Optimization of accessible catalase activity in polyacrylamide gel-immobilized Saccharomyces cerevisiae

The permeabilization of Saccharomyces cerevisiae (baker's yeast), either before or after immobilization in polyacrylamide gel (PAG), has been examined as a means to increase the catalase activity of PAG-immobilized yeast cells. Prior permeabilization of the cells resulted in large losses of catalase activity during immobilization, but permeabilization after immobilization produced increases in the catalase activity of yeast/PAG particles. A dependence of the accessible catalase activity on the concentration of polyacrylamide in permeabilized yeast/PAG particles, and on the method of permeabilization of the immobilized cells, was observed. Optimal levels of stable catalase activity (1000-2000 IU/g PAG particles; ca. 5%-10% of total available activity) were obtained by immobilizing yeast cells (0.5 g wet cells/mL gel) in 10% (w/v) PAG, followed by permeabilization of the entrapped cells with either cetyltrimethylammonium bromide, Triton X-100 and one freeze-thaw, or five freeze-thaw cycles. (c) 1992 John Wiley & Sons, Inc.     

Oscillatory metabolism of Saccharomyces cerevisiae in continuous culture

Short-period (40-50 min) synchronized metabolic oscillation was found in a continuous culture of yeast Saccharomyces cerevisiae under aerobic conditions at low-dilution rates. During oscillation, many parameters changed cyclically, such as dissolved oxygen concentration, respiration rate, ethanol and acetate concentrations in the culture, glycogen, ATP, NADH, pyruvate and acetate concentrations in the cells. These changes were considered to be associated with glycogen metabolism. When glycogen was degraded, the respiro-fermentative phase was observed, in which ethanol was produced and the respiration rate decreased. In this phase, the levels of intracellular pyruvate and acetate became minimum, ATP became high and intracellular pH at its lowest level. When glycogen metabolism changed from degradation to accumulation, the respiratory phase started, during which ethanol was re-assimilated from the culture and the respiration rate increased. Intracellular pyruvate and acetate became maximum, ATP decreased and the intracellular pH appeared high. These findings may indicate new aspects of the control mechanism of glycogen metabolism and how respiration and ethanol fermentation are regulated together under aerobic conditions.     

重组酿酒酵母催化二氢大豆苷元生产雌马酚

雌马酚是大豆异黄酮的代谢产物,是一种天然的选择性雌激素受体调节剂,稳定性和生物学活性高。为实现雌马酚的微生物合成,采用模块途径工程策略,构建编码雌马酚合成关键酶基因 orf-1、orf-2和orf-3 的表达载体,成功用于转化酿酒酵母BY4741,得到工程菌株。结果表明,工程菌株有效表达了外源基因,并可将大豆异黄酮代谢中间体二氢大豆苷元转化为雌马酚。为构建从头合成雌马酚的微生物细胞工厂提供了重要科学参考。     

The presence of Saccharomyces cerevisiae DNA in various media used to propagate yeasts and its removal by ethidium monoazide

Aims:  In this study we demonstrate the interference of yeast extract in enumeration of Saccharomyces cerevisiae using real-time PCR and develop a method for its removal from the media using ethidium monoazide (EMA).
Methods and Results:  Using real-time PCR and primers to S. cerevisiae we demonstrate the presence of yeast DNA in various media as well as the media impact on S. cerevisiae real-time PCR standard curves. By pretreatment with EMA, we were able to remove this interference.
Conclusions:  Saccharomyces cerevisiae DNA can be found in a number of common laboratory media and may impact the enumeration of this yeast by real-time PCR. However, pretreatment with EMA eliminates this concern.
Significance and Impact of the Study:  We have developed a method for removal of contaminating DNA in yeast growth media.     

Glucose metabolism and cell size in continuous cultures of Saccharomyces cerevisiae

A detailed analysis of the cell size, monitored as protein content, has been performed in glucose-limited continuous cultures, so as to obtain the values of the average protein content for various subpopulations at different cell cycle stages, as a function of the growth rate. Glucose metabolism appears to affect cell size, since there is an increase of the average protein content of the population when cells produce ethanol above the critical dilution rate. If the production of ethanol is forced at low growth rates by the addition of formate, the average protein content increases. These results indicate a link between glucose metabolism and cell size in budding yeast, as observed for mammalian cells.     

