Search for Yeast Producers of Brassylic and Sebacic Fatty Acids

Yeast cultures belonging to the genera Candida, Torulopsis, Saccharomyces, Debaryomyces, Hansenula, Pichia, and Yarrowia, capable of synthesizing brassylic and sebacic fatty acids, were screened. Overall about 200 cultures grown in media containing decane or tridecane as a sole source of carbon were tested. On the medium with tridecane, yeasts synthesized insignificant amounts of brassylic acid. Sebacic acid was produced more intensively in the medium with n-decane. The culture Candida tropicalis, displaying the highest ability to synthesize sebacic acid, was selected.     

Selection of high brassylic acid producing strains of Torulopsis candida by single-cell cloning and by mutation

Single-cell colonies of Torulopsis candida NC-3-58 varied in their productivity of brassylic acid (1,11-undecanedicarboxylic acid). Several strains such as M-4-2 and M-5-1 with higher yields of brassylic acid have been isolated by repeated single-cell cloning.Mutagenesis of M-5-1 with UV irradiation gave strains such as Bu-1, and Bu-1-2 that produced much brassylic acid. Brassylic acid and n-tridecane degradation among the parent and mutant strains was compared.     

Yeast contaminants of micropropagated plant cultures

L eifert , C., W aites , W.M., N icholas , J.R. & K eetley , J.W. 1990. Yeast contaminants of micropropagated plant cultures. Journal of Applied Bacteriology 69 , 471–476.
Of 36 yeast strains isolated from contaminated plant cultures 78% were Candida, 20% Rhodotorula and 2% were Cryptococcus species. Strains of Candida guilliermon-dii represented 45% of all yeasts isolated. Yeasts grew rapidly on plant growth media with a pH of between 2.5 and 6.0 and decreased the medium pH to values of between 2.0 and 3.0. Candida yeasts growing on plant medium for 28 d metabolized about 75% of the sucrose and produced fermentation products such as ethanol and acetic acid. In contrast, Rhodotorula did not produce ethanol or acetic acid and only metabolized about 20% of the sucrose. Possible sources of contamination are discussed.     

Metabolic engineering for the production of dicarboxylic acids and diamines

Microbial production of chemicals and materials from renewable carbon sources is becoming increasingly important to help establish sustainable chemical industry. In this paper, we review current status of metabolic engineering for the bio-based production of linear and saturated dicarboxylic acids and diamines, important platform chemicals used in various industrial applications, especially as monomers for polymer synthesis. Strategies for the bio-based production of various dicarboxylic acids having different carbon numbers including malonic acid (C3), succinic acid (C4), glutaric acid (C5), adipic acid (C6), pimelic acid (C7), suberic acid (C8), azelaic acid (C9), sebacic acid (C10), undecanedioic acid (C11), dodecanedioic acid (C12), brassylic acid (C13), tetradecanedioic acid (C14), and pentadecanedioic acid (C15) are reviewed. Also, strategies for the bio-based production of diamines of different carbon numbers including 1,3-diaminopropane (C3), putrescine (1,4-diaminobutane; C4), cadaverine (1,5-diaminopentane; C5), 1,6-diaminohexane (C6), 1,8-diaminoctane (C8), 1,10-diaminodecane (C10), 1,12-diaminododecane (C12), and 1,14-diaminotetradecane (C14) are revisited. Finally, future challenges are discussed towards more efficient production and commercialization of bio-based dicarboxylic acids and diamines.     

Hydrolytic degradation of ricinoleic-sebacic-ester-anhydride copolymers

The degradation process of poly(ricinoleic-co-sebacic-ester-anhydride)s in buffer solution was investigated by following the composition of the degradation products released into the degradation medium and the degraded polymer. The first week of degradation was characterized by the hydrolysis of the anhydride bonds and significant release of sebacic acid (SA). The remaining oligoesters of SA and ricinoleic acid (RA) degraded into shorter oligoesters composed of RA ester dimers, trimers, and tetramers as well as dimers of RA-SA. To confirm and determine the hydrolytic behavior of the degradation products, short oligoesters of sebacic and ricinoleic acid were synthesized and degraded. It was established that during the hydrolysis under physiological conditions the degradation products have a composition and water absorption similar to those of the synthesized oligoesters.     

