Carnitine acetyltransferase activity in oleaginous yeasts

Abstract The highest activities of carnitine acetyltransferase (CAT) were found in non-oleaginous yeasts ( Candida utilis and Saccharomyces cerevisiae ); lower activities, ranging from 50% down to 3% of the highest values, were found in various strains of oleaginous yeasts ( Candida curvata, Lipomyces starkeyi, Rhodosporidium toruloides and Trichosporon cutaneum ). Supply of acetyl units into the cytosol of the latter, but not of the former yeasts, was therefore necessarily reliant on the action of ATP: citrate lyase (ACL), which was present in all oleaginous yeasts. There was no correlation, however, between the amount of lipid in the oleaginous yeasts and the specific activities of either CAT or ACL. Activity of CAT was increased up to 30-fold by growing yeasts on a triacylglycerol.     

Temperature adaptation in Lactobacillus fermentum: interconversions of oleic, vaccenic and dihydrosterulic acids.

The interchange of octadecenoic acids and dihydrosterulic acid was a response of aerobically growing Lactobacillus fermentum to changes in growth temperature. Oleic and vaccenic acid contents decreased both at temperatures below 20 degrees C and above 26 degrees C, showing mirror image behaviour, with a concomitant increase in dihydrosterulic acid. A temperature-dependent shift from vaccenic to oleic acid synthesis, and the conversion of the latter to dihydrosterulic acid was responsible for the overall change. Consequently, the degree of fatty acid unsaturation decreased at temperatures above 26 degrees C, whereas the degree of cyclization increased. The converse occurred below 20 degrees C. The relative amount of lactobacillic acid, total cellular fatty acid content, and mean fatty acid chain length were practically temperature-independent. The occurrence of oleic acid is thought to be related to aerobic growth conditions.     

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.     

Possible involvement of acetyl coenzyme A carboxylase as well as fatty acid synthetase in the temperature-controlled synthesis of fatty acids in Saccharomyces cerevisiae

Fatty acid synthetase (FAS) preparations from Saccharomyces cerevisiae cells grown at either 35 or 10 degrees C produced the same products at different temperatures and showed quite similar temperature-dependencies in Arrhenius plots, with break points at 25 degrees C. This break point does not appear to reflect a phase transition of phospholipids present in the purified FAS preparations but rather is associated with protein conformational changes. S. cerevisiae cells grown at 35 degrees C and then shifted to 10 degrees C produced fatty acids with a shorter average chain length than those fatty acids synthesized at 10 degrees C by cells already adapted to 10 degrees C (hyper response). Acetyl-CoA carboxylase activity was relatively higher in the cells grown at 35 degrees C than in the cells grown at 10 degrees C; moreover, fatty acids with longer average chain lengths were synthesized in vitro at higher malonyl-CoA concentrations, which was consistent with the difference in the average chain lengths of newly synthesized fatty acids in cells grown at 35 and 10 degrees C. However, the activity levels of acetyl-CoA carboxylase and fatty acid synthetase alone did not account for the hyper response phenomena.     

广谱碳源产油酵母菌的筛选

对10株酵母菌利用不同单糖为碳源条件下菌体内积累油脂的能力进行了初步考察,并对菌油进行了分离和脂肪酸组成分析。实验发现,以葡萄糖为唯一碳源时有9株菌油脂含量超过自身细胞干重的20%,可以界定为产油微生物。其中6#菌(T.cutaneumAS2.571)利用葡萄糖发酵菌体油脂含量达到65%(W/W)。所有实验菌株都能同化多种单糖,其中1#菌(L.starkeyiAS2.1390)、4#菌(R.toruloidesAS2.1389)和11#菌(L.starkeyiAS2.1608)表现出对碳源利用的广谱性,能转化五碳糖木糖和阿拉伯糖并在菌体内积累油脂,油脂含量最高达到26%。脂肪酸组成分析结果表明,菌油富含饱和及低度不饱和长链脂肪酸,其中棕榈酸、油酸和亚油酸三者之和占总脂肪酸组成的90%以上,脂肪酸组成分布类似于常见的植物油。这些结果对利用产油微生物转化木质纤维素水解混合糖获取油脂资源的研究具有重要意义。     

Alterations of the composition and size of the free fatty acid pool of Tetrahymena responding to low-temperature stress

