Higher fatty acids of Candida lipolytica grown in n-alkanes by the industrial method ("Toprina")]

The present study deals with the composition of superior fatty acids of total lipids, polar lipids, and neutral lipids from dried biomass of Candida lipolytica grown by industrial process ("Toprina") on n-alkanes (C10-C20) extracted from petroleum. The data related to our knowledge about yeast and Candida lipolytica, lead to the conclusion that fatty acids feature of "Toprina" are similar to the Candida lipolytica ones grown in batch culture at the same conditions. In addition, a possible physiologic role of 17:1 and 17:2 is considered, in the perspective of the utilization of "Toprina" in animal food.     

Different fatty acids in the egg, blood and feces of laying hens fed a diet containing "Toprina"

Total lipids (TL, phospholipids (PL) and fatty acid composition of TL and PL (the latter in blood and eggs only) were estimated in Candida lipolytica cultivated on n-alkanes by industrial method ("Toprina") in 0%, 10% and 15% "Toprina" diets and in eggs, blood and faeces of laying hens fed these diets. With increasing concentrations of dietary "Toprina" 15:0 and 17:0 fatty acids increased, 17:1 and 17:2, typical of the product, appeared and increased in diets, eggs, blood and faeces. Other fatty acid values were not affected by dietary yeast.     

Unusual fatty acids in turkeys fed a diet containing "Toprina". I. Composition of the higher fatty acids in "Toprina", the diet, feces and blood]

Totals lipids (TL) phospholipids (PL) and fatty acid composition of TL and PL (the latter in blood only) were estimated in Candida lipolytica cultivated on n-alkanes by industrial method ("Toprina"), in 0%, 10% and 15% "Toprina" diets and in faeces and blood of turkeys, males and females, fed these diets. With increasing concentrations of dietary "Toprina" phospholipids and 15:0 and 17:0 fatty acids increased, 17:1 and 17:2, typical of the product, appeared and increased in diets and blood. Other lipid and fatty acids values were not affected by dietary yeast.     

Unusual fatty acids in turkeys fed a diet containing "Toprina". III. Composition of higher fatty acids in the heart and skeletal muscle

Total lipids (TL), phospholipids (PL) and fatty acid composition of TL and PL were estimated in breast, leg and cardiac muscles of turkeys, males and females, fede diets containing 0%, 10% and 15% of Candida lipolytica cultivated on n-alkanes by industrial method ("Toprina"). LT and PL were also valued in "Toprina" and diets. With increasing concentration of dietary yeasts it was observed that: a) Pl increased in diets, breast and leg muscles; b) fatty acids 15:0 and 17:0 increased, 17:1 and 17:2, typical of the product, appeared and increased in the tissues.     

Mixed cultures of different yeasts species and yeasts with filamentous fungi in the SCP production. I. Production of single cell protein by mixed cultures Candida lipolytica and Candida tropicalis.

The aim of this study was to determine the application of mixed cultures Candida lipolytica and Candida tropicalis in the SCP production. N-paraffin fraction of crude oil and individual n-alkanes C:7--C:17 and glucose were used as carbon sources. The cultures were grown on laboratory scale in shaking flasks and in a 7 1 fermentor. It was found that the mixed cultures gave about 18% higher yield of biomass than the individual cultures.     

Yeast growth on solid natural oil]

Two strains of the yeast Candida lipolytica with a specific response to n-alkanes could grow on a medium with paraffins only in the case of contact of the cells with the particles of hydrocarbons. A mixture of paraffins with a solidification point of 35 degrees C contained 41.6% of n-alkanes with the carbon chain from C8 to C36. Assimilation of n-alkanes was studied along with the yeast growth in the course of fermentation. Penetration of the hydrocarbons into the yeast cells and the specificity of the substrates for both yeast strains are discussed.     

Acetyl-coenzyme-A carboxylase of Candida lipolytica. 2. Regulation of cellular content and synthesis of the enzyme.

