Carbon assimilation and extracellular antigens of some yeast-like fungi

Many yeast-like fungi assimilated n-hexadecane, butylamine and putrescine as sole carbon sources. Methanol was not assimilated. This points to a physiological similarity to endomycetous, hydrocarbon-utilizing yeasts. Stephanoascus ciferrii assimilated uric acid, adenine and allantoin as sole source of carbon and nitrogen. All strains of Geotrichum candidum and many other yeast-like fungi assimilated acetoin and butan-2,3-diol. Assimilation tests for adenine, uric acid, allantoin, acetoin and butan-2,3-diol were found to be suitable for taxonomic purposes.Extracellular antigens immunologically related to those produced by Geotrichum candidum were detected in the cell-free culture liquids of several yeast-like fungi. The extracellular antigen excreted by Stephanoascus ciferrii was species-specific.     

The effect of aminopterin (4-amino folic acid) on growth and cell division of fermentative versus oxidative yeasts

In a related brewing study detailed characteristics of fermentations displaying effective yeastaminopterin interaction were presented.Fermentative yeast types (certain Saccharomyces species and Selenotila intestinalis) proved effective aminopterin reactors whereas oxidative yeasts (certain Candida, Cryptococcus, Pichia, Rhodotorula, Saccharomyces, and Trigonopsis species) proved ineffective reactors. In general effective reactors were polyploids characterized by the lack of film or pellicle formation and ineffective reactors the opposite. In stationary fermentations the Fleischmann 139 strain of S. cerevisiae proved a fair reactor. When aerated it proved an ineffective reactor and aminopterin or products there-of stimulated growth. Conversely aeration enhanced aminopterin activity of effective reactor yeasts.The positive effect of biotin on aminopterin activity and the negative effect of yeast extract, L-asparagine, adenine and thymine is shown and compared and contrasted with earlier reported studies.These findings supported by outside data suggest that oxidative yeasts (and bacteria) can readily elicit enzymes capable of inactivating aminopterin whereas fermentative types are lacking in this capability. Finally that past yeast-aminopterin studies were conducted with oxidative yeast types.Advantages of effective aminopterin reactor yeasts to be published elsewhere include improved ultrastructure using KMnO₄–OsO₄ fixation, a yeast bioassay procedure for detecting aminopterin in plasma and urine, and cell synchronization.Non-Standard Abbreviation apt aminopterin     

The influence of pH on sulphite formation by yeasts

The influence of the initial pH of the substrate on the sulphite formation of three low-sulphite-and five high-sulphite-forming yeasts is described. Four distinctly different groups become apparent. The need for better evaluation of pure culture wine yeasts is stressed.     

Effects of growth conditions on mitochondrial morphology inSaccharomyces cerevisiae

Effects of growth conditions on mitochondrial morphology were studied in livingSaccharomyces cerevisiae cells by vital staining with the fluorescent dye dimethyl-aminostyryl-methylpyridinium iodine (DASPMI), fluorescence microscopy, and confocal-scanning laser microscopy. Cells from respiratory, ethanol-grown batch cultures contained a large number of small mitochondria. Conversely, cells from glucose-grown batch cultures, in which metabolism was respiro-fermentative, contained small numbers of large, branched mitochondria. These changes did not significantly affect the fraction of the cellular volume occupied by the mitochondria. Similar differences in mitochondrial morphology were observed in glucose-limited chemostat cultures. In aerobic chemostat cultures, glucose metabolism was strictly respiratory and cells contained a large number of small mitochondria. Anaerobic, fermentative chemostat cultivation resulted in the large, branched mitochondrial structures also seen in glucose-grown batch cultures. Upon aeration of a previously anaerobic chemostat culture, the maximum respiratory capacity increased from 10 to 70 µmole.min^–1.g weight^–1 within 10 h. This transition resulted in drastic changes of mitochondrial number, morphology and, consequently, mitochondrial surface area. These changes continued for several hours after the respiratory capacity had reached its maximum. Cyanide-insensitive oxygen consumption contributed ca. 50% of the total respiratory capacity in anaerobic cultures, but was virtually absent in aerobic cultures. The response of aerobic cultures to oxygen deprivation was qualitatively the reverse of the response of anaerobic cultures to aeration. The results indicate that mitochondrial morphology inS. cerevisiae is closely linked to the metabolic activity of this yeast: conditions that result in repression of respiratory enzymes generally lead to the mitochondrial morphology observed in anaerobically grown, fermenting cells.     