Cells of Tetrahymena mimbres (formerly T. pyriformis NT-1) in midlogarithmic growth under isothermal conditions (at 39 degrees C) contained a very small, compositionally discrete pool of free fatty acids, principally (60.6% of the total free fatty acid mass) palmitic and stearic acids. The composition, degree of unsaturation, and size of this free fatty acid pool were rapidly (15 min or less) altered in response to chilling. During the acclimation period following chilling to 15 degrees C, the size of the free fatty acid pool increased from a mean value of 15.5 nmol free fatty acid/mumol lipid phosphorus in 39 degrees C cells to 24.2 nmol free fatty acid/mumol lipid phosphorus. The degree of free fatty acid saturation rapidly increased over the initial hour following the onset of hypothermal conditions, but by 24 h the unsaturated free fatty acid/saturated free fatty acid ratio was 0.35 (equivalent to a 2.7-fold increase in unsaturation relative to 39 degrees C controls (unsaturated/saturated ratio = 0.13) and 4.4-fold greater than cells acclimated for 1 h (unsaturated/saturated ratio = 0.08)). By 24 h the percentage of palmitic and stearic acids had decreased to 45.6%. Similar, and in some instances more pronounced, changes were observed to occur in triacylglycerol-bound fatty acids. Modulation of steady-state free fatty acid composition could also be achieved by the addition of exogenous fatty acids to the growth medium. The ability to manipulate the level of intracellular free fatty acids should prove to be a valuable experimental tool in determining how specific fatty acids regulate various lipid-modifying enzymes.     

Changes in fatty acid branching and unsaturation of Streptomyces griseus and Brevibacterium fermentans as a response to growth temperature.

Streptomyces griseus showed three different modes of changing fatty acids in response to temperature change. In Brevibacterium fermentans, two such responses were found. The responses involved changes in fatty acid branching, unsaturation, or chain length, depending on growth temperature range. Changes in unsaturation of branched-chain acids were characteristic at low growth temperatures.     

Changes in fatty acid branching and unsaturation of Streptomyces griseus and Brevibacterium fermentans as a response to growth temperature.

Streptomyces griseus showed three different modes of changing fatty acids in response to temperature change. In Brevibacterium fermentans, two such responses were found. The responses involved changes in fatty acid branching, unsaturation, or chain length, depending on growth temperature range. Changes in unsaturation of branched-chain acids were characteristic at low growth temperatures.     

Fatty acid specificity of hormone-sensitive lipase. Implication in the selective hydrolysis of triacylglycerols.

The selective mobilization of fatty acids from white fat cells depends on their molecular structure, in particular the degree of unsaturation. The present study was designed to examine if the release of fatty acids by hormone-sensitive lipase (HSL) in vitro i) is influenced by the amount of unsaturation, ii) depends on the temperature, and iii) could explain the selective pattern of fatty acid mobilization and notably the preferential mobilization of certain highly unsaturated fatty acids. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 35 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation from 0 to 6 double bonds was measured. Fatty acid composition of in vitro released NEFA was compared with that of fat cell triacylglycerols (TAG), the ratio % NEFA/% TAG being defined as the relative hydrolysis. The relative hydrolysis of individual fatty acids differed widely, ranging from 0.44 (24:1n-9) to 1.49 (18:1n-7) with rat HSL, and from 0.38 (24:1n-9) to 1.67 (18:1n-7) with human HSL. No major difference was observed between rat and human HSL. The relative release was dependent on the number of double bonds according to chain length. The amount of fatty acid released by recombinant rat HSL was decreased but remained robust at 4 degrees C compared with 37 degrees C, and the relative hydrolysis of some individual fatty acids was affected. The relative hydrolysis of fatty acids moderately, weakly, and highly mobilized by adipose tissue in vivo was similar and close to unity in vitro. We conclude that i) the release of fatty acids by HSL is only slightly affected by their degree of unsaturation, ii) the ability of HSL to efficiently and selectively release fatty acids at low temperature could reflect a cold adaptability for poikilotherms or hibernators when endogenous lipids are needed, and iii) the selectivity of fatty acid hydrolysis by HSL does not fully account for the selective pattern of fatty acid mobilization, but could contribute to explain the preferential mobilization of some highly unsaturated fatty acids compared with others.     

Analysis of fatty acid composition of Candida species by gas-liquid chromatography using a polar column

The fatty acid composition of representative Candida species was examined by gas-liquid chromatography (GLC) using a polar column. The major fatty acids were C14:0, C16:0, C18:0 saturated, C16:1 and C18:1 monoenoic series, with or without C18 polyunsaturated acids (C18:2 and C18:3). In Torulopsis glabrata and Saccharomyces cerevisiae the C18:2 and C18:3 acids were not found, but the C10:0 and C12:0 acids were detected in S. cerevisiae. These results indicated that the Candida genus could be distinguished from Torulopsis and Saccharomyces genera by GLC analysis of fatty acids. Quantitative differences in the fatty acid composition between cells grown at high temperature (37 degrees C) and low temperature (25 degrees C) were found generally in Candida species, and the amounts of C18 polyunsaturated acids (C18:2 and C18:3) increased in the cells grown at 25 degrees C. Each Candida species showed a characteristic profile in fatty acid composition. Determination of the cellular fatty acid composition in Candida species is likely to be useful for the grouping or chemotaxonomy of newer isolates of Candida species.     