The level of acetyl-coenzyme-A carboxylase activity in Candida lipolytica undergoes large variations depending upon the carbon source on which the yeast is grown. Cells grown on n-alkanes or fatty acids exhibit a lower activity level than do cells grown on glucose. Among the n-alkanes and fatty acids tested, n-heptadecane, n-octadecane, oleic acid and linoleic acid reduce the enzyme activity to the lowest levels, which are 16-18% of the activity level in glucose-grown cells. Immunochemical titrations and Ouchterlony double-diffusion analysis with specific antibody as well as kinetic studies have indicated that the observed decrease in the level of acetyl-CoA carboxylase activity is due to a reduction in the cellular content of the enzyme. Furthermore, isotopic leucine incorporation studies with the use of the immunoprecipitation technique have demonstrated that the relative rate of synthesis of the enzyme in oleic-acid-grown cells is diminished to 12% of that in glucose-grown cells. Evidence has also been obtained to support the view that the enzyme in this yeast is not degraded at a rate high enough to contribute to the marked decrease in the cellular content of the enzyme. Thus, it is concluded that the reduction in acetyl-CoA carboxylase content in fatty-acid-grown cells is due to diminished synthesis of the enzyme.     

Control of fatty-acid synthetase levels by exogeneous long-chain fatty acids in the yeasts Candida lipolytica and Saccharomyces cerevisiae.

Endogeneous fatty acid biosynthesis in the two yeast species, Saccharomyces cerevisiae and Candida lipolytica is completely repressed by the addition of long-chain fatty acids to the growth medium. In Candida lipolytica, this repression is accompanied by a corresponding loss of fatty acid synthetase activity in the cell homogenate, when the cells were grown on fatty acids as the sole carbon source. The activity of the Saccharomyces cerevisiae fatty acid synthetase, however, remains unaffected by the addition of fatty acids to a glucose-containing growth medium. From fatty-acid-grown Candida lipolytica cells no fatty acid synthetase complex can be isolated, nor is there any immunologically cross-reacting fatty acid synthetase protein detectable in the crude cell extract. From this it is concluded that Candida lipolytica, but not Saccharomyces cerevisiae, is able to adapt to the growth on fatty acids either by repression of fatty acid synthetase biosynthesis or by a fatty-acid-induced proteolytic degradation of the multienzyme complex. Similarly, the fatty acid synthetase complex disappears rapidly from stationary phase Candida lipolytica cells even after growth in fatty-acid-free medium. Finally, it was found that the fatty acid synthetase complexes from Saccharomyces cerevisiae and Candida lipolytica, though very similar in size and subunit composition, were immunologically different and had no common antigenic determinants.     

Toxic effects of fatty acids on yeast cells: dependence of inhibitory effects on fatty acid concentration.

The yeasts Saccharomyces cerevisiae, Candida utilis, and Candida lipolytica were used to investigate the action of different concentrations of fatty acids (from acetic to myristic acid) on cell growth, division, uptake of inorganic phosphate, and substrate oxidation. The former two yeasts were found to undergo an inhibition of growth, cell division, and phosphate uptake at lower acid concentrations and to experience the inhibition of substrate oxidation at higher acid concentrations. The concentration dependence of the action of fatty acids can be classified into four categories: 1) subthreshold concentrations which do not inhibit growth and have either no effect on, or stimulate, oxygen consumption; 2) threshold concentrations which lower the rate of growth, cell division, and phosphate uptake but do not inhibit the oxidation of carbon substrate; 3) above-threshold concentrations which inhibit partially even substrate oxidation, and 4) microbicide concentrations. Candida lipolytica displays the same sensitivity toward the action of fatty acids as the above yeast species; however, the threshold concentrations are higher and can be quickly lowered owing to oxidation by the yeast. The concentrations of fatty acids found in the medium after cultivations of yeast with n-alkanes are of the same order as limiting concentrations; the formation of acids with twelve and less carbons in the molecule can thus be assumed to be one of the basic reasons for lowering of biomass yields during cultivations on these hydrocarbons.     

