Triacylglycerol lipases of the yeast

All eukaryotes including the yeast contain a lipid storage compartment which is named lipid particle, lipid droplet or oil body. Lipids accumulating in this subcellular fraction serve as a depot of energy and building blocks for membrane lipid synthesis. In the yeast, the major storage lipids are triacylglycerols (TGs) and steryl esters (SEs). An important step in the life cycle of these non-polar lipids is their mobilization from their site of storage and channeling of their degradation components to the appropriate metabolic pathways. A key step in this mobilization process is hydrolysis of TG and SE which is accomplished by lipases and hydrolases. In this review, we describe our recent knowledge of TG lipases from the yeast based on biochemical, molecular biological and cell biological information. We report about recent findings addressing the versatile role of TG lipases in lipid metabolism, and discuss non-polar lipid homeostasis and its newly discovered links to various cell biological processes in the yeast.     

Triacylglycerol biosynthesis in everted sacs of rat intestinal mucosa.

The molecular specificity of the biosynthesis of triacylglycerols by rat intestinal mucosa was examined by means of radioactive and mass tracers, and thin-layer chromatography with silver nitrate and gas-liquid chromatography with radioactivity monitoring. Bile salt micelles of alternately labeled monoacylglycerols and free fatty acids were incubated with everted sacs of intestinal mucosa for various periods of time and the triacylglycerols isolated by solvent extraction and thin-layer chromatography. Analyses of the molecular species of the triacylglycerols labeled from monoacylglycerols showed that the 2-monoacylglycerol pathway was responsible for the biosynthesis of a maximum of 90% and the X-1-monoacylglycerol pathway for about 10% of the total radioactive triacylglycerols. Detailed analyses of the molecular species of triacylglycerols labeled fro free fatty acids showed that the phosphatidic acid pathway contributed a minimum of 20-30% of the total labeled triacylglycerol formed. There was a preferential utilization in triacylglycerol biosynthesis of the more unsaturated diacylglycerols arising from the monoacylglycerol pathway and of the more saturated diacylglycerols originating from the phosphatidic acid pathway. The above experiments do not allow a demonstration of the utilization of the sn-2,3-diacylglycerols in triacylglycerol biosynthesis but are not inconsistent with it.     

Triacylglycerol biosynthesis in bovine liver and subcutaneous adipose tissue.

1. Adipose tissue from Angus and Brahman steers incubated with [1-14C]palmitate in the absence and presence of glucose exhibited a greater rate of lipid production than liver (P < 0.05). 2. Homogenates of adipose tissue used in the glycerol-3-phosphate acyltransferase assay exhibited a greater glycerolipid specific activity (nmol lipid/mg protein/30 min) when compared to liver (P < 0.05). 3. The inverse was true for liver homogenates when calculated for tissue activity (nmol lipid/g tissue/30 min). 4. Lysophosphatidate was produced in greater (P < 0.05) amounts than all other glycerolipids in the glycerol-3-phosphate acyltransferase assay. 5. The activity of phosphatidate phosphohydrolase in liver homogenates displayed greater rates than their respective adipose tissue homogenates. 6. Diacylglycerol acyltransferase activity was greater in adipose tissue homogenates compared to liver homogenates.     

Triacylglycerol biosynthesis in the adipose tissue of the obese-hyperglycaemic mouse.

Obesity in obese-hyperglycaemic mouse is associated with an increase in number and size of adipocytes. Adipocytes from the obese mouse showed increased incorporation of [14C]acetate and[14C]glucose into triacylglycerol. This increased capacity of triacylglycerol formation was correlated with increased activities of various triacylglycerol-forming enzymes measured in the microsomal fraction of adipose tissue from obese mice. Microsomal fractions from lean and obese mice contained sn-glycerol 3-phosphate acyltransferase, phosphatidate phosphohydrolase and diacylglycerol acyltransferase. Phosphatidate phosphohydrolase was also detected in the soluble fraction. In the presence of Mg2+, the phosphatidate phsophohydrolase from the soluble and the microsomal fractions was active towards membrane-bound phosphatidate. Among the three enzymes studied here, the increase in Mg2+-dependent phosphatidate phosphohydrolase was most prominent in adipose tissue of obese mice.     

Fatty acid biosynthesis in yeast

Summary Fatty acid synthetase and acetyl CoA carboxylase mutants have been used to study several aspects of fatty acid biosynthesis in yeast: the contribution of the various enzymes of fatty acid biosynthesis and modification to the overall cellular fatty acid composition, the mechanism of fatty acyl chain elongation in yeast, the molecular structure and the reaction mechanism of the fatty acid synthetase complex and the genetic control of the biosynthesis of this multi-enzyme system. Genetic and biochemical evidence suggest an ₆₆ molecular structure of this complex, where and are multifunctional proteins comprising, respectively, 3 and 5 of the various fatty acid synthetase component functions. The two subunits and are synthesized on two different, unliked genes, fas 2 and fas 1. The biosynthesis of both is coordinated. The various component enzyme activities reside in distinct domains on the multifunctional chains. While most domains appear to be functionally independent, the three acyl transferases exhibit extensive mutual interactions. It is suggested that the biosynthesis of a multifunctional protein is favoured on the grounds of kinetics and regulation as compared with the formation of a complex of the corresponding individual enzymes.     