Role of phospholipid fatty acids on the kinetics of high and low affinity sites of cytochrome c oxidase

The nature of the interactions between cytochrome c oxidase and the phospholipids in mitochondrial membranes has been investigated by varying the nature of the fatty acyl components of Saccharomyces cerevisiae. A double fatty acid yeast mutant, FAI-4C, grown in combinations of unsaturated (oleic, linoleic, linolenic, and eicosenoic) and saturated (lauric and palmitic) fatty acids, was employed to modify mitochondrial membranes. The supplemented fatty acids constituted a unique combination of different acyl chain lengths with varying degrees of unsaturation which were subsequently incorporated into mitochondrial phospholipids. Phosphatidylethanolamine and cardiolipin, the predominant phospholipids of the inner mitochondrial membrane, were characterized by their high levels of supplemented unsaturated fatty acids. Increasing the chain length or the degree of unsaturation of mitochondrial membrane phospholipids had no effect on altering the nature of the phospholipid polar head group but did result in a profound change on the specific activity of cytochrome c oxidase. When studied under conditions of different ionic strengths and pHs the enzyme's activity, as documented by Eadie-Hofstee plots, showed biphasic kinetics. The kinetic parameters for the low affinity reaction were greatly influenced by the changes in the membrane fatty acids and only marginal effects were noted at the high affinity reaction site. The discontinuities in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, monitored at increasing temperatures, suggested that changes in membrane fluidity were conditioned by alterations in mitochondrial membrane fatty acid constituents. These results indicate that the lipid changes affecting the low affinity binding site of cytochrome c oxidase may be the result of lipid-protein interactions which lead to enzyme conformational changes or may be due to gross changes in membrane fluidity. It may, therefore, follow that this enzyme site may be embedded in or be juxtaposed to the outer surface of the inner mitochondrial membrane bilayer in contrast to the high affinity site which has been shown to be significantly above the membrane plane.     

Effect of cyclic changes in culture conditions on the growth kinetics and physiological characteristics of yeasts

Candida utilis, Saccharomyces cerevisiae and Candida scottii were used to study the effect of cyclic changes in the pH and pO2 within a range of 1 to 60 min on their growth kinetics and physiological properties. These changes were shown to increase the specific growth rate from 0.33 to 0.5-0.6 h-1 without decreasing the economic coefficient and the quantity of budding cells during 2-3 generations of the exponentially growing batch culture of C. utilis. Optimal conditions of cyclic changes in the pO2 (minutes) were found to increase the specific growth rate of C. scottii and S. cerevisiae. The authors discuss a hypothesis for the formation of intermediate products in the substrate oxidation in the course of pulse aeration by the yeasts during the aerobic stage and the utilization of the products at the anaerobic stage of cyclic regimes. The paper describes a mathematical model for the yeast growth under the nonsteady-state conditions of pH and pO2, which accounts for the formation and utilization of possible intermediate biosynthetic products within the studied time intervals.     

Total fatty acid content of the plasma membrane of Saccharomyces cerevisiae is more responsible for ethanol tolerance than the degree of unsaturation

The effect of change in unsaturated fatty acid composition on ethanol tolerance in Saccharomyces cerevisiae overexpressing ScOLE1 (?9 fatty acid desaturase gene of S. cerevisiae), CaFAD2 (?12 fatty acid desaturase gene of Candida albicans), or CaFAD3 (ω3 fatty acid desaturase gene of C. albicans) was examined. ScOLE1 over-expression increased the total unsaturated fatty acid content and enhanced ethanol tolerance, compared with a control strain. In contrast, overexpression of CaFAD2 and CaFAD3, which led to production of linoleic acid (18:2) and α-linolenic acid (18:3), respectively, neither changed total unsaturated fatty acids nor enhanced ethanol tolerance. The total unsaturated fatty acid content rather than the degree of unsaturation is thus an important factor for ethanol tolerance.     

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.     