Relation between phylogeny and physiology in some ascomycetous yeasts

The question of whether yeasts with similar physiological properties are closely related has been examined using recently published phylogenetic analyses of 26S domain D1/D2 rDNA nucleotide sequences from all currently recognized ascomycetous yeasts. When apparently unique metabolic pathways are examined, some relationships between physiology and rDNA phylogeny are evident. Most Candida and Pichia species that are able to assimilate methanol as the sole carbon source are in a clade delimited by C. nanospora and C. boidinii. Exceptions are P. capsulata and P. pastoris which are phylogenetically separated from the other methanol-assimilating yeasts. Yeasts subject to the petite mutation, resulting in respiratory deficiency, belong to three different clades, viz. a Saccharomyces clade delimited by S. cerevisiae and S. rosinii,the Dekkera/Brettanomyces clade, and some Schizosaccharomyces species (‘Archiascomycete’ clade). However, petite mutants were also found in Zygosaccharomyces fermentati and some other more distantly related species. Yeasts able to assimilate n-hexadecane, uric acid or amines as sole carbon source are broadly distributed over the ascomycetous phylogenetic tree. However, species that assimilate adenine as sole carbon source are closely related. Most of these species also assimilated glycine, uric acid, n-hexadecane, putrescine and branched-chain aliphatic compounds such as isobutanol, leucine and isoleucine. Among the Saccharomycetales, species utilizing all or the great majority of these eight compounds are in the Stephanoascus/Arxula/Blastobotrys clade. Candida blankii, which is distantly related to this clade, proved to be an exception and assimilated six of eight of these compounds. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of growth conditions on production of capsular and extracellular polysaccharides byRhizobium leguminosarum

The influence of growth rate and medium composition on exopolymer production byRhizobium leguminosarum was studied. When grown in medium containing 10g/l mannitol and 1g/l glutamic acid,Rhizobium leguminosarum biovartrifolii TA-1 synthesized up to 2.0g/l of extracellular polysaccharide (EPS), and up to 1.6g/l of capsular polysaccharide (CPS). Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1g/l, while EPS-production remained relatively low (0.8g/l). Maximal CPS-yield was 2.9g CPS/l medium in a medium containing 20g/l mannitol and 2g/l glutamic acid. TheEPS-deficient strain R. leguminosarum RBL5515,exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets. With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500mg cell protein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h^–1). The production rates were 50–100mg/g protein/h for EPS and 15–20mg/g protein/h for CPS. Only low amounts of cyclic -(1,2)-glucans were excreted (10–30 mg/l) over the entire range of growth rates.Abbreviations bv biovar - CPS capsular polysaccharide - EPS extracellular polysaccharide - HM_r high molecular mass - LM_r low molecular mass - YEMCR Yeast Extract-Mannitol-Congo Red agar     

非常规酵母的分子遗传学及合成生物学研究进展

先进的合成生物学技术与传统的分子遗传学技术的结合更有助于实现酵母底盘细胞的快速改造和优化。酵母合成生物学研究最早开始于常规酵母——酿酒酵母(Saccharomyces cerevisiae),近些年来又迅速扩展至一些非常规酵母,包括巴斯德毕赤酵母(Pichiapastoris)、解脂耶氏酵母(Yarrowialipolytica)、乳酸克鲁维酵母(Kluyveromyces lactis)和多形汉逊酵母(Hansenula polymorpha)等。借助合成生物学技术与工具,目前科学家们已经成功开发出了能够高效生产生物材料、生物燃料、生物基化学品、蛋白质制剂、食品添加剂和药物等工业产品的重组非常规酵母工程菌株。本文系统总结了合成生物学工具(主要是基因组编辑工具)、合成生物学组件(主要是启动子和终止子)和相关分子遗传学方法在上述非常规酵母系统(底盘细胞)中的最新研究进展和应用情况,并讨论了其他合成生物学技术在这些非常规酵母表达系统中的潜在适用性和应用前景。这为研究人员利用合成生物学方法在这一新型非模式微生物底盘细胞中设计和构建各种高附加值工业产品的异源合成模块并最终实现目标化合物的高效生物合成提供了科学的理论指导。     

Fermentation of maltotriose by brewer's and baker's yeasts

Two brewer's yeasts and one baker's yeast grew with 95% (w/w) pure maltotriose as carbon source in the presence of antimycin A to block respiration. Biomass yields (0.15 and 0.24 g dry yeast g^–1 sugar, respectively, with and without antimycin A) were similar for growth on maltose and maltotriose, and yields of ethanol were 80% of stoichiometric. Yeasts harvested during growth on glucose and containing low maltose transport activity did not begin to use maltotriose in the presence of antimycin A until after a long lag phase (up to 50 h), but yeast harvested during growth on maltose, and containing high maltose transport activity, began to use maltotriose after about 25 h. Much shorter lags were observed before growth started in the absence of antimycin A.     

