Effects of the anti-lipogenic antibiotic cerulenin on growth and fatty acid composition of n-alkane-utilizingCandida lipolytica

Summary The effects of cerulenin, an anti-lipogenic antibiotic, on the growth and cellular fatty acid composition ofCandida lipolytica were investigated by changing the chain length of n-alkane, the growth substrate. The antibiotic inhibited almost completely the growth of the yeast on glucose, n-undecane and n-dodecane, but partly that on n-tridecane. The yeast growth on longer alkanes, e.g., from n-tetradecane to n-octadecane, was not affected by this antibiotic, indicating that a chain elongation system and/or intact incorporation system predominantly operate in the formation of cellular fatty acids from such longer chain n-alkanes. Comparison of the fatty acid profiles between the cells grown on n-alkanes of different chain lengths, especially on n-pentadecane, in the presence and absence of cerulenin, supported the supposition that only the de novo synthesis system of the yeast would be affected by the antibiotic, whereas the chain elongation system would not.     

Metabolism of aromatic hydrocarbons by yeasts

Six yeasts were examined for their ability to metabolize naphthalene, biphenyl and benzo(a)pyrene. All of the organisms tested oxidized these aromatic hydrocarbons. Candida lipolytica oxidized naphthalene to 1-naphthol, 2-naphthol, 4-hydroxy-1-tetralone and trans-1,2-dihydroxy-1,2-dihydronaphthalene. The major metabolite was 1-naphthol. C. lipolytica oxidized biphenyl to produce 2-, 3-, and 4-hydroxybiphenyl, 4,4′-dihydroxybiphenyl and 3-methoxy-4-hydroxybiphenyl. 4-Hydroxybiphenyl was the predominant metabolite formed. C. lipolytica oxidized benzo(a)pyrene to 3-hydroxybenzo(a)pyrene and 9-hydroxybenzo(a)pyrene. Metabolites were isolated and identified by absorption spectrophotometry, mass spectrometry and thin-layer, gasliquid and high-pressure liquid chromatography. Where possible the structures of these metabolites were confirmed by comparison with authenic compounds.     

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.     

Cinétique de dégradation des hydrocarbures par Candida lipolytica

Résumé Le mécanisme d'assimilation des hydrocarbures par une levure, Candida lipolytica est étudié au moyen de l'analyse cinétique de la croissance du microorganisme et de la disparition du substrat hydrocarboné. Les hydrocarbures utilisés sont des n-paraffines. On ajoute au milieu soit un seul hydrocarbure (n-tetradécane ou n-hexadécane), soit un mélange binaire (n-dodécane et n-heptadécane), soit un mélange complexe (du n-undécane au n-octadécane). Contrairement à d'autres auteurs, nous pensons qu'il est peu probable que l'essentiel de la réaction s'effectue par contact des gouttes de substrat et des microorganismes puisque l'on observe des retards d'assimilation de certains hydrocarbures: ceux de faible poids moléculaire (les plus solubles) sont assimilés plus rapidement. Il semble donc que l'assimilation se fasse en grande partie à partir d'hydrocarbures préablement solubilisés.

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Kinetics of hydrocarbon assimilation by Candida lipolytica

Summary The mechanism of hydrocarbon uptake by a yeast, Candida lipolytica has been studied by means of the kinetic analysis of micro-organism growth and substrate assimilation. Hydrocarbons used as only source of carbon are normal alkanes either pure (n-tetradecane or n-hexadecane) or in mixture of two paraffins (n-dodecane and n-heptadecane) or eight paraffins (n-undecane to n-octadecane). In these last cases delays in n-alkanes consumption are observed. They show that the most soluble substrates (lower molecular weight) are first consumed. In opposition to other authors we think that there is little probability for main reaction occurring by direct contact between drops and micro-organisms. The evidence indicates that n-alkanes are mainly utilized in the dissolved state.

