Inhibition of yeast phosphatidic-acid synthesis by free fatty acids

Particulate preparations obtained from cells of yeast Saccharomyces sake have been shown to possess glycerolphosphate acyltransferase and 1-acylglycerolphosphate acyltransferase activities. Glycerolphosphate acyltransferase exhibits a high specificity for saturated and monoenoic fatty acyl-CoA thioesters. When palmitoyl-CoA is employed as sole acyl group donor, the major lipid product is lysophosphatidic acid. 1-Acylglycerolphosphate acyltransferase of this yeast species has a rather strict specificity for monoenoic fatty acyl-CoA thioesters as acyl donor. These two acyltransferases are strongly inhibited in vitro by low concentrations of free fatty acids. 1-Acylglycerolphosphate acyltransferase is much more susceptible to fatty acid inhibition than glycerolphosphate acyltransferase. The inhibition is dependent not only on the concentration of fatty acid, but also on the length of exposure to fatty acid. Both saturated and unsaturated fatty acids inhibit the acyltransferase activities. The inhibitory effects of fatty acids cannot be ascribed to a nonspecific surfactant action of fatty acids. The present results support the view that free fatty acid serves as a regulator of glycerolipid synthesis.     

Utilization of C20-polyunsaturated fatty acids by a yeast fatty acid desaturase mutant

A study was made of the utilization of C₂₀-polyunsaturated fatty acids by the $\text{S. cerevisiae}$ fatty acid desaturase mutant $\text{olel-1}$, Arachidonic acid, 8,11,14-eicosatrienoic acid, and 5,8,11,14,17-eicosapentaenoic acid were about equally effective in supporting growth with lactate as the carbon source. The relative proportion of these fatty acids in total cell fatty acids was $\text{ca}$. 50%. 5,8,11-eicosatrienoic acid synthesized from oleate was less effective. Very little growth occurred with 11,14,17-eicosatrienoic acid or with 11,14-eicosadienoic acid. These results indicate the usefulness of the yeast mutant as a eucaryotic model for study of membrane systems enriched in specific C₂₀-polyunsaturated fatty acids.     

Selective denaturation of several yeast enzymes by free fatty acids.

The denaturation of eight purified yeast enzymes, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, alcohol dehydrogenase, beta-fructosidase, hexokinase and glucose-6-phosphate isomerase, promoted under controlled conditions by the free fatty acids myristic and oleic, is selective. Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1 oxidoreductase, EC 1.1.1.49) is extremely sensitive to destabilization and was studied in greater detail. Results show that chain length and degree of unsaturation of fatty acids are important to their destabilizing effect, and that ligands of the enzyme can afford protection. The denaturation process results in more than one altered form. These results can be viewed in the perspective of the possibility that amphipathic substances, and in particular free fatty acids, may play a role for enzyme degradation in vivo, by initiating steps of selective denaturation.     

Synthesis of unsaturated fatty acids

This article reviews synthetic routes leading to polyunsaturated fatty acids having “skipped” double bonds. Emphasis is placed on the “acetylenic approach”.The suitability of building blocks, their condensation reactions as well as the controlled reduction of triple bonds to cis double bonds are discussed. In addition, the application of the various methods to the preparation of polyunsaturated fatty acids labelled with ³H and/or ¹⁴C at distinct positions of the molecules is reviewed.     

Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast

To create a conditional system for molecular analysis of effects of polyunsaturated fatty acids (PUFA) on cellular physiology, we have constructed a strain of yeast (Saccharomyces cerevisiae) that functionally expresses, under defined conditions, the Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This strain produces up to 15% PUFA, exclusively under inducing conditions resulting in production of 4-hydroxy-2-nonenal, one of the major end products of n-6 polyunsaturated fatty acid peroxidation. The PUFA-producing yeast was initially more sensitive to oxidative stress than the wild-type strain. However, over extended time of cultivation it became more resistant to hydrogen peroxide indicating adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, PUFA-producing strain showed an increased concentration of endogenous ROS, while initially increased hydrogen peroxide sensitivity was followed by an increase in catalase activity and adaptation to oxidative stress. The deletion mutants constructed to be defective in the catalase activity lost the ability to adapt to oxidative stress. These data demonstrate that the cellular synthesis of PUFA induces endogenous oxidative stress which is overcome by cellular adaptation based on the catalase activity.     

