which prompted us to search for a more suitable strain in which to study the role of Dga1∆Np in lipid accumulation. We found that the overexpression of Dga1∆Np in the ∆dga1 mutant effectively increased the lipid content up to about 45 % in the medium containing 10 % glucose. The high lipid content of the transformant was dependent on glucose concentration, nitrogen limitation, and active leucine biosynthesis. To better understand the effect of dga1 disruption on the ability of Dga1∆Np to stimulate lipid accumulation, the ∆dga1-1 mutant, in which the 3'-terminal 36 bp of the dga1 open reading frame (ORF) remained, and the ∆dga1-2 mutant, in which the 3'-terminal 36 bp were also deleted, were prepared with URA3 disruption cassettes. Surprisingly, the overexpression of Dga1∆Np in the ∆dga1-1 mutant had a lower lipid content than the original ∆dga1 mutant, whereas overexpression in the ∆dga1-2 mutant led to a high lipid content of about 45 %. These results indicated that deletion of the 3' terminal region of the dga1 ORF, rather than abrogation of genomic Dga1p expression, was crucial for the effect of Dga1∆Np on lipid accumulation. To investigate whether dga1 disruption affected gene expression adjacent to DGA1, we found that the overexpression of Esa1p together with Dga1∆Np in the ∆dga1 mutant reverted the lipid content to the level of the wild-type strain overexpressing Dga1∆Np. In addition, RT-qPCR analysis revealed that ESA1 mRNA expression in the ∆dga1 mutant was decreased compared to the wild-type strain at the early stages of culture, suggesting that lowered Esa1p expression is involved in the effect of dga1 disruption on Dga1∆Np-dependent lipid accumulation. These results provide a new strategy to engineer S. cerevisiae for optimal lipid production.

Surface plasmon resonance analysis of interactions between diacylglycerol acyltransferase and its interacting molecules.

To measure the interactions of diacylglycerol acyltransferase (DGAT) by surface plasmon resonance (SPR), we immobilized Saccharomyces cerevisiae DGAT2 encoded by DGA1 on a BIACORE sensor chip surface. We used N-terminally truncated Dga1p with a FLAG tag at the C-terminus, which was purified to apparent homogeneity, maintaining significant DGAT activity (Kamisaka et al., Appl. Microbiol. Biotechnol., 88, 105-115 (2010)). Truncated Dga1p with a FLAG tag was immobilized with an anti-FLAG antibody that had been coupled with an L1 chip surface consisting of a carboxymethyl dextran matrix with additional hydrophobic alkane groups. The Dga1p-immobilized chip surface was analyzed for interactions of Dga1p with oleoyl-CoA, its substrate, and anti-Dga1p IgG, its interacting protein, by SPR. The binding of these analytes with the Dga1p-immobilized chip surface was specific, because butyryl-CoA, which cannot be used as a substrate for DGAT, and anti-glyceraldehyde-3-phosphate dehydrogenase IgG, did not induce any signals on SPR. Furthermore, injection of organic compounds such as xanthohumol, a DGAT inhibitor, into the Dga1p-immobilized chip surface induced significant SPR signals, probably due to interaction with DGAT. Another DGAT inhibitor, piperine, did not induce SPR signals on application, but induced them due to piperine on application together with oleoyl-CoA, in which piperine can be incorporated into the micelles of oleoyl-CoA. The results indicate that the Dga1p-immobilized L1 chip surface recognized DGAT inhibitors. Taking all this together, SPR measurement using the Dga1p-immobilized L1 chip surface provided a useful system to elucidate the structure-function relationships of DGAT and screen DGAT inhibitors.

Efficient accumulation of oleic acid in Saccharomyces cerevisiae caused by expression of rat elongase 2 gene (rELO2) and its contribution to tolerance to alcohols.

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.

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

Since Saccharomyces cerevisiae contains Delta9 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 Delta12 (KlFAD2) and omega3 (KlFAD3) fatty acid desaturase genes enabled S. cerevisiae to make also polyunsaturated fatty acids (PUFAs), linoleic (18:2n-6), and alpha-linolenic (18:3n-3) acids. Unlike Delta9 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 degrees C), it was marginal at 30 degrees C. Overexpression of cytochrome b5 at 20 degrees C did not affect the fatty acid synthesis so much, but it significantly enhanced the synthesis of PUFA at 30 degrees C.

Activation of diacylglycerol acyltransferase expressed in Saccharomyces cerevisiae: overexpression of Dga1p lacking the N-terminal region in the Deltasnf2 disruptant produces a significant increase in its enzyme activity.

