Retargeting of heterologous enzymes results in improved β-carotene synthesis in Saccharomyces cerevisiae.

Aims: Carotenoids are a class of hydrophobic substances that are important as food and feed colorants and as antioxidants. The pathway for β-carotene synthesis has been expressed in various yeast species, albeit with rather low yields and titers. The inefficient conversion of phytoene to lycopene is often regarded as a bottleneck in the pathway.

Phosphatidic acid biosynthesis in the model organism yeast Saccharomyces cerevisiae - a survey.

Phosphatidic acid biosynthesis represents the initial part of de novo formation of all glycerophospholipids (membrane lipids) as well as triacylglycerols (storage lipids), and is thus the centerpiece of glycerolipid metabolism. The universal route of phosphatidic acid biosynthesis starts from the precursor glycerol-3-phosphate and comprises two consecutive acylation reactions which are catalyzed by a glycerol-3-phosphate acyltransferase and a 1-acyl glycerol-3-phosphate acyltransferase. In addition, yeast and mammals harbor a set of enzymes which can synthesize phosphatidic acid from the precursor dihydroxyacetone phosphate.

Phosphorylation of the lipid droplet localized glycerol‑3‑phosphate acyltransferase Gpt2 prevents a futile triacylglycerol cycle in yeast.

The proteome of lipid droplets, storage compartments of triacylglycerols (TAGs), comprises TAG synthesizing and TAG degrading enzymes. Thus, to prevent a futile cycle the activity of enzymes catalyzing key steps in TAG turnover has to be strictly coordinated. The first and committed reaction of TAG synthesis is catalyzed by a glycerol‑3‑phosphate acyltransferase (GPAT).

The impact of nonpolar lipids on the regulation of the steryl ester hydrolases Tgl1p and Yeh1p in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae degradation of steryl esters is catalyzed by the steryl ester hydrolases Tgl1p, Yeh1p and Yeh2p. The two steryl ester hydrolases Tgl1p and Yeh1p localize to lipid droplets, a cell compartment storing steryl esters and triacylglycerols. In the present study we investigated regulatory aspects of these two hydrolytic enzymes, namely the gene expression level, protein amount, stability and enzyme activity of Tgl1p and Yeh1p in strains lacking both or only one of the two major nonpolar lipids, steryl esters and triacylglycerols.

Involvement of a putative substrate binding site in the biogenesis and assembly of phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) produces the largest amount of cellular phosphatidylethanolamine (PE). Psd1p is synthesized as a larger precursor on cytosolic ribosomes and then imported into mitochondria in a three-step processing event leading to the formation of an α-subunit and a β-subunit. The α-subunit harbors a highly conserved motif, which was proposed to be involved in phosphatidylserine (PS) binding.

Regulation of the yeast triacylglycerol lipases Tgl4p and Tgl5p by the presence/absence of nonpolar lipids.

Tgl3p, Tgl4p, and Tgl5p are the major triacylglycerol lipases of the yeast Saccharomyces cerevisiae Recently we demonstrated that properties of Tgl3p are regulated by the formation of nonpolar lipids. The present study extends these investigations to the two other yeast triacylglycerol lipases, Tgl4p and Tgl5p. We show that Tgl4p and Tgl5p, which are localized to lipid droplets in wild type, are partially retained in the endoplasmic reticulum in cells lacking triacylglycerols and localize exclusively to the endoplasmic reticulum in a mutant devoid of lipid droplets.

Defects in triacylglycerol lipolysis affect synthesis of triacylglycerols and steryl esters in the yeast.

Tgl3p, Tgl4p and Tgl5p are the major triacylglycerol lipases of the yeast Saccharomyces cerevisiae catalyzing degradation of triacylglycerols stored in lipid droplets. Previous results from our laboratory (Athenstaedt and Daum, 2005, J. Biol.

Regulation of the yeast triacylglycerol lipase TGl3p by formation of nonpolar lipids.