固定化脂肪酶合成二元酸酯

The syntheses of dicarboxylic esters by immobilized lipase from Candida sp. -1619 were investigated. The reaction system was composed of 1 mmol dicarboxylic acid, 2 mmol alcohol, 3 mL hexane and 15 mg celite-adsorbed im mobilized lipase(300 u), in a closed 100 mL Erlenmeyer flask, shaken at 40°C for 5h. Sebacic acid was the best substrate among nine dicarboxylic acids selected. Among the 18 saturated fatty n-alcohols, the alcohols with carbon chain length rangin from C4～C18 had good reactivity. The primary alcohols had much better reactivities than corresponding secondary alcohols and multihydroxy-alcohols. Tertiary alcohols showed no reactivity. Hydrocarbons, benzene, toluene, xylene and te trachloride were favorite reactants among 15 organic solvents selected, in none-solvent stationary system, (5 mmol sebacic acid, 10 mmol dndecanol, 150 mg immobilized lipase(3000 u))reacted without plug for 3.5h, the optimum temperature was 60°C. The conversion degree was over 92% when reaction carried out at 50～90°C for 17h. The suitable reaction pH ranged from 6～8. The reactant was developed on GF254 plate(hexane ethyl ether acetic acid = 30201 ( V V V).There were three spots with different Rf value at 0.96, 0.55 and 0 corresponding to product, oleyl alcohol and sebacic acids, respectively.     

Activity and substrate specificity of the alcohol dehydrogenases of n-alkane oxidizing yeasts

The activity and substrate specificity of alcohol dehydrogenases (ADH) in the fractions of cytosol and membrane particles were compared in the yeasts Torulopsis candida, Candida lipolytica and Candida tropicalis grown in media with glucose and hexadecane. In all studied yeast cultures growing in the medium with hexadecane, NAD-dependent ADH specifically dehydrogenating only medium and higher alcohols are induced in the membrane structures of the cells. Soluble ADH are found in the cytosol of the cultures grown either on glucose or on hexadecane. These ADH oxidize all alcohols with the carbon chain length from C2 to C16. As was found by electrophoresis in polyacrylamide gel, the number of ADH molecular forms in the cytosol fraction of the cultures depends on the carbon growth substrate being used and the peculiarities of yeast culture.     

The genusCandida berkhout

Basic nutritional conditions were studied in 30 strains ofCandida albicans comprising slightly, moderately and highly pathogenic cultures. It was found that the given strains had no special requirements as far as growth substances were concerned and that even after tenfold transfer they continued to reproduce on minimum medium. A mixture of the most important amino acids only slightly stimulated growth, but tended to stimulate alcohol and total acid synthesis. Its stimulating effect was strongest in the group of highly pathogenic strains. The only treatment which markedly stimulated and accelerated reproduction, especially in the initial phase of growth of the culture, was a combination of four vitamins—biotin, calcium pantothenate, nicotinic acid and thiamine. Differences were also found in the dry weight increase; cultures with a rapid, slow and moderate rate of growth were differentiated, according to the size of their economic coefficient. The group of slightly pathogenic strains also had different economic coefficients from moderately and highly pathogenic strains. There was no significant difference between moderately and highly pathogenic strains. One strain differed from all the rest by reproducing well in minimum medium without amino acids or vitamins; its rate of reproduction in minimum medium was the same as in medium enriched with amino acids.     

Makeup of free intracellular amino acids in Cunninghamella elegans growing on media with hydrocarbons]

The rate of growth of Cunninghamella elegans (--) 1204 is higher on a mineral medium with glucose (6.56 g/litre) than on a mineral medium containing undecane, tridecane, and pentadecane (0.72--0.87 g/litre); all glutamic acid is consumed only from the medium with glucose. The cells contain 15--16 free amino acids and 1--2 amides, glutamic and aspartic acids and alanine prevailing. The culture grown on the medium with glucose contains asparagine, and the cells cultivated on the medium with alkanes contain histidine. Non-proteinogenous aminobutyric acids were found in the pool of the cells grown on all tested media with an exception of the medium containing undecane.     

Urinary excretion of dicarboxylic acids from patients with the Zellweger syndrome. Importance of peroxisomes in beta-oxidation of dicarboxylic acids

The urinary excretion of adipic acid, suberic acid and sebacic acid from two patients with the cerebrohepato-renal syndrome of Zellweger was studied. The patients had a complete lack of peroxisomes in the liver as judged by electron microscopy. In the non-ketotic state, the total excretion of free and conjugated adipic acid, suberic acid and sebacic acid was increased by about 100%, 200% and 350%, respectively, as compared to the corresponding excretion from six healthy infants of the same age. The excretion of free dicarboxylic acid was increased to a considerably lesser extent than the free + conjugated dicarboxylic acid. In view of the presence of adipic acid in urine of the Zellweger patients, it is concluded that peroxisomes are not obligatory for beta-oxidation of medium-chain dicarboxylic acids in vivo. The relative accumulation of suberic acid and sebacic acid as compared to adipic acid is, however, consistent with a relative block in the conversion of suberic acid and sebacic acid into adipic acid in patients with the Zellweger syndrome.     