We previously found that overexpression of DGA1 encoding diacylglycerol acyltransferase (DGAT) in the Deltasnf2 disruptant of Saccharomyces cerevisiae caused a significant increase in lipid accumulation and DGAT activity. The present study was conducted to investigate how Dga1p is activated in the Deltasnf2 disruptant. To analyze the expression of Dga1p in wild type and the Deltasnf2 disruptant, we overexpressed Dga1p with a 6x His tag at the N-terminus and a FLAG tag at the C-terminus. Immunoblotting using anti-6x His and anti-FLAG antibodies revealed that, in addition to full-length protein, Dga1p lacking the N-terminus was produced only in the Deltasnf2 disruptant. Full-length Dga1p and N-terminally truncated Dga1p were separated and purified from the lipid body fraction by using anti-FLAG M2 agarose and TALON metal affinity resin. Major DGAT activity was recovered in the purified fraction of N-terminally truncated Dga1p, indicating that proteolytic cleavage at the N-terminal region is involved in DGAT activation in the Deltasnf2 disruptant. Analysis of the cleavage site of N-terminally truncated Dga1p revealed a major site between Lys-29 and Ser-30. We then overexpressed truncated Dga1p variants that lacked different N-terminal amino acids and had a FLAG tag at the C-terminus. The homogenate and lipid body fraction of the Deltasnf2 disruptant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1DeltaN2p) had higher DGAT activity than that overexpressing Dga1p, indicating that Dga1DeltaN2p is activated Dga1p. Dga1DeltaN2p-FLAG(C-terminus) was purified to near homogeneity by anti-FLAG M2 agarose chromatography and maintained significant DGAT activity. These results provide a new strategy to engineer expression of DGAT.

A molecular model for diacylglycerol acyltransferase from Mortierella ramanniana var. angulispora.

Acyl CoA diacylglycerol acyltransferase (DGAT, EC 2.3.120) is recognized as a key player of cellular diacylglycerol metabolism. It catalyzes the terminal, yet the committed step in triacylglycerol synthesis using diacylglycerol and fatty acyl CoA as substrates. The protein sequence of diacylglycerol acyltransferse (DGAT) Type 2B in Moretierella ramanniana var. angulispora (Protein_ID = AAK84180.1) was retrieved from GenBank. However, a structure is not yet available for this sequence. The 3D structure of DGAT Type 2B was modeled using a template structure (PDB ID: 1K30) obtained from Protein databank (PDB) identified by searching with position specific iterative BLAST (PSI-BLAST). The template (PDB ID: 1K30) describes the structure of DGAT from Cucurbita moschata. Modeling was performed using Modeller 9v2 and protein model is hence generated. The DGAT type 2B protein model was subsequently docked with six inhibitors (sphingosine; trifluoroperazine; phosphatidic acid; lysophospatidylserine; KCl; 1, 2-diolein) using AutoDock (a molecular docking program). The binding of inhibitors to the protein model of DGAT type 2B is discussed.

Improvement of Stearidonic acid production in Oleaginous Saccharomyces cerevisiae.

Rat Delta6 desaturase was introduced in "oleaginous" Saccharomyces cerevisiae genetically modified by the snf2 disruption, DGA1 overexpression, and LEU2 expression to improve stearidonic acid (18:4n-3 (Delta6, 9, 12, 15)) production. When this transformed yeast was cultured in the presence of 0.7 g/l alpha-linolenic acid for 7 d, 0.4 g of alpha-linolenic acid was accumulated and 44 mg of stearidonic acid was produced, mainly as a triacylglycerol in the 1 liter broth.

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.

Isolation and characterization of a Delta5-desaturase from Oblongichytrium sp.

We isolated a cDNA clone with homology to known desaturase genes from Oblongichytrium sp., recently classified as a new genus of thraustochytrids (Labyrinthulomycetes), and found that it encoded Delta5-desaturase by its heterologous expression in yeast. The enzyme had higher activity toward 20:4n-3 than 20:3n-6, indicating that this Delta5-desaturase can be used in the production of n-3 polyunsaturated fatty acids in transgenic organisms.

Heterologous production of dihomo-gamma-linolenic acid in Saccharomyces cerevisiae.

To make dihomo-gamma-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Delta12 fatty acid desaturase, rat Delta6 fatty acid desaturase, and rat elongase genes. Because Fad2p is able to convert the endogenous oleic acid to linoleic acid, this allowed DGLA biosynthesis without the need to supply exogenous fatty acids on the media. Medium composition, cultivation temperature, and incubation time were examined to improve the yield of DGLA. Fatty acid content was increased by changing the medium from a standard synthetic dropout medium to a nitrogen-limited minimal medium (NSD). Production of DGLA was higher in the cells grown at 15 degrees C than in those grown at 20 degrees C, and no DGLA production was observed in the cells grown at 30 degrees C. In NSD at 15 degrees C, fatty acid content increased up until day 7 and decreased after day 10. When the cells were grown in NSD for 7 days at 15 degrees C, the yield of DGLA reached 2.19 microg/mg of cells (dry weight) and the composition of DGLA to total fatty acids was 2.74%. To our knowledge, this is the first report describing the production of polyunsaturated fatty acids in S. cerevisiae without supplying the exogenous fatty acids.

DGA1 (diacylglycerol acyltransferase gene) overexpression and leucine biosynthesis significantly increase lipid accumulation in the Deltasnf2 disruptant of Saccharomyces cerevisiae.