Peptidase profiles from non-albicans Candida spp. isolated from the blood of a patient with chronic myeloid leukemia and another with sickle cell disease

Candida lipolytica and Candida rugosa were isolated from blood samples from a patient with chronic myeloid leukemia (31 years old) and a patient with sickle cell disease (1-year-old), respectively. Isolates were grown for 48 h at 37 degrees C in either Sabouraud or tryptone soy broth (TSB). Peptidases were tested for using substrate sodium dodecyl sulfate-polyacrylamide gels with gelatin, casein, bovine serum albumin (BSA) or hemoglobin. Enzymography analyses were made on the following substrates: human albumin, IgG and human fibrinogen, which had been incubated with the concentrated supernatants. For C. lipolytica, a approximately 60-kDa gelatin-degrading serine proteolytic activity was found in the TSB supernantant as well as a metallopeptidase activity capable of hydrolysing human albumin, IgG and human fibrinogen. With C. rugosa, albumin, IgG and human fibrinogen substrates were degraded by an aspartyl-like peptidase activity. Supernatants from C. rugosa also showed three serine proteolytic activities towards gelatin (approximately 50 kDa, TSB), casein ( approximately 94 kDa, TSB) and BSA ( approximately 120-kDa, Sabouraud), in addition to a metallopeptidase capable of degrading casein ( approximately 110 kDa, Sabouraud). Little is known about peptidases of C. rugosa and C. lipolytica and this preliminary data may prove useful for future work on host-parasite relationship and antifungal agents.     

Control of triglyceride synthesis by the intracellular level of long-chain acyl coenzyme A for lipid synthesis

Candida lipolytica mutants defective in acyl coenzyme A synthetase I synthesized triglyceride to a markedly less extent than did the wild-type yeast, when grown on oleic acid. The synthesis of triglyceride was controlled by the level of long-chain acyl coenzyme A available for lipid synthesis, whereas the synthesis of phospholipids was hardly affected.     

Degradation of Hydrocarbons by Members of the Genus Candida III. Oxidative Intermediates from 1-Hexadecene and 1-Heptadecene by Candida lipolytica

Candida lipolytica (Phaff) was grown in a mineral-salts medium amended with either 1-hexadecene or 1-heptadecene as substrate. Intermediates of the same chain length as the substrate were isolated and identified by various analytical procedures. The following intermediates of 16 and 17 carbon atoms were identified: omega-unsaturated acids, omega-unsaturated primary and secondary alcohols, 1,2-epoxides, 1,2-diols, and 2-hydroxy acids. Based on the chemical structure of these compounds, three oxidative mechanisms are proposed for the degradation of long-chain 1-alkenes by this yeast: (i) methyl-group oxidation, (ii) double-bond oxidation, and (iii) subterminal oxidation.     

Evolutionary relationships among pathogenic Candida species and relatives.

Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida.     

Studies on the metabolism of unsaturated fatty acids. XVI. Purification and general properties of 2,4-dienoyl-CoA reductase from Candida lipolytica

2,4-Dienoyl-CoA reductase has been purified to homogeneity from Candida lipolytica cultivated in the presence of linoleic acid. The native enzyme had a molecular weight close to 360,000 as estimated by gel filtration on Sepharose CL-4B, whereas the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 33,000. The purified 2,4-dienoyl-CoA reductase from C. lipolytica gave a single precipitin line with antibodies raised against the purified enzyme from C. lipolytica. The general properties of the 2,4-dienyl-CoA reductase from C. lipolytica were examined. The enzyme had optimal pH at 6.5 and was inactivated by heat treatment at 50 degrees C for 10 min. trans-2,trans-4-Octadienoyl-CoA was the most active substrate of the dienoyl-CoA esters examined.     