Tgl3p, the major triacylglycerol lipase of the yeast Saccharomyces cerevisiae, is a component of lipid droplets but is also present in the endoplasmic reticulum in a minor amount. Recently, it was shown that this enzyme can also serve as a lysophospholipid acyltransferase (Rajakumari, S., and Daum, G.

Controlling lipid fluxes at glycerol-3-phosphate acyltransferase step in yeast: unique contribution of Gat1p to oleic acid-induced lipid particle formation.

The ability to channel excess fatty acids into neutral lipids like triacylglycerol (TAG) is a critical strategy used by cells to maintain lipid homeostasis. Upon activation to acyl-CoA, fatty acids become readily available as substrates for acyltransferases involved in neutral lipid synthesis. Neutral lipids are then packed into organelles derived from the endoplasmic reticulum called lipid particles (LPs).

YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encode major triacylglycerol synthases of the oleaginous yeast Yarrowia lipolytica.

The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce and store triacylglycerols resembling adipocytes of higher eukaryotes. Here, the identification of two genes YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encoding major triacylglycerol synthases of Yarrowia lipolytica is reported. Heterologous expression of either DGA1 or LRO1 in a mutant of the budding yeast Saccharomyces cerevisiae defective in triacylglycerol synthesis restores the formation of this neutral lipid.

Lipid storage and mobilization pathways in yeast.

Biochemistry, cell biology and molecular biology of lipids can be properly studied using the yeast Saccharionyces cerevisiae as a model system. We employ this microorganism to investigate pathways of neutral lipid (triacylglycerol, steryl ester) synthesis, storage and mobilization and to identify major gene products involved in these processes. The steryl ester synthases Are1p and Are2p were shown to catalyze steryl ester formation, and Dgalp and Lro1p were identified as major enzymes of triacylglycerol synthesis.

Dynamics of neutral lipid storage and mobilization in yeast.

We make use of the yeast Saccharomyces cerevisiae as a flexible experimental system to investigate coordinate pathways of neutral lipid synthesis, storage and mobilization with special emphasis on the role of different organelles in these processes. Recently, a number of new gene products involved in triacylglycerol (TAG) and steryl ester (STE) metabolism were identified in our laboratory and by other groups. STE are synthesized by the two STE synthases Are1p and Are2p, whereas TAG are formed mainly through the action of the two TAG synthases Dga1p and Lro1p with minor contributions of Are1p and Are2p.

Synthesis, storage and degradation of neutral lipids in yeast.

The single cell eukaryote Saccharomyces cerevisiae is an attractive model to study the complex process of neutral lipid (triacylglycerol and steryl ester) synthesis, storage and turnover. In mammals, defects in the metabolism of these lipids are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Since the yeast harbors many counterparts of mammalian enzymes involved in these pathways, conclusions drawn from research with the microorganism can be readily applied to the higher eukaryotic system.

The life cycle of neutral lipids: synthesis, storage and degradation.

Triacylglycerols (TAGs), steryl esters (SEs) and wax esters (WEs) form the group of neutral lipids. Whereas TAGs are present in all types of cell, the occurrence of SEs in prokaryotes is questionable, and the presence of WEs as storage molecules is restricted to plants and a few bacteria. Here, we summarize recent knowledge on the formation, storage and degradation of TAGs and SEs in various cell types.

Lipid particle composition of the yeast Yarrowia lipolytica depends on the carbon source.

Lipid particles (LP) of all types of cells are a depot of neutral lipids. The present investigation deals with the isolation of LP from the yeast Yarrowia lipolytica and the characterization of their lipid and protein composition. Properties of LP varied depending on the carbon source.

Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae are localized to lipid particles.

Triacylglycerol (TAG) lipases are required for mobilization of TAG stored in lipid particles. Recently, Tgl3p was identified as a major TAG lipase of the yeast Saccharomyces cerevisiae (Athenstaedt, K., and Daum, G.