Removal of heavy metals using a brewer's yeast strain of Saccharomyces cerevisiae: advantages of using dead biomass

Aim:  The capacities of live and heat-killed cells of Saccharomyces cerevisiae at 45°C for the removal of copper, nickel and zinc from the solution were compared.
Methods and Results:  Kinetic studies have shown a maximum accumulation of Ni²⁺ and Zn²⁺ after 10 min for both types of cells, while for Cu²⁺ this was attained after 30 and 60 min for dead and live cells, respectively. Equilibrium studies have shown that inactivated biomass displayed a greater Zn²⁺ and Ni²⁺ accumulation than live yeasts. For Cu²⁺, live and dead cells showed similar accumulation. Fluorescence, scanning electron microscopy and infrared spectroscopy studies have shown that no appreciable structural or molecular changes occurred in the cells during the killing process. The increased metal uptake observed in dead cells can be most likely explained by the loss of membrane integrity, which allows the exposition of further metal-binding sites present inside the cells.
Conclusions:  Heat-killed cells showed a higher degree of heavy metal removal than live cells, being more suitable for further bioremediation works.
Significance and Impact of the Study:  Dead flocculent cells can be used in a low cost technology for detoxifying metal-bearing effluents as this approach combines an efficient metal removal with the ease of cell separation.     

The metabolic response of Saccharomyces cerevisiae to continuous heat stress

A study has been initiated to integrate molecular and physiological responses of Saccharomyces cerevisiae to heat stress conditions. We focus our research on a quantification of the energetics of the stress response. A series of continuous heat stresses was applied to exponentially growing cells of the strain X2180-1A at 28°C, by increasing the growth temperature to 37, 39, 40, 41, 42, or 43°C. Here, the results on cell growth and viability, as well as on anabolic and catabolic rates are presented. We observed a surprisingly thin line for the cells between growing, surviving, and dying, with regard to growth temperature. The heat stress showed a dual effect on catabolism: immediately after the temperature increase a strong peak was seen, after which a new, steady level was reached. In addition, the yield on glucose decreased with increasing temperature. Our results indicate that life at elevated temperatures is energetically unfavourable and a non-lethal heat stress invokes a redistribution of catabolic and anabolic fluxes.     

固定化酵母乙醇发酵及固化小球微生态的研究

比较了SA-PVA-SiO₂固定化酿酒酵母和游离酿酒酵母的乙醇发酵能力,采用批次发酵试验研究固定化酿酒酵母的发酵稳定性。结果表明,SA-PVA-SiO₂固定化酿酒酵母的发酵速度比游离酿酒酵母快,发酵周期短;发酵稳定性很好,30℃,橡胶塞、90 r/min摇床培养24 h时,乙醇体积分数均在3%~3.5％之间,连续发酵14批次后,固化小球的形态依然完好,不发粘。通过扫描电镜对酿酒酵母包埋微生态环境进行了分析,图像表明固定化小球的内部环境非常有利于酵母细胞的厌氧发酵产乙醇,充分证明了SA-PVA-SiO₂固定化酿酒酵母乙醇发酵的优越性。     

Purification and Characterization of an Endo-Polygalacturonase from a Mutant of Saccharomyces cerevisiae

An extracellular endo-polygalacturonase (PGase) produced by a mutant of Saccharomyces cerevisiae was isolated. The enzyme was regarded, immunologically, as a PGase belonging to the Kluyveromyces marxianus group. The enzyme had properties similar to the PGase from K. marxianus in heat and pH stability, and N-terminal amino acid sequence. However, the enzyme showed different properties in optimum pH and temperature, molecular weight, and reactivity in antiserum against PGase from K. marxianus, indicating that the enzyme has a different molecular structure from the PGase from K. marxianus.     

Effects of xylulokinase activity on ethanol production from D-xylulose by recombinant Saccharomyces cerevisiae

AIMS: Recombinant Saccharomyces cerevisiae strains harbouring different levels of xylulokinase (XK) activity and effects of XK activity on utilization of xylulose were studied in batch and fed-batch cultures. METHODS AND RESULTS: The cloned xylulokinase gene (XKS1) from S. cerevisiae was expressed under the control of the glyceraldehyde 3-phosphate dehydrogenase promoter and terminator. Specific xylulose consumption rate was enhanced by the increased specific XK activity, resulting from the introduction of the XKS1 into S. cerevisiae. In batch and fed-batch cultivations, the recombinant strains resulted in twofold higher ethanol concentration and 5.3- to six-fold improvement in the ethanol production rate compared with the host strain S. cerevisiae. CONCLUSIONS: An effective conversion of xylulose to xylulose 5-phosphate catalysed by XK in S. cerevisiae was considered to be essential for the development of an efficient and accelerated ethanol fermentation process from xylulose. SIGNIFICANCE AND IMPACT OF THE STUDY: Overexpression of the XKS1 gene made xylulose fermentation process accelerated to produce ethanol through the pentose phosphate pathway.     