     

Fatty Acid Composition of Cladosporium resinae Grown on Glucose and on Hydrocarbons

Cladosporium resinae was grown in submerged cultures on glucose; on Jet-A commercial aviation fuel; and on a series of n-alkanes, n-decane through n-tetradecane. Cell yield was greatest on glucose and least on Jet-A; n-alkanes were intermediate. Among n-alkanes cell yield decreased as chain length increased, except for n-dodecane, which supported less growth than n-tridecane or n-tetradecane. The total fatty acids of stationary-phase cells were analyzed by gas-liquid chromatography. In all cases the predominant fatty acids were 16:0, 18:1, and 18:2. The fatty acid composition of glucose-grown cells was similar to that of hydrocarbon-grown cells. Cells grown on n-tridecane or n-tetradecane yielded small amounts of acids homologous to the carbon source, but a similar correlation was not noted for n-decane, n-undecane, or n-dodecane. Cells grown on n-undecane or n-tridecane contained more odd-carbon fatty acids than cells grown on the other substrates, and the effect was more pronounced in n-tridecane-grown cells. Thus, the fatty acids of this organism are derived chiefly from de novo synthesis rather than from direct incorporation of oxidized hydrocarbons. The extent of direct incorporation increases as the chain length of the hydrocarbon growth substrate is increased.     

Degradation of Hydrocarbons by Members of the Genus Candida II. Oxidation of n-Alkanes and 1-Alkenes by Candida lipolytica

Candida lipolytica ATCC 8661 was grown in a mineral-salts hydrocarbon medium. n-Alkanes and 1-alkenes with 14 through 18 carbon atoms were used as substrates. Ether extracts of culture fluids and cells obtained from cultures grown on the various substrates were analyzed by thin-layer and gas-liquid chromatography. Analyses of fluids from cultures grown on n-alkanes indicated a predominance of fatty acids and alcohols of the same chain length as the substrate. In addition, numerous other fatty acids and alcohols were present. Analyses of saponifiable and nonsaponifiable material obtained from the cells revealed essentially the same products. The presence of primary and secondary alcohols, as well as fatty acids, of the same chain length as the n-alkane substrate suggested that attack on both the methyl and α-methylene group was occurring. The significance of these two mechanisms in the degradation of n-alkanes by this organism was not evident from the data presented. Analyses of fluids from cultures grown on 1-alkenes indicated the presence of 1,2-diols, as well as ω-unsaturated fatty acids, of the same chain length as the substrate. Alcohols present were all unsaturated. Saponifiable and nonsaponifiable material obtained from cells contained essentially the same products. The presence of 1,2-diols and ω-unsaturated fatty acids of the same chain length as the substrate from cultures grown on 1-alkenes indicated that both the terminal methyl group and the terminal double bond were being attacked.     

Comparative lipid content ofCandida lipolytica grown on glucose and onn-hexadecane

Candida lipolytica, grown onn-hexadecane as the sole source of carbon and energy, contained 17.1% lipids in the logarithmic phase of growth, and 7.3% lipids in the stationary phase of growth. When the yeast was grown on glucose, it contained 6.2% lipids in the logarithmic phase of growth, and 3.6% lipids in the stationary phase of growth. Fatty acids, that could be extracted by petroleum ether after saponification, constituted the major part of the fatty acids ofC. lipolytica in its logarithmic phase of growth on glucose. They constituted only a minor amount of the fatty acids in the stationary phase of growth on glucose. The reverse was true when the yeast was grown onn-hexadecane. The broth contained more free, petroleum ether-soluble fatty acids when the cellular lipid content was high than when it was low. Overnight starvation ofC. lipolytica grown onn-hexadecane in a carbon-free nutrient medium, removed the residual cell-bound hydrocarbon, increased the cell population by one half and decreased the cellular lipid content (as % of dry yeast) by one third. Various methods for the determination of lipids, described as appropriate for yeasts were compared. The highest yields were obtained by extraction of the freeze-dried paste, at room temperature, with a 1:1 chloroform-methanol mixture.     

Fermentation of 1-hexadecene by Candida lipolytica

Candida lipolytica, strain Phaff, was grown on 1.0% 1-hexadecene as sole source of carbon. Several oxidative intermediates were isolated and identified. Based on these intermediates two pathways are proposed for the degradation of the 1-alkene via the methyl group and the double bond. Subterminal oxidation of the 1-alkene was also indicated. Cell yield, lipid content, fatty acid profile and 1, 2-diol concentration are given for various rates of aeration during growth in a fermentor.     