Flux of sterol intermediates in a yeast strain deleted of the lanosterol C-14 demethylase Erg11p.

Lanosterol C-14 demethylase Erg11p of the yeast Saccharomyces cerevisiae catalyzes the enzymatic step following formation of lanosterol by the lanosterol synthase Erg7p in lipid particles (LP). Localization experiments employing microscopic inspection and cell fractionation revealed that Erg11p in contrast to Erg7p is associated with the endoplasmic reticulum (ER). An erg11Delta mutation in erg3Delta background, which is required to circumvent lethality of the erg11 defect, did not only change the sterol pattern but also the sterol distribution within the cell.

Loss of Swiss cheese/neuropathy target esterase activity causes disruption of phosphatidylcholine homeostasis and neuronal and glial death in adult Drosophila.

The Drosophila Swiss cheese (sws) mutant is characterized by progressive degeneration of the adult nervous system, glial hyperwrapping, and neuronal apoptosis. The Swiss cheese protein (SWS) shares 39% sequence identity with human neuropathy target esterase (NTE), and a brain-specific deletion of SWS/NTE in mice causes a similar pattern of progressive neuronal degeneration. NTE reacts with organophosphate compounds that cause a paralyzing axonal degeneration in humans and has been shown to degrade endoplasmic reticulum-associated phosphatidylcholine (PtdCho) in cultured mammalian cells.

Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica.

Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal beta-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation.

A yeast strain lacking lipid particles bears a defect in ergosterol formation.

Lipid particles of the yeast Saccharomyces cerevisiae are storage compartments for triacylglycerols (TAG) and steryl esters (STE). Four gene products, namely the TAG synthases Dga1p and Lro1p, and the STE synthases Are1p and Are2p contribute to storage lipid synthesis. A yeast strain lacking the four respective genes is devoid of lipid particles thus providing a valuable tool to study the physiological role of storage lipids and lipid particles.

Engineering of Saccharomyces cerevisiae for the production of L-glycerol 3-phosphate.

L-glycerol 3-phosphate (L-G3P) was accumulated in Saccharomyces cerevisiae by pathway engineering. Intracellular concentration of this metabolic intermediate could be increased more than 20 times compared to the wild type by overexpressing GPD1 encoding the glycerol 3-phosphate dehydrogenase in a gpp1 Delta gpp2 Delta mutant which lacks both isoenzymes of glycerol 3-phosphatase. Investigation of cellular pattern of triacylglycerols and glycerophospholipids did not reveal considerable changes due to accumulation of their precursor L-G3P.

In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity.

In Saccharomyces cerevisiae, the 3-keto reductase (Erg27p) encoded by ERG27 gene is one of the key enzymes involved in the C-4 demethylation of the sterol intermediate, 4,4-dimethylzymosterol. The oxidosqualene cyclase (Erg7p) encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study, we found that erg27 strains grown on cholesterol- or ergosterol-supplemented media did not accumulate lanosterol or 3-ketosterols but rather squalene, oxidosqualene, and dioxidosqualene intermediates normally observed in ERG7 (oxidosqualene cyclase) mutants.

YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae.

Previous work from our laboratory (Athenstaedt, K., Zweytick, D., Jandrositz, A.

The neurodegeneration mutant löchrig interferes with cholesterol homeostasis and Appl processing.

The novel Drosophila mutant löchrig (loe) shows progressive neurodegeneration and neuronal cell death, in addition to a low level of cholesterol ester. loe affects a specific isoform of the gamma-subunit of AMP-activated protein kinase (AMPK), a negative regulator of hydroxymethylglutaryl (HMG)-CoA reductase and cholesterol synthesis in vertebrates. Although Drosophila cannot synthesize cholesterol de novo, the regulatory role of fly AMPK on HMG-CoA reductase is conserved.

Yeast oxidosqualene cyclase (Erg7p) is a major component of lipid particles.

Oxidosqualene cyclase of the yeast encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study (Athenstaedt, K., Zweytick, D.