Genetic identification of naturalSaccharomyces sensu stricto yeasts from Finland,Holland and Slovakia

Genetic and karyotypic studies of naturalSaccharomyces sensu stricto yeasts from Finland, Holland and Slovakia revealed three wild sibling-species:Saccharomyces cerevisiae, Saccaromyces bayanus andSaccharomyces paradoxus.     

Kinetics of growth and sugar consumption in yeasts

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts.Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called Crabtree effect probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect inS. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast.S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.Non-Saccharomyces yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeastCandida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.     

The effect of growth factors on anoxic chemostat cultures of two Saccharomyces cerevisiae strains

In anoxic chemostat cultures of Saccharomyces cerevisiae ATCC 4126 and CBS 8066 grown in a medium containing yeast extract, a sharp increase in the steady-state residual glucose concentration occurred at relatively low dilution rates, contrary to the expected Monod kinetics. However, supplementation with vitamins and amino acids facilitated efficient glucose uptake. This enhanced requirement for growth factors under anoxic conditions and at high growth rates could explain the exceptionally high apparent k _s values for S. cerevisiae reported in the literature.     

The effect of ethanol on cell wall antigens ofSaccharomyces cerevisiae and specific isolation of high ethanol producing strains of this yeast,making use of a serological technique

Generally, natural isolates of high ethanol producingSaccharomyces cerevisiae obtained by screening are used in alcoholic industries. The methods involved in their isolation and identification are elaborate. Antigenic analysis using antibodies raised against wholeSaccharomyces cells indicated species specificity of cell wall surface thermostable antigens. By affinity purification, the specific antibodies could be obtained and used for specific isolation ofS. cerevisiae. Antigenic studies using antibodies raised against isolated cell walls of fermentatively grownS. cerevisiae indicated the occurrence of thermolabile antigens common toSaccharomyces species. Higher concentrations of these antigens could be detected in thoseS. cerevisiae that had the ability for high ethanol production. The concentrations of these cell wall common antigens increased with increasing culture age and ethanol accumulation in culture broths. In younger yeast cells, the concentration could be increased by growing the cells in a medium containing added ethanol. Using dilutions of cross absorbed antibody specific for common antigens and Ouchterlony test, high ethanol producingS. cerevisiae could be identified.     

Interruption of glycerol pathway in industrial alcoholic yeasts to improve the ethanol production

The two homologous genes GPD1 and GPD2, encoding two isoenzymes of NAD⁺-dependent glycerol-3-phosphate dehydrogenase in industrial yeast Saccharomyces cerevisiae CICIMY0086, had been deleted. The obtained two kinds of mutants gpd1Δ and gpd2Δ were studied under alcoholic fermentation conditions. gpd1Δ mutants exhibited a 4.29% (relative to the amount of substrate consumed) decrease in glycerol production and 6.83% (relative to the amount of substrate consumed) increased ethanol yield while gpd2Δ mutants exhibited a 7.95% (relative to the amount of substrate consumed) decrease in glycerol production and 7.41% (relative to the amount of substrate consumed) increased ethanol yield compared with the parental strain. The growth rate of the two mutants were slightly lower than that of the wild type under the exponential phase whereas ANG1 (gpd1Δ) and the decrease in glycerol production was not accompanied by any decline in the protein content of the strain ANG1 (gpd1Δ) but a slight decrease in the strain ANG2 (gpd2Δ). Meanwhile, dramatic decrease of acetate acid formation was observed in strain ANG1 (gpd1Δ) and ANG2 (gpd2Δ) compared to the parental strain. Therefore, it is possible to improve the ethanol yield by interruption of glycerol pathway in industrial alcoholic yeast.     