Triacylglycerol synthesis in lipid particles from baker's yeast (Saccharomyces cerevisiae).

Triacylglycerol synthesis has been studied in a lipid particle preparation of baker's yeast (Saccharomyces cerevisiae), and compared with the synthesis in other subcellular fractions. Fatty acid-CoA ligase (AMP) (EC 6.2.1.3) activity and sn-glycerol 3-phosphate acyltransferase activity (EC 2.3.1.15) were present in all the subcellular fractions tested but the highest specific activities of both enzymes were observed with the lipid particle fraction. The products of the glycerol 3-phosphate acylation indicate that triacyglycerol synthesis proceeds through the phosphatidic acid pathway. However, only a small and nearly constant amount of lysophosphatidic acid was found with the lipid particle fraction while the other subcellular fraction produced lysophosphatidic and phosphatidic acid with a more pronounced precursor/product relationship. Triacylglycerol synthesis from endogenous diacylglycerol present in the lipid particle was also demonstrated.     

Control of lysine biosynthesis in yeast

The inhibition of the pathway of biosynthesis of lysine in yeast has been studied in vivo. The site sensitive to inhibition at low concentrations of lysine is after α-aminoadipate on the pathway. A test was made for a branch point in the pathway, but no evidence was obtained to demonstrate a branch point.     

Triacylglycerol and phospholipid fatty acids of the silverleaf whitefly: composition and biosynthesis

The identification and composition of the fatty acids of the major lipid classes (triacylglycerols and phospholipids) within Bemisia argentifolii Bellows and Perring (Homoptera: Aleyrodidae) nymphs were determined. Comparisons were made to fatty acids from the internal lipids of B. argentifolii adults. The fatty acids, as ester derivatives, were analyzed by capillary gas chromatography (CGC) and CGC-mass spectrometry (MS). All lipid classes contained variable distributions of eight fatty acids: the saturated fatty acids, myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), arachidic acid (20:0); the monounsaturated fatty acids, palmitoleic acid (16:1), oleic acid (18:1); the polyunsaturated fatty acids, linoleic acid (18:2), linolenic acid (18:3). Fourth instar nymphs had 5-10 times the quantities of fatty acids as compared to third instar nymphs and 1-3 times the quantities from adults. The fatty acid quantity differences between fourth and third instar nymphs were related to their size and weight differences. The percentage compositions for fatty acids from each lipid class were the same for the pooled groups of third and fourth instar nymphs. For nymphs and adults, triacylglycerols were the major source of fatty acids, with 18:1 and 16:0 acids as major components and the majority of the polyunsaturated fatty acids, 18:2 and 18:3 were present in the two phospholipid fractions, phosphatidylethanolamine and phosphatidylcholine. Evidence was obtained that whiteflies indeed synthesize linoleic acid and linolenic acid de novo: radiolabel from [2-(14)C] acetate was incorporated into 18:2 and 18:3 fatty acids of B. argentifolii adults and CGC-MS of pyrrolidide derivatives established double bonds in the Delta(9,12) and Delta(9,12,15) positions, respectively.     

Tailored biosynthesis of gibberellin plant hormones in yeast

The application of small amounts of natural plant growth hormones, such as gibberellins (GAs), can increase the productivity and quality of many vegetable and fruit crops. However, gibberellin growth hormones usage is limited by the high cost of their production, which is currently based on fermentation of a natural fungal producer Fusarium fujikuroi that produces a mix of several GAs. We explored the potential of the oleaginous yeast Yarrowia lipolytica to produce specific profiles of GAs. Firstly, the production of the GA-precursor ent-kaurenoic acid (KA) at 3.75 mg/L was achieved by expression of biosynthetic enzymes from the plant Arabidopsis thaliana and upregulation of the mevalonate (MVA) pathway.We then built a GA₄-producing strain by extending the GA-biosynthetic pathway and upregulating the MVA-pathway further, resulting in 17.29 mg/L GA₄. Additional expression of the F. fujikoroi GA-biosynthetic enzymes resulted in the production of GA₇ (trace amounts) and GA₃ (2.93 mg/L). Lastly, through protein engineering and the expression of additional KA-biosynthetic genes, we increased the GA₃-production 4.4-fold resulting in 12.81 mg/L. The developed system presents a promising resource for the recombinant production of specific gibberellins, identifying bottlenecks in GA biosynthesis, and discovering new GA biosynthetic genes.ClassificationBiological Sciences, Applied Biological Sciences.