Jasminka Rupčić

Cell lipids of the Candida lipolytica yeast grown on methanol

Candida lipolytica yeast, grown on 1% methanol as the only carbon and energy source, synthesized 4.9% of dry cell mass as lipids, 52.3% of which were polar lipids. Polar lipids consisted mainly of phospholipids and sphingolipids as their minor components. The total long-chain bases content has been found to account only for 0.7% of the polar lipids. The long-chain bases composition determined by thin-layer and gas chromatography shows a preponderance of trihydroxy bases and a small amount of dihydroxy bases. The striking finding was the high content of 19-phytosphingosine (90.8% of total long-chain bases). Fatty acid (FA) composition of polar lipids was characterized by the relatively high concentration of unsaturated fatty acids (66.4% of total FA) and by the predominance of fatty acids with 16 carbon atoms (85.0% of total FA).

Isolation and chemical composition of the ceramide of the Candida lipolytica yeast

Candida lipolytica yeast was grown batchwise on glucose medium. Ceramide was quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography. Fatty acids and long-chain bases, obtained after acid methanolysis, were analyzed by gas-liquid chromatography. Fatty acid composition was characterized by predominance of fatty acids shorter than 20 carbon atoms (72.2% of total fatty acids) and by high concentration of fatty acids with 16 carbon atoms (43.3% of total fatty acids). The dominant fatty acids were h16:0 and 18:1. The major fatty acid with more than 20 carbon atoms was h24:0. Hydroxylated fatty acids quantitatively represented almost half of the total fatty acids. The ceramide contained an unusual long-chain base composition. Besides the well-known trihydroxy bases, 18- and 20-phytosphingosine, which is a common characteristic for yeast sphingolipids, a high proportion of more polar bases than the trihydroxy ones, was found. The structures of 4,5-dihydroxyicosasphinanine (1,3,4,5-tetrahydroxy-2-aminooctadecane) and 4,5-dihydroxyicosasphinganine (1,3,4,5-tetrahydroxy-2-aminoeicosane) were tentatively assigned to these bases. Their chromatographic properties were identical to those of synthetic bases.

Lipid analysis of the plasma membrane and mitochondria of brewer’s yeast

The plasma membrane and mitochondria of bottom fermenting brewer’s yeast obtained as a by-product of industrial beer production were isolated and the lipid fraction was analyzed. The phospholipid content accounted for 78 mg/g protein in the plasma membrane and 59 mg/g protein in the mitochondria. Major phospholipids in both preparations were phosphatidylinositol, phosphatidylcholine and phosphatidyl-ethanolamine but their proportions differed significantly. In the plasma membrane phosphatidy linositol, and in the mitochondria phosphatidylcholine were present in the highest concentration (37 and 30 %, respectively). The main classes of neutral lipids (triacylglycerols, ergosterol, squalene and steryl esters) were twice more abundant in the plasma membrane than in the mitochondria (61 and 33 mg/g protein, respectively). A characteristic of the neutral lipid composition of both organelles was the low content of ergosterol (12 and 7 mg/g protein, respectively) and a high content of squalene (25 and 22 mg/g protein). The main feature of the fatty acid composition of both organelles was the preponderance of saturated fatty acids (78 and 79 %, respectively), among which palmitic acid was the principal one. The most expressed characteristics of lipid fractions of the analyzed plasma membranes and mitochondria, high concentration of squalene and preponderance of saturated fatty acids are the consequences of anaerobic growth conditions. The lack of oxygen had possibly the strongest effect on the lipid composition of the plasma membranes and mitochondria of bottom fermenting brewer’s yeast.

The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast

Candida lipolytica yeast was grown batchwise on two different carbon sources, glucose and n-hexadecane. Free ceramides were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography. Their composition, after acid methanolysis, was analysed by gas-liquid chromatography. The ceramide content accounted for 2.6% of the total cell lipids in hexadecane-grown cells, which was 1.5 times higher than in glucose-grown cells. The fatty acid composition of ceramides was characterized by the predominance of fatty acids shorter than 20 carbon atoms and by high concentrations of fatty acids with 16 carbon atoms after growth on both carbon sources. The dominant fatty acid was hydroxylated 16:0 in the glucose-grown cells and 16:0 in the hexadecane-grown cells. The striking finding was the low degree of fatty acid hydroxylation and relatively high proportion of odd-numbered fatty acids in ceramide of the n-hexadecane-grown cells. The ceramides contained an unusual long-chain base composition. In hexadecane-grown cells more than 60% of the long-chain bases were C19 phytosphingosine. In glucose-grown cells more than one-half of the total long-chain bases were tetrahydroxy bases, 4,5-dihydroxysphinganine and 4,5-dihydroxyeicosasphinganine.

Influence of stressful fermentation conditions on neutral lipids of a Saccharomyces cerevisiae brewing strain.

The neutral lipid fraction of the aerobically grown starter yeast culture of a Saccharomyces cerevisiae brewing strain, and three-first recycled yeast generations exposed to multiple stress factors during beer fermentation was studied. No pronounced changes in the cellular neutral lipid content between the non-stressed starter and stressed recycled cells were found. However, it was found that recycled yeast generations modulate their neutral lipid composition during fermentation. The ergosterol content was increased at the expense of steryl esters (SEs) and squalene, which resulted in a higher ergosterol/SEs molar ratio and a slightly higher ergosterol/squalene molar ratio. In addition, the proportion of unsaturated fatty acids, mainly palmitoleic acid increased in the neutral lipid fraction of the stressed recycled yeast generations. These results suggest that some specific neutral lipid species and fatty acids stored in the neutral lipid fraction are involved in the adaptive response of the brewer’s yeast to stressful fermentation conditions. The striking finding was a high squalene content in the neutral lipid fraction of both the starter yeast culture and recycled yeast generations (22.4 vs. 19–20%, respectively), implying a possible biotechnological exploitation of this biologically active molecule from the yeast biomass.

Effect of Growth Phase on the Content and Composition of Ceramides of the Hydrocarbon-assimilating Yeast Candida lipolytica

Candida lipolytica yeast was grown batchwise on n-hexadecane as the carbon and energy source. Ceramides were quantitatively isolated from total lipids of exponential and stationary phase cells by a combination of column chromatography and preparative high-performance thin-layer chromatography. After acid methanolysis their composition was analyzed by gas-liquid chromatography. The ceramide content of the exponential phase cells was two times higher than the one of the stationary phase cells. The composition of long-chain base moiety of ceramides did not change significantly during the growth. In both growth phases 19-phytosphingosine was the major long-chain base. However, the fatty acid composition of ceramides changed greatly during the growth. In the exponential growth phase, ceramides contained predominantly fatty acids greater than 20 carbon atoms, while fatty acids shorter than 20 atoms predominated in ceramides of the stationary phase, 16:0 being the main one. In the exponential growth phase fatty acid moiety of ceramides was characterized by unusually high degree of unsaturation and relatively high proportion of odd-numbered fatty acids. However, the proportion of both, unsaturated and odd-numbered fatty acid decreased significantly in ceramides of the stationary phase. The unexpected finding was the absence of fatty acid hydroxylation of ceramides in the exponential phase cells and unusually low degree of hydroxylation in the stationary phase.