Comparison of promoters suitable for regulated overexpression of β-galactosidase in the alkane-utilizing yeastYarrowia lipolytica

Promoters of the genesG3P, ICL1, POT1, POX1, POX2 andPOX5 of the yeastY. lipolytica were studied in respect to their regulations and activities during growth on different carbon sources. The aim of this study was to select suitable promoters for high expression of heterologous genes in this yeast. For this purpose the promoters were fused with the reporter genelacZ ofE. coli and integrated as single copies into the genome ofY. lipolytica strain PO1d. The measurement of expressed activities of β-galactosidase revealed thatpICL1, pPOX2 andpPOT1 are the strongest regulable promoters available forY. lipolytica, at present.pPOX2 andpPOT4 were highly induced during growth on oleic acid and were completely repressed by glucose and glycerol.pICL1 was strongly inducible by ethanol besides alkanes and fatty acids, however, not completely repressible by glucose or glycerol. Ricinoleic acid methyl ester appeared as a very strong inducer forpPOT1 andpPOX2, in spite of that it inhibited growth ofY. lipolytica transformants.     

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.     

Fermentation of n-Paraffins by Yeast

α-Ketoglutarate productivity from n-paraffins of 141 strains of identified yeasts was studied. Among the strains tested, only strains of Candida lipolytica exclusively showed a high ability to produce α-ketoglutarale.

It was also observed that these strains of Candida lipolytica required thiamine for their growth and that exegenous thiamine stimulated the activity of α-ketoglutarate dehydrogenase of Candida lipolytica AJ 5004.

From these results, relationship between thiamine requirement and α-ketoglutarate productivity of Candida lipolytica was discussed.

α-Ketoglutarate fermentation by representative strains of Candida lipolytica was also carried out.     

褐飞虱共生解脂假丝酵母抗吡虫啉菌株的驯化

为进一步研究共生菌在褐飞虱对吡虫啉产生抗性中的生理生化机制,在稻田杀虫剂对褐飞虱共生解脂假丝酵母生长影响的基础上,选用不同吡虫啉浓度进行抗药性菌株的驯化。结果表明,褐飞虱共生解脂假丝酵母在不同吡虫啉浓度(2 000、1 000和500 mg/L)的固体培养基上继代培养,经过20代后2 000 mg/L培养基上的共生菌菌落数量,与未加吡虫啉的培养基上的菌落数量差异不明显,并且连续3代稳定后定为抗2 000 mg/L吡虫啉的共生菌菌株。在光镜下比较不同抗感吡虫啉菌株假菌丝的形态变化,发现抗吡虫啉菌株的假菌丝出现畸形,而且假丝变短,部分出现了膨大。     

Comparative analysis of dufour's gland secretions of two carpenter bees (xylocopinae: anthophoridae) with different nesting habits

• 1.1. The chemistry and function of Dufour's gland secretions of two carpenter bees were studied.
• 2.2. Dufour's gland of Proxylocopa olivieri, a ground nesting bee, produces long chain hydrocarbons that are utilized to line its brood cells and are mixed with its bee bread.
• 3.3. Dufour's gland secretion of Xylocopa sulcatipes, on the other hand, is dominated by ethyl eicosanoate and ethyl docosanoate accompanied by the corresponding methyl esters and high boiling hydrocarbons.
• 4.4. This wood nesting bee apparently does not use Dufour's gland secretion to line its nest.
• 5.5. The relationship between the respective chemistry and function of Dufour's gland secretion and the nesting ecology of the two bees is discussed.

     

The metabolism of long-chain fatty acids and alcohols byCandida tropicalis andSaccharomyces cerevisiae

The factors affecting the growth ofCandida tropicalis andSaccharomyces cerevisiae on medium- and long-chain fatty acids and alcohols in batch culture were investigated. Growth on solid acids and alcohols dispersed in the medium is a maximum for tetradecanoic acid and tetradecanol. The poorer growth observed on shorter chain lengths can be ascribed to their toxicity to the yeasts, whilst the fall off in growth on the higher members is explained by their increasing insolubility in the medium. When the longer-chain-length acids are dissolved in a non-metabolisable hydrocarbon, the growth ofC. tropicalis is improved, but that ofS. cerevisiae is unaffected. This suggests that acids can enter the cells of the former organism by direct contact with the hydrocarbon droplets. The surface ofS. cerevisiae is too hydrophilic for this transfer mechanism to be possible. Fatty acids dissolved in gas oil are utilized as substrates for the growth ofCandida tropicalis in competition with then-paraffins contained in the gas oil. Each fatty acid contributes to a constant proportion of yeast produced, but this proportion decreases as the chain is lengthened. Thus, in mixtures of gas oil with dodecanoic acid, 65% of the yeast is produced from metabolism of the acid, while with octadecanoic acid only 15% is produced. The log specific rates of utilization of the fatty acids within this range diminish linearly with increasing chain length.     