Poly(sebacic acid-co-ricinoleic acid) biodegradable injectable in situ gelling polymer

The investigated polymers, poly(sebacic acid-co-ricinoleic acid) containing > or =70% ricinoleic acid, may be injected via a 22 gauge needle and become gel upon contact with aqueous medium, both in vitro and in vivo. Various properties of the polymers including viscosity, thermal analysis, and in vivo behavior, before and after exposure to aqueous medium, were determined. These polymers were observed using scanning electron microscopy (SEM) at dry and wet states. It was found that the viscosity and melting temperature of P(SA:RA) increased after exposure to buffer. The viscosity at 37 degrees C of P(SA:RA)3:7 had the highest increase: from 4200 cP before to 8940 cP after exposure to buffer; in the case of P(SA:RA)25:75 before exposure to buffer the viscosity was 1150 cP while after it raised to 3200 cP. The viscosity of P(SA:RA)2:8 also increased from 400 cP before exposure to buffer to 1000 cP after. On the other hand polymer without sebacic acid, (poly(ricinoleic acid)), did not show gelation properties. Thermal analysis also showed an increase in the melting point of the polymers exposed to the aqueous medium during the first 24 h of incubation. Images obtained by SEM showed formation of a three-dimensional network in polymers exposed to buffers. When injected into animals, P(SA:RA) forms a solid implant in the injection site already at 8 h postinjection.     

Studies on the formation of long-chain dicarboxylic acids from puren-alkanes by a mutant ofCandida tropicalis

Summary Individualn-alkanes, from C₁₁–C₁₆, were metabolized by a mutant ofCandida tropicalis. This strain was selected for its inability to grow in the presence of dodecanedioic acid and dodecane as the sole carbon source. Transformations were studied in fed-batch cultures. Undecane was only poorly transformed, but from dodecane to hexadecane high transformation yields were achieved. Maximum yield of acid-precipitable long-chain dioic acids was obtained with tridecane as substrate. All the products were mixtures of different acids. Besides the ,-alkanedioic acids, the 3-hydroxy derivatives of long-chain ,-alkanedioic acids and dioic acids with a shortened carbon chain were found.     

Detection of interactions between yeasts and lactic acid bacteria isolated from sugary kefir grains

Different techniques were tested for studying the synergism between the micro-organisms of sugary kefir grains. Agar cultures in Petri dishes did not give reproducible results. In sequential cultures, i.e. growing one organism, sterile filtering and then inoculating the other, 10 of 18 selected lactic acid bacteria/yeast pairs revealed stimulation of bacterial growth in a poor glucose medium. In mixed culture, Saccharomyces florentinus supported better survival of Lactobacillus hilgardii and a significant increase in lactic acid production; at the same time, the growth and alcoholic fermentation of S. florentinus were drastically reduced. The inter-relationships between these two strains were the same when immobilized in calcium alginate beads, even though total metabolite production was always lower than with free cells. The stimulation of Lact. hilgardii by Candida lambica in sequential culture was not confirmed in mixed culture, where the two organisms grew as in pure culture, and bacterial growth and lactic acid production were inhibited in the immobilized system.     

Effect of fatty acids on aflatoxin production byAspergillus parasiticus

The effect of saturated and unsaturated fatty acids on aflatoxin production was studied in a synthetic medium. The aflatoxin production decreased (10-75%) in the presence of lauric acid and palmitic acid but the addition of behenic and sebacic acid stimulated aflatoxin production by 125-541%. Linolenic and linoleic acids effected aflatoxin production and mycelium growth. An 34-fold increase in aflatoxin production was observed with 50 mM linoleic acid. An inverse relationship was observed between aflatoxin production and mycelium mass, irrespective of the nature of the fatty acid.     

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.     

Inhibition of Histoplasma capsulatum by Candida albicans and Other Yeasts on Sabouraud''s Agar Media

The inhibition of growth of Histoplasma capsulatum by Candida albicans and other yeasts on Sabouraud's agar was investigated. Histoplasma (yeast-phase inoculum) was grown alone and in mixtures with yeasts at 25 C for 4-week periods. As few as 10 colonies of C. albicans completely inhibited the growth of approximately 50,000 potential colonies of Histoplasma. The pH was determined in cultures of 36 colonies of Candida on media containing 1, 2, and 4% glucose by spotting the agar with pH indicators. A drop in the pH became noticeable in all three media about the 3rd day of incubation, and a pH of 3.5 was reached in about 7 days. Subsequently, the pH remained almost stationary in the 4% glucose-agar, rose slowly in the 2% glucose-agar, and rose sharply in the 1% glucose-agar. The growth of Histoplasma was inhibited completely at pH 4 and below. When the pH was controlled in mixed cultures, some growth of Histoplasma was obtained. Substitution of maltose for glucose delayed the development of acidity and allowed the appearance of numerous mycelial colonies in the presence of Candida. This growth was arrested as soon as the medium became acid. Four other species which also acidified the Sabouraud's medium effected similar inhibition. It was thus shown that severe and prolonged acidity produced by some yeasts in the sugar-rich Sabouraud's media is alone sufficient to completely inhibit Histoplasma during the standard 4-week incubation of specimens such as sputum.     

