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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Inhibition of lipid accumulation and lipid body formation in oleaginous yeast by effective components in spices, carvacrol, eugenol, thymol, and piperine.

We screened natural organic compounds, which affected the lipid accumulation and the lipid body formation in oleaginous yeast, Lipomyces starkeyi, generating large lipid bodies. We found that four natural components in spices, carvacrol, thymol, eugenol, and piperine, inhibited the lipid accumulation at concentrations of 20-50 mg/L with a slight growth inhibition. The inhibitory effects were quantitatively represented by the total lipid accumulation amount, the triacylglycerol accumulation amount, and the average lipid body size.

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.

Identification of genes affecting lipid content using transposon mutagenesis in Saccharomyces cerevisiae.

Genes involved in lipid accumulation were identified in Saccharomyces cerevisiae using transposon insertion mutagenesis. Five ORFs, such as SNF2, IRA2, PRE9, PHO90, and SPT21 were found from the analysis of the insertion sites in transposon insertion mutants with higher lipid content. Since these ORFs are not directly involved in storage lipid biosynthesis, we speculate that they are involved in carbon fluxes into storage lipids in response to nutrient conditions.

Appearance of smaller lipid bodies and protein kinase activation in the lipid body fraction are induced by an increase in the nitrogen source in the Mortierella fungus.

We studied the regulation of lipid body biogenesis in the oleaginous fungus Mortierella ramanniana var. angulispora by investigating culture conditions to modulate lipid body size, which we found was affected by the carbon-to-nitrogen ratio (C/N ratio) in the culture medium. Increasing the nitrogen source or decreasing the C/N ratio from 38 to 9 induced the appearance of lipid bodies with diameters less than 2-3 micro m, which are usually found at a C/N ratio of 38 in this fungus.

Biosynthesis of triacylglycerol molecular species in an oleaginous fungus, Mortierella ramanniana var. angulispora.

The incorporation of radiolabeled lipid precursors into triacylglycerol (TG) molecular species in Mortierella ramanniana var. angulispora, an oleaginous fungus, was studied to determine the biosynthetic pathways for TG molecular species. Radiolabeled TG molecular species were separated and quantified by reverse-phase high performance liquid chromatography with a radioisotope detector.