Lipid composition of Cunninghamella elegans cultivated on n-alkanes]

The ability to oxidize n-alkanes was studied with various species of fungi belonging to the Cunninghamella genus. These fungi are able to assimilate hydrocarbons and to accumulate up to 1.5 g/litre of biomass. The most active strain was Cunninghamella elegans (-) 1204. The amount of lipids formed, and their composition, depended on the length of the carbon chain of oxidized alkane. The content of fat in the cells increased with the length of the hydrocarbon chain. The following lipid fractions have been detected: phospholipids, monoglycerides, diglycerides, triglycerides, sterols, free fatty acids, sterol esters, and hydrocarbons. The qualitative composition of the fractions depended, to a considerable extent, on the n-alkane utilized. Investigation of the fatty-acid composition of intracellular lipids has shown that fatty acids with an even number of carbon atoms are formed from hydrocarbons with an even number of these atoms, while fatty acids both with an even and odd number of carbon atoms are synthesized from hydrocarbons with an odd number of these atoms. The relative content of the acids with the same number of carbon atoms as that of the alkane being utilized increased with the length of the carbon chain.     

Mating Responses in Candida lipolytica

Culture medium that restricted cell multiplication increased fertility in selected heterothallic stocks of Candida lipolytica and triggered sporulation in newly formed diploids; a medium that supported vigorous cell growth prevented sporulation and permitted the newly formed diploids to bud.     

The teleomorph state of Candida deformans Langeron & Guerra and description of Yarrowia yakushimensis comb. nov.

Yarrowia lipolytica is the only known species in the teleomorph (i.e. sexual) genus Yarrowia and has its anamorph (i.e. asexual state) classified in the genus Candida Berkhout as Candida lipolytica. This species can be found readily in nature, has significant industrial value and is important to the food and medical fields. Candida deformans was first considered a variety of C. lipolytica and was later listed as a synonym of this species. More recent studies based on sequence variation in the nuclear rRNA gene sequences suggested C. deformans to be a separate species with no known teleomorph linked to it. In this study we show that C. deformans strains, obtained from South Africa, can mate with strains present in the CBS yeast collection and produce a Yarrowia teleomorph, described here as Yarrowia deformans. Strains of Candida yakushimensis nom. inval., a species also belonging to the Yarrowia phylogenetic clade, were also studied and described as a novel species of Yarrowia.     

The absence of energy conservation coupled with electron transfer via the alternative pathway in cyanide-resistant yeast mitochondria

Electron transfer via the alternative pathway in cyanide-resistant mitochondria of the yeast Candida lipolytica is not coupled with ATP synthesis, generation of membrane potential or energy-dependent reverse electron transport in the main respiratory chain. We conclude that during transfer via the alternative pathway no accumulation of energy in the form of high-energy compounds or membrane potential occurs.     

The substrate specificity of two yeast strains utilizing hydrocarbons

StrainsCandida lipolytica 4-1 andCandida lipolytica K were compared in their growth and dawaxing capacities during batch growth on model gas oil. The model gas oil was composed of a mixture of even-numbered puren-alkanes (n-decane ton-dotriacontane) dissolved in dewaxed gas oil. The results show that both strains differ in their substrate specificity and in the sequence of utilization of individualn-alkanes. Strain K, previously used for dewaxing of mineral oil, has its substrate specificity shifted toward the highern-alkanes.     

Mode of Metabolism of 1-Tetradecene by Candida Yeasts and Citrates Production

Metabolic pathways for the oxidation of 1-tetradecene by Candida lipolytica were investigated and compared with those for Candida tropicalis in order to elucidate the difference in the productivity of citrates reported in the previous paper. No difference was found in the pathways for the initial stage of oxidation of 1-tetradecene between the two strains, indicating that the difference in the productivity of citrates is not attributable to the metabolic pathways. The metabolic rate of 1-tetradecene with C. lipolytica was found to be much lower than that with C. tropicalis. The production of citrates was much enhanced in the presence of surfactants which were known to be stimulative for microbial metabolism of hydrocarbons and 11 ~ 15 g/liters was attained on the 6th day of cultivation.