We previously found that SNF2, a gene encoding a transcription factor forming part of the SWI/SNF (switching/sucrose non-fermenting) chromatin-remodelling complex, is involved in lipid accumulation, because the Deltasnf2 disruptant of Saccharomyces cerevisiae has a higher lipid content. The present study was conducted to identify other factors that might further increase lipid accumulation in the Deltasnf2 disruptant. First, expression of LEU2 (a gene encoding beta-isopropylmalate dehydrogenase), which was used to select transformed strains by complementation of the leucine axotroph, unexpectedly increased both growth and lipid accumulation, especially in the Deltasnf2 disruptant. The effect of LEU2 expression on growth and lipid accumulation could be reproduced by adding large amounts of leucine to the culture medium, indicating that the effect was not due to Leu2p (beta-isopropylmalate dehydrogenase) itself, but rather to leucine biosynthesis. To increase lipid accumulation further, genes encoding the triacylglycerol biosynthetic enzymes diacylglycerol acyltransferase (DGA1) and phospholipid:diacylglycerol acyltransferase (LRO1) were overexpressed in the Deltasnf2 disruptant. Overexpression of DGA1 significantly increased lipid accumulation, especially in the Deltasnf2 disruptant, whereas LRO1 overexpression decreased lipid accumulation in the Deltasnf2 disruptant. Furthermore, the effect of overexpression of acyl-CoA synthase genes (FAA1, FAA2, FAA3 and FAA4), which each supply a substrate for Dga1p (diacylglycerol acyltransferase), was investigated. Overexpression of FAA3, together with that of DGA1, did not further increase lipid accumulation in the Deltasnf2 disruptant, but did enhance lipid accumulation in the presence of exogenous fatty acids. Lastly, the total lipid content in the Deltasnf2 disruptant transformed with DGA1 and FAA3 overexpression vectors reached approx. 30%, of which triacylglycerol was the most abundant lipid. Diacylglycerol acyltransferase activity was significantly increased in the Deltasnf2 disruptant strain overexpressing DGA1 as compared with the wild-type strain overexpressing DGA1; this higher activity may account for the prominent increase in lipid accumulation in the Deltasnf2 disruptant with DGA1 overexpression. The strains obtained have a lipid content that is high enough to act as a model of oleaginous yeast and they may be useful for the metabolic engineering of lipid production in yeast.

Isolation of Delta12 and omega3-fatty acid desaturase genes from the yeast Kluyveromyces lactis and their heterologous expression to produce linoleic and alpha-linolenic acids in Saccharomyces cerevisiae.

Two clones with homology to known fatty acid desaturase genes were isolated from the yeast Kluyveromyces lactis. The first gene, which we designate KlFAD2, consists of 411 amino acids with an overall identity of 73.0% to FAD2 from Saccharomyces kluyveri. It exhibited Delta12 fatty acid desaturase activity when expressed in S. cerevisiae under the control of ADH1 promoter and produced endogenous linoleic acid. The second clone, which we designate KlFAD3, consists of 415 amino acids with an overall identity of 79.3% to FAD3 from S. kluyveri. It exhibited omega3 fatty acid desaturase activity in S. cerevisiae when expressed under the control of ADH1 promoter in the presence of the exogenous substrate linoleic acid and produced alpha-linolenic acid. Co-expression of KlFAD2 and KlFAD3 resulted in the endogenous production of both linoleic and alpha-linolenic acids. The yield of alpha-linolenic acid reached 0.8% of total fatty acids and its production was not increased by adding exogenous oleic acid; alpha-linolenic acid reached 8.7% when exogenous linoleic acid was available.

Peroxisome proliferator-activated receptor gamma ligands stimulate myeloid differentiation and lipogenensis in human leukemia NB4 cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in adipocyte and macrophage differentiation. Pioglitazone (Actos, AD4833), an antidiabetic drug, and 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2) have recently been identified as synthetic and natural ligands for PPARgamma, respectively. In this study, we examined the effects of PPARgamma ligands on differentiation and lipogenesis in promyelocytic leukemia NB4 cells, in which PPARgamma protein was expressed and ligand-stimulated PPARgamma-specific transcription of adipocyte fatty-acid binding protein was confirmed. Treatment with PPARgamma ligand (AD4833 or PGJ2) alone markedly suppressed proliferation but did not induce differentiation. The combined treatment of the cells with PPARgamma ligand and all-trans retinoic acid (ATRA) synergistically induced myelocytic differentiation, as determined by nitroblue tetrazolium reducing ability and cell morphology. During these processes of differentiation, we observed marked accumulation of lipid droplets in the cytoplasm. The cellular triacylglycerol levels increased 2.7-fold after treatment with the inducers. Simultaneously, BODIPY-fatty acid was incorporated into the cytosol and concentrated in lipid droplets. The biosynthesis of triacylglycerol-containing BODIPY-fatty acids was increased twofold in differentiated cells. These findings clearly demonstrate that treatment with PPARgamma ligands not only induced differentiation but also stimulated lipogenesis in NB4 cells, indicating a close association between differentiation and lipogenesis in PPARgamma-stimulated human myeloid cells.