Relationship between molecular and enological features of Patagonian wine yeasts: relevance in selection protocols

Summary In this paper, a study of the relationship between genetic patterns, obtained by the combination of mtDNA-RFLP and PCR-amplified inter-δ sequence DNA polymorphism analysis, and relevant enological phenotypic data (fermentative power, specific productivity, volatile and total acidity) was carried out on Argentinean Saccharomyces cerevisiae isolates from north Patagonia. The use of a powerful statistical tool, Generalized Procrustes analysis, allowed us to weigh the relationship for each isolate in particular, denoting a good enough degree of agreement between molecular and physiological data for most of the population analysed. The inclusion of a physiological feature, as the killer sensitivity biotype, within identification methods resulted in a higher degree of discrimination among isolates and in better correlation between both characterizations. The combined use of methods based on molecular polymorphisms and killer biotype could be applied so as not to miss any isolate with differential enological properties in selection protocols.     

Interaction of yeasts and some nitrogen fixing bacteria on nodulation of legumes

Summary Combined inoculation ofRhizobium trifolii withSaccharomyces cerevisiae and other yeasts generally enhanced the number of nodules, length of plants and dry weight of Egyptian clover (Trifolium alexandrinum) seedlings grown on agar slopes. Similar effects were observed when seedlings were inoculated withR. trifolii in the presence of dialyzed culture filtrate ofS. cerevisiae.     

Influence of culture conditions on glutathione production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20–30°C), agitation rate (100–300 rpm), initial pH (5.0–7.0), inoculum concentration (5–15%), and glucose concentration (30–70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2^5–2), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2²) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20°C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.     

Glycerol production in relation to the ATP pool and heat production rate of the yeasts Debaryomyces hansenii and Saccharomyces cerevisiae during salt stress

Changes in glycerol production and two parameters related to energy metabolism i. e. the heat production rate and the ATP pool, were assayed during growth of Saccharomyces cerevisiae and Debaryomyces hansenii in 4 mM and 1.35 M NaCl media. For both of the yeasts, the specific ATP pool changed during the growth cycle and reached maximum values around 10 nmol per mg dry weight in both types of media. The levels of glycerol were markedly enhanced by high salinity. In the presence of 1.35 M NaCl, D. hansenii retained most of its glycerol produced intracellularly, while S. cerevisiae extruded most of the glycerol to the environment. The intracellular glycerol level of S. cerevisiae equalled or exceeded that of D. hansenii, however, with values never lower than 3 mol per mg dry weight at all phases of growth. When D. hansenii was grown at this high salinity the intracellular level of glycerol was found to correlate with the specific heat production rate. No such correlation was found for S. cerevisiae. We concluded that during salt stress, D. hansenii possesses the capacity to regulate the metabolism of glycerol to optimize growth, while S. cerevisiae may not be able to regulate when exposed to different demands on the glycerol metabolism.     

Oxidation of amines by yeasts grown on 1-aminoalkanes or putrescine as the sole source of carbon,nitrogen and energy

The maximum growth rate of Trichosporon cutaneum CBS 8111 in chemostat cultures was 0.185 h^-1 on ethylamine and 0.21 h^-1 on butylamine, that of Candida famata CBS 8109 was 0.32 h^-1 on putrescine.The amine oxidation pattern of the ascomycetous strains studied, viz. Candida famata CBS 8109, Stephanoascus ciferrii CBS 4856 and Trichosporon adeninovorans CBS 8244 was independent of the amine that had been used as the growth substrate. It resembled that of benzylamine/putrescine oxidase found in other ascomycetous yeasts. However, differences in pH optimum and substrate specificity were observed between the amine-oxidizing systems of these three species.The amine oxidation pattern of cell-free extracts of Trichosporon cutaneum CBS 8111 varied with the amine that was used as growth substrate. The enzyme system produced by Cryptococcus laurentii CBS 7140 failed to oxidize isobutylamine and benzylamine, and showed a high pH optimum.The synthesis of amine oxidase in the four yeast strains studied was not repressed by ammonium chloride and was weakly repressed by glucose but was strongly repressed if both compounds were present in the growth medium.     

A possible application of ale brewery strains ofSaccharomyces cerevisiae in lager beer production

The physiological characteristics of two strains of brewery ale yeasts,Saccharomyces cerevisiae, with sedimentation abilities, were investigated to see if the strains were suitable for lager beer production. Compared with typical industrial ale strains ofS. cerevisiae and lager strains ofS. uvarum (nowS. cerevisiae), the investigated strains differ in fermentation dynamics, as well as in biological properties. The differences, however, particularly between the two strains and the lager brewing yeasts, were not significant.     

Inhibitory effect of ethanol on growth and solute accumulation by Saccharomyces cerevisiae as affected by plasma-membrane lipid composition

Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-³H] glucose, d-[1-¹⁴C] glucosamine, l-[U-¹⁴C] lysine or arginine, or KH₂ ³²PO₄ lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.