阴道白色念珠菌致病株与携带株两相性与毒力关系研究

目的探讨阴道白色念珠菌致病株和携带株菌丝相和酵母相分泌型酸性蛋白酶和细胞外磷脂酶活性以及与其毒力的关系。方法分别采用牛奶平板和卵黄培养基法检测白色念珠菌致病株和携带株200株分泌型酸性蛋白酶和细胞外磷脂酶的活力,分别将致病产酶株的菌丝相和孢子相菌悬液（5×10^6CFU／ral）注射小鼠尾静脉,1个月内观察小鼠死亡率及平均存活时间,以平均存活时间评价菌株毒力。结果白色念珠菌致病株和携带株分泌型酸性蛋白酶检出率分别为83．3％和35．7％（P〈0．01）;细胞外磷脂酶阳性率分别为87．5％和39．3％（P〈0．01）。动物实验结果表明,白色念珠菌致病株菌丝相分泌型酸性蛋白酶和细胞外磷脂酶的活力均显著高于孢子相（P〈0．01,P〈0．005）;注射菌丝相白色念珠菌的小鼠死亡率高于注射孢子相的小鼠（P〈0．01）,且平均存活期短于注射孢子相的小鼠（P〈0．01）。结论分泌型酸性蛋白酶和细胞外磷脂酶是白色念珠菌重要毒力因子,致病株毒力高于携带株,菌丝相毒力高于酵母相。     

Enrichment of butteroil with conjugated linoleic acid via enzymatic interesterification (acidolysis) reactions

Microbial lipases from Candida cylindracea, Pseudomonas sp., Mucor miehei and Candida antarctica were screened for their ability to incorporate conjugated linoleic acid into butteroil triacylglycerides. The lipase from Candida antarctica was employed in a substrates-only medium to increase the conjugated linoleic acid content of the acylglycerides from 0.6 to 15 g/100 g fat.     

Process for the Stereoselective Biotransformation of Acetoacetic Acid Esters Using Yeasts

A process for the stereospecific reduction of acetoacetic acid esters to the 3-(S)-hydroxy-butanoic acid esters by the yeasts Saccharomyces cerevisiae and Candida utilis grown on glucose and ethanol media was developed. A continuous single stage steady state production system was found to be superior to pulse-, batch- and fed-batch systems in terms of optical product purity, biomass concentration and production rates.

Optical purity of 3-(S)-hydroxybutanoic acid esters produced with Saccharomyces cerevisiae and Candida utilis was dependent on pH. A maximal optical purity was obtained at pH2.2 from S. cerevisiae growing on ethanol medium. The specific product formation rate of the chemostat cultures was 0.02…0.05 gg^?1 h^?1. C. utilis was more productive than S. cerevisiae but it reconsumed the product under carbon limited growth conditions.     

The lipid composition and permeability to the triazole antifungal antibiotic ICI 153066 of serum-grown mycelial cultures of Candida albicans

The total lipid content of Candida albicans (serotype A: NCPF 3153) exponential-phase mycelial cultures grown in tissue-culture medium 199 (containing 10%, v/v, foetal calf serum) was 29.8 +/- 8 mg (g dry weight)-1 (mean +/- SD). The weight ratios of phospholipid to neutral lipid and phospholipid to non-esterified sterol were 2.6 +/- 0.4 and 24.9 +/- 0.5, respectively. The major phospholipid was phosphatidylcholine with smaller amounts of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and diphosphatidylglycerol; the most abundant fatty acids were palmitic, palmitoleic, oleic and linoleic acids. The major neutral lipids comprised esterified sterol, triacylglycerol and non-esterified fatty acid with a smaller amount of non-esterified sterol. The fatty acid compositions of the three fatty-acid-containing neutral lipids were distinct from each other and the phospholipids. Comparison with previous data on yeast cultures of C. albicans A grown in glucose broth shows that mycelial cultures have a larger lipid content, lower phospholipid to neutral lipid ratio and higher phospholipid to non-esterified sterol ratio. We now show that mycelial cultures were more permeable to a [14C]triazole antifungal antibiotic compared with exponentially growing yeast cultures of several azole-sensitive strains. Taken together these data are consistent with there being a relationship between the phospholipid/non-esterified sterol ratio of a culture and its ability to accumulate a triazole.