Analysis of the β-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway

2005 ◽  
Vol 1734 (2) ◽  
pp. 169-177 ◽  
Author(s):  
Julien Robert ◽  
Silvia Marchesini ◽  
Syndie Delessert ◽  
Yves Poirier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Unsaturated Fatty Acid ◽  
Pha Synthase ◽  
Recombinant Saccharomyces Cerevisiae ◽  
Dependent Pathway

scholarly journals Engineering the Monomer Composition of Polyhydroxyalkanoates Synthesized in Saccharomyces cerevisiae

2006 ◽  
Vol 72 (1) ◽  
pp. 536-543 ◽  
Author(s):  
Bo Zhang ◽  
Ross Carlson ◽  
Friedrich Srienc
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Chain Length ◽  
Fatty Acid Metabolism ◽  
Acid Metabolism ◽  
Pha Synthase ◽  
Fatty Acid Degradation ◽  
Medium Chain ◽  
Acid Degradation ◽  
Wide Range

ABSTRACT Polyhydroxyalkanoates (PHAs) have received considerable interest as renewable-resource-based, biodegradable, and biocompatible plastics with a wide range of potential applications. We have engineered the synthesis of PHA polymers composed of monomers ranging from 4 to 14 carbon atoms in either the cytosol or the peroxisome of Saccharomyces cerevisiae by harnessing intermediates of fatty acid metabolism. Cytosolic PHA production was supported by establishing in the cytosol critical β-oxidation chemistries which are found natively in peroxisomes. This platform was utilized to supply medium-chain (C6 to C14) PHA precursors from both fatty acid degradation and synthesis to a cytosolically expressed medium-chain-length (mcl) polymerase from Pseudomonas oleovorans. Synthesis of short-chain-length PHAs (scl-PHAs) was established in the peroxisome of a wild-type yeast strain by targeting the Ralstonia eutropha scl polymerase to the peroxisome. This strain, harboring a peroxisomally targeted scl-PHA synthase, accumulated PHA up to approximately 7% of its cell dry weight. These results indicate (i) that S. cerevisiae expressing a cytosolic mcl-PHA polymerase or a peroxisomal scl-PHA synthase can use the 3-hydroxyacyl coenzyme A intermediates from fatty acid metabolism to synthesize PHAs and (ii) that fatty acid degradation is also possible in the cytosol as β-oxidation might not be confined only to the peroxisomes. Polymers of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers can be controlled by feeding the appropriate substrates. This ability should permit the rational design and synthesis of polymers with desired material properties.

scholarly journals Synthesis of Polyhydroxyalkanoate in the Peroxisome of Saccharomyces cerevisiae by Using Intermediates of Fatty Acid β-Oxidation

2001 ◽  
Vol 67 (11) ◽  
pp. 5254-5260 ◽  
Author(s):  
Yves Poirier ◽  
Nadine Erard ◽  
Jean MacDonald-Comber Petétot
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Isocitrate Lyase ◽  
Dry Weight ◽  
Pha Synthase ◽  
Recombinant Yeast ◽  
Medium Chain Length ◽  
The Media

ABSTRACT Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads.Saccharomyces cerevisiae was transformed with thePseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinantS. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the β-oxidation of fatty acids present in the media. S. cerevisiaecan thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.

Effects of unsaturated fatty acid deprivation on neutral lipid synthesis in Saccharomyces cerevisiae

1982 ◽  
Vol 152 (2) ◽  
pp. 747-756
Author(s):  
T M Buttke ◽  
A L Pyle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Lipid Metabolism ◽  
Cell Growth ◽  
Unsaturated Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Lipid Synthesis ◽  
Yeast Cells

The effects of unsaturated fatty acid deprivation on lipid synthesis in Saccharomyces cerevisiae strain GL7 were determined by following the incorporation of [14C]acetate. Compared to yeast cells grown with oleic acid, unsaturated fatty acid-deprived cells contained 200 times as much 14C label in squalene, with correspondingly less label in 2,3-oxidosqualene and 2,3;22,23-dioxidosqualene. Cells deprived of either methionine or cholesterol did not accumulate squalene, demonstrating that the effect of unsaturated fatty acid starvation on squalene oxidation was not due to an inhibition of cell growth. Cells deprived of olefinic supplements displayed additional changes in lipid metabolism: (i) an increase in 14C-labeled diacylglycerides, (ii) a decrease in 14C-labeled triacylglycerides, and (iii) increased levels of 14C-labeled decanoic and dodecanoic fatty acids. The changes in squalene oxidation and acylglyceride metabolism in unsaturated fatty acid-deprived cells were readily reversed by adding oleic acid. Pulse-chase studies demonstrated that the [14C]squalene and 14C-labeled diacylglycerides which accumulated during starvation were further metabolized when cells were resupplemented with oleic acid. These results demonstrate that unsaturated fatty acids are essential for normal lipid metabolism in yeasts.

scholarly journals Saccharomyces cerevisiae CWH43Is Involved in the Remodeling of the Lipid Moiety of GPI Anchors to Ceramides

2007 ◽  
Vol 18 (11) ◽  
pp. 4304-4316 ◽  
Author(s):  
Mariko Umemura ◽  
Morihisa Fujita ◽  
Takehiko Yoko-o ◽  
Akiyoshi Fukamizu ◽  
Yoshifumi Jigami
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Unsaturated Fatty Acid ◽  
Terminal Region ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Lipid Moiety

The glycosylphosphatidylinositol (GPI)-anchored proteins are subjected to lipid remodeling during their biosynthesis. In the yeast Saccharomyces cerevisiae, the mature GPI-anchored proteins contain mainly ceramide or diacylglycerol with a saturated long-fatty acid, whereas conventional phosphatidylinositol (PI) used for GPI biosynthesis contains an unsaturated fatty acid. Here, we report that S. cerevisiae Cwh43p, whose N-terminal region contains a sequence homologous to mammalian PGAP2, is involved in the remodeling of the lipid moiety of GPI anchors to ceramides. In cwh43 disruptant cells, the PI moiety of the GPI-anchored protein contains a saturated long fatty acid and lyso-PI but not inositolphosphorylceramides, which are the main lipid moieties of GPI-anchored proteins from wild-type cells. Moreover, the C-terminal region of Cwh43p (Cwh43-C), which is not present in PGAP2, is essential for the ability to remodel GPI lipids to ceramides. The N-terminal region of Cwh43p (Cwh43-N) is associated with Cwh43-C, and it enhanced the lipid remodeling to ceramides by Cwh43-C. Our results also indicate that mouse FRAG1 and C130090K23, which are homologous to Cwh43-N and -C, respectively, share these activities.

scholarly journals Ethanol Tolerance in the Yeast Saccharomyces cerevisiae Is Dependent on Cellular Oleic Acid Content

2003 ◽  
Vol 69 (3) ◽  
pp. 1499-1503 ◽  
Author(s):  
Kyung Man You ◽  
Claire-Lise Rosenfield ◽  
Douglas C. Knipple
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Unsaturated Fatty Acid ◽  
Palmitoleic Acid ◽  
Ethanol Tolerance ◽  
Vaccenic Acid ◽  
Yeast Cells ◽  
Oleic Acid Content ◽  
Yeast Saccharomyces Cerevisiae

ABSTRACT In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Δ9Z-C16:1) and oleic acid (Δ9Z-C18:1), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C16:0) and stearic acid (C18:0), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Δ9 and Δ11) and substrate chain-length preferences (i.e., C16:0 and C18:0); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Δ11Z-C18:1), whereas neither Δ11Z-C16:1 nor palmitoleic acid (Δ9Z-C16:1) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.

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