Synthesis of green note aroma compounds by biotransformation of fatty acids using yeast cells coexpressing lipoxygenase and hydroperoxide lyase

Green notes are substances that characterize the aroma of freshly cut grass, cucumbers, green apples, and foliage. In plants, they are synthesized by conversion of linolenic or linoleic acid via the enzymes lipoxygenase (LOX) and hydroperoxide lyase (HPL) to short-chained aldehydes. Current processes for production of natural green notes rely on plant homogenates as enzyme sources but are limited by low enzyme concentration and low specificity. In an alternative approach, soybean LOX2 and watermelon HPL were overexpressed in Saccharomyces cerevisiae. After optimization of the expression constructs, a yeast strain coexpressing LOX and HPL was applied in whole cell biotransformation experiments. Whereas addition of linolenic acid to growing cultures of this strain yielded no products, we were able to identify high green note concentrations when resting cells were used. The primary biotransformation product was 3(Z)-hexenal, a small amount of which isomerized to 2(E)-hexenal. Furthermore, both aldehydes were reduced to the corresponding green note alcohols by endogenous yeast alcohol dehydrogenase to some extent. As the cosolvent ethanol was the source of reducing equivalents for green note alcohol formation, the hexenal/hexenol ratio could be influenced by the use of alternative cosolvents. Further investigations to identify the underlying mechanism of the rather low biocatalyst stability revealed a high toxicity of linolenic acid to yeast cells. The whole cell catalyst containing LOX and HPL enzyme activity described here can be a promising approach towards a highly efficient microbial green note synthesis process.     

Synthesis of ordinary and -hydroxy fatty acids by heart mitochondria

The fatty acids (FA) synthesized from acetate by intact rabbit heart mitochondria were identified. These FA were mainly 12 to 16 carbons long. One-half were β-hydroxy FA, and mass spectrometric analysis after [1-¹³C)acetate incorporation showed them to be synthesized de novo. The latter were oxidized by the mitochondria with an ADP pulse, which means that they were L(+) isomers. β-Hydroxymyristate was the predominant endogenous saturated β-hydroxy FA detected in heart mitochondria.     

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.     

Synthesis of fatty acids from malonyl-CoA and NADPH by pigeon liver fatty acid synthetase

Pigeon liver fatty acid synthetase has been found to catalyze the formation of palmitic acid from malonyl-CoA and NADPH in the absence of acetyl-CoA. Radio-chemical and spectral assays show that the activity of the complex in the absence of acetyl-CoA is about 25–30% of the activity in the presence of this compound. Initial velocities were determined for a series of reactions in which the malonyl-CoA concentration was varied over a range of 5–200 μm at a fixed NADPH concentration of 100μm and vice versa. No inhibitory effects of one substrate over the other were found. However, when the synthesis of fatty acids was studied in the presence of acetyl-CoA, a significant inhibitory effect of malonyl-CoA was observed. It has also been shown that the fatty acid synthetase synthesizes triacetic lactone from malonyl-CoA in the absence of NADPH and acetyl-CoA. No evidence was obtained for the direct decarboxylation of malonyl-CoA to acetyl-CoA in this reaction. Hence it is proposed that decarboxylation of the malonyl moiety bound covalently to 4′-phosphopantetheine occurs to yield acetyl-4′-phosphopantetheine. Further, it is proposed that the acetyl moiety of the latter compound is transferred to the cysteine site of the enzyme complex and that fatty acid synthesis proceeds in the presence of NADPH as proposed by Phillips et al. [Arch. Biochem. Biophys.138, 380 (1970)]. In the absence of NADPH triacetic lactone is formed.