Assimilation of chlorinated alkanes by hydrocarbon-utilizing fungi.

The fatty acid compositions of two filamentous fungi (Cunninghamella elegans and Penicillium zonatum) and a yeast (Candida lipolytica) were determined after the organisms were grown on 1-chlorohexadecane or 1-chlorooctadecane. These organisms utilized the chlorinated alkanes as sole sources of carbon and energy. Analyses of the fatty acids present after growth on the chlorinated alkanes indicated that 60 to 70% of the total fatty acids in C. elegans were chlorinated. Approximately 50% of the fatty acids in C. lipolytica were also chlorinated. P. zonatum contained 20% 1-chlorohexadecanoic acid after growth on either substrate but did not incorporate C18 chlorinated fatty acids.     

Disruption of the SCS2 ortholog in the alkane-assimilating yeast Yarrowia lipolytica impairs its growth on n-decane, but does not impair inositol prototrophy

Disruption of an SCS2 ortholog impaired the growth of the alkane-assimilating yeast Yarrowia lipolytica on n-alkanes, particularly on n-decane, although the mRNA level of the ALK1 gene encoding a highly inducible cytochrome P450ALK was not much affected. The same disruption did not cause inositol auxotrophy, implying that Y. lipolytica SCS2 has a different function from its Saccharomyces cerevisiae counterpart.     

Cell surface topography of Candida and Leucosporidium yeasts as revealed by scanning electron microscopy.

The cell surface topography of the following yeast strains was examined by scanning electron microscopy: Candida slooffii, C. lipolytica, Leucosporidium frigidum, and L. nivalis. Multipolar and lateral budding were observed in the Candida yeasts in contrast to bipolar budding in the Leucosporidium species. The cell surface topography and the morphology of the bud and birth scars in these yeasts differed markedly. Apart from the bud and birth scars, the cells of C. slooffii showed a relatively smooth topography. The bud scars were seen as a circular ridge of wall material surrounding a markedly convex scar plug. Birth scars were raised, rounded structures, which appeared to distend upon cell growth. In contrast, bud scars of C. lipolytica were platelike, lacked a distinct annulus of wall material, and were much less protuberent than those of C. slooffii. Birth scars were a more permanent feature of these cells. The topography of Leucosporidium yeasts was characterized by the presence of numerous protrusions on the cell surface. In some cases, the entire cell surface was covered by these protrusions. There appeared to be some correlations between the age of the cell and the extent of surface protrusions and degree of surface convolution...     

Mechanism of action of some quinoline alkaloids on respiratory chain of mitochondria]

The mechanism of action of some quinoline alkaloids and their derivatives on respiratory chain of rat liver and Candida lipolytica yeast mitochondria was studied. The alkaloids were shown to inhibit electron transfer in the respiratory chain. The site of their action is localized between b and c cytochromes. Besides their ability to inhibit electron transfer in the respiratory chain, alkaloids are shown to be specific inhibitors of "exogenous" NADH-dehydrogenase of C. lipolytica yeast mitochondria. In addition to their inhibiting properties alkaloids can stimulate ATPase activity of mitochondria. O-alkylation of pseudane-IX permits to differentiate the inhibiting and uncoupling properties of this alkaloid.     

Esterase activity of Candida species isolated from immunocompromised hosts

A total of 149 clinical isolates of Candida species isolated from immunocompromised patients were examined to ascertain their esterase activity by the Tween 80 opacity test, which is a biochemical test used mainly to differentiate between Candida albicans and Candida dubliniensis. Our results showed that C. albicans (92.3%), Candida tropicalis (92.3%), Candida parapsilosis (25%), C. dubliniensis (16.6%), Candida inconspicua (100%), and Candida lipolytica (100%) produced opacity halos through the 10-day post-inoculation period. The remaining Candida species did not produce a positive test response. These findings indicate that Tween 80 opacity test cannot be used as the sole phenotypic trait in the differentiation of C. albicans and C. dubliniensis.