Survey of nonconventional yeasts for lipid and hydrocarbon biotechnology

Nonconventional yeasts have an untapped potential to expand biotechnology and enable process development necessary for a circular economy. They are especially convenient for the field of lipid and hydrocarbon biotechnology because they offer faster growth than plants and easier scalability than microalgae and exhibit increased tolerance relative to some bacteria. The ability of industrial organisms to import and metabolically transform lipids and hydrocarbons is crucial in such applications. Here, we assessed the ability of 14 yeasts to utilize 18 model lipids and hydrocarbons from six functional groups and three carbon chain lengths. The studied strains covered 12 genera from nine families. Nine nonconventional yeasts performed better than Saccharomyces cerevisiae, the most common industrial yeast. Rhodotorula toruloides, Candida maltosa, Scheffersomyces stipitis, and Yarrowia lipolytica were observed to grow significantly better and on more types of lipids and lipid molecules than other strains. They were all able to utilize mid- to long-chain fatty acids, fatty alcohols, alkanes, alkenes, and dicarboxylic acids, including 28 previously unreported substrates across the four yeasts. Interestingly, a phylogenetic analysis showed a short evolutionary distance between the R. toruloides, C. maltosa, and S. stipitis, even though R. toruloides is classified under a different phylum. This work provides valuable insight into the lipid substrate range of nonconventional yeasts that can inform species selection decisions and viability of lipid feedstocks.     

Sequential utilization of mixed monosaccharides by yeasts

Four yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilus, and Rhodotorula toruloides) were tested for their ability to grow and consume D-glucose, D-xylose, D-xylulose, and D-xylitol. Sequential utilization of substrates was observed when D-glucose as mixed with D-xylulose as the carbon source. Catabolite inhibition was tentatively concluded to be responsible for this regulatory mechanism. D-Glucose was also found to inhibit the utilization of D-xylose and D-xylitol in C. utilus and R. toruloides. D-Xylose, D-xylitol, and D-xylulose were consumed simultaneously by R. toruloides and C. utilus.     

Accumulation of sulphite by Saccharomyces cerevisiae and Zygosaccharomyces bailii as affected by phospholipid fatty-acyl unsaturation and chain length

Analyses were made of the fatty-acyl composition of phospholipids from each of two strains of Saccharomyces cerevisiae and Zygosaccharomyces bailii grown aerobically. Residues of C16:0, C16:1 and C18:1 predominated in phospholipids from strains of the first yeast, while phospholipids from Z. bailii contained mainly C16:0, C18:1 and C18:2 residues. S. cerevisiae NCYC 431, grown anaerobically in media supplemented with ergosterol and C14:1, C16:1, C18:1, C18:2, C18:3 or C20:1 fatty acids, contained phospholipids enriched with residues of the exogenously provided acid, to a greater extent with shorter chain than longer chain acids. A plot of the permeability coefficient for sulphite, derived from Woolf-Eadie plots, against the degree of unsaturation in phospholipids (expressed as delta mol-1 value) showed that the coefficient was greater the lower the degree of unsaturation in the phospholipids. A plot of the permeability coefficient against values for the mean fatty-acyl chain length divided by the delta mol-1 value, which is an approximation of the cross-section surface area of a phospholipid molecule, showed that the permeability coefficient tended to increase the greater the surface-area value.     

Effect of variou cultural conditions on the fatty acid and lipid composition of Choanephora cucurbitarum.

The fatty acid composition of the total and polar lipid fractions of Choanephora cucurbitarum grown under different cultural conditions were analyzed by thin-layer and gas-liquid chromatography. It was observed that temperature, age, pH, and light influenced the degree of unsaturation, this being due mainly to changes in the gamma-linolenic acid concentration. The conditions used in this study did not alter the qualitative profile of fatty acids normally present in the organism. Neither did these conditions stimulate the production of further long-chain fatty acids (C20-C26) beyond gamma-linolenic acid (C18:3) as reported earlier using growth media containing glutamic acid. The fatty acid pattern of lipid fractions though the same qualitatively, differed quantitatively. The polar lipid fractions, phosphatidyl choline, phosphatidyl ethanolamine, and diphosphatidyl glycerol showed an appreciable variation in gamma-linolenic acid content under different cultural conditions. The degree of unsaturation of the various lipid fractions decreased with increases in temperature, light intensity, and pH, but within each treatment the same pattern of decreasing degree of unsaturation with increasing age was observed. The significance of these observations is discussed.     

Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Delta(9) position. We expressed two sunflower (Helianthus annuus) oleate Delta(12) desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Delta(9,12), the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15 degrees C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp(+) or Trp(-) strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30 degrees C or 15 degrees C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

Competition for glucose between the yeasts Saccharomyces cerevisiae and Candida utilis.

The competition between the yeasts Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 for glucose was studied in sugar-limited chemostat cultures. Under aerobic conditions, C. utilis always successfully completed against S. cerevisiae. Only under anaerobic conditions did S. cerevisiae become the dominant species. The rationale behind these observations probably is that under aerobic glucose-limited conditions, high-affinity glucose/proton symporters are present in C. utilis, whereas in S. cerevisiae, glucose transport occurs via facilitated diffusion with low-affinity carriers. Our results explain the frequent occurrence of infections by Crabtree-negative yeasts during bakers' yeast production.