Protective Effect of a Fermented Substance from Saccharomyces cerevisiae on Liver Injury in Mice Caused by Acetaminophen

The protective effect of a fermented substance from Saccharomyces cerevisiae (FSSC) on liver injury caused by acetaminophen (AAP) was studied in mice. Mice were pretreated with FSSC (0.5–2.0 g/kg, p.o.) for 4 d, and on the fourth day, the mice received an overdose of AAP (500 mg/kg, i.p.). Subsequently, they were sacrificed at 7 h, and blood was drawn from the abdominal vein and liver samples were collected. Histological and biochemical examinations revealed that the administration of AAP caused liver injury in the mice, including increases in plasma alanine aminotransferase and asparate aminotransferase activities and decreases in the hepatic reduced form of glutathione (GSH) content and antioxidant enzyme activities. Prior to AAP treatment, the mice pretreated with FSSC showed significantly reduced levels of alanine aminotransferase (ALT) and aspirate aminotransferase (AST) activity. Liver histology in the FSSC-pretreated mice was significant. In these mice, pretreatment with FSSC also served to reduce hepatic GSH depletion and the inhibition of antioxidant enzyme activity caused by AAP overdose. In conclusion, oral administration of FSSC significantly reduced AAP-induced hepatic injury in the mice.     

酿酒酵母转化木糖生产化学品的研究进展

木糖的有效利用是木质纤维素生产生物燃料或化学品经济性转化的基础.30年来,通过理性代谢改造和适应性进化等工程策略,显著提高了传统乙醇发酵微生物——酿酒酵母Saccharomyces cerevisiae的木糖代谢能力.因此,近年来在酿酒酵母中利用木糖生产化学品的研究逐步展开.研究发现,酿酒酵母分别以木糖和葡萄糖为碳源时...     

The role of trehalose in dehydration resistance of Saccharomyces cerevisiae

Abstract High levels of intracellular trehalose in stationary-phase cells of Saccharomyces cerevisiae or cells incubated in the absence of a nitrogen source were found to increase the resistance of the cells to dehydration. Exponential-phase cells showed negligible dehydration resistance. When stationary-phase cells were inoculated into fresh medium, trehalose was rapidly broken down, and this was correlated with a rapid loss of dehydration resistance. It appeared that a minimum internal concentration of 120 mM trehalose was required before there was a significant increase in dehydration resistance. Exogenous trehalose increased the dehydration resistance of S. cerevisiae : this effect was most marked for stationary-phase cells, where almost 100% survival was obtained at trehalose concentrations of 500 mM and above while maximum survival for exponential cells was less than 10%, even at 1000 mM external trehalose.     

Use of microcalorimetric monitoring in establishing continuous energy balances and in continuous determinations of substrate and product concentrations of batch-grown Saccharomyces cerevisiae

Energy balance calculations were performed for different physiological states during batch growth of Saccharomyces cerevisiae with glucose as carbon and energy source. For the different physiological states, energy recoveries close to one were obtained, which permitted a continuous control that the constantly changing growth process was quantified accurately. During the respiro-fermantative phase of growth, during which glucose served as the carbon and energy source, a low-heat-yield value (DeltaQ(x)) of -8.6 kJ/g dry biomass formed was obtained. This low-heat-yield value was due to the mainly fermentative metabolism during the middle of this phase of growth. After a transition phase, the ethanol produced during the respiro-fermentative growth was respired. During this respiratory phase, the heat yield values increased markedly, resulting in a lowest value of -42.7 kJ/g. The low-heat-yield values of the respiro-fermentative growth is not a reflection of the most efficient metabolism of S. cerevisiae. On the contrary, during the middle of this phase, 74% of the energy input was dissipated as ethanol, 6% was dissipated as heat, and the energy conserved as biomass was just 13%, while during the early respiratory phase, 69% of the energy input was dissipated as heat, and 22% of the energy input was conserved as biomass. By mathematical modeling and direct monitoring on-line of the rate of heat production, continuous calculations of (1) glucose consumption, and (3) biomass production were performed, and were shown to correlate closely with measured values for the continuously changing growth process.     

酿酒酵母单倍体菌株分离筛选及其产腺苷甲硫氨酸的初步研究

获得产腺苷甲硫氨酸的二倍体酿酒酵母CGMCC 2842遗传育种单倍体亲本。采用不同产孢培养基考察了酿酒酵母的产孢率,并对酿酒酵母CGMCC 2842进行了生孢培养分离子囊孢子得到单倍体菌株,确定单倍体配型,测定不同单倍体菌株腺苷甲硫氨酸含量。从分离的七株单倍体菌株(6株a型和1株α型)中筛选出一株产腺苷甲硫氨酸较高的a配型的单倍体菌株,经250 m L摇瓶发酵48 h后产腺苷甲硫氨酸1.10 g/L。筛选得到了一株产腺苷甲硫氨酸较高a型的单倍体菌株,为菌株的进一步遗传育种改良和腺苷甲硫氨酸微生物发酵法规模化生产奠定了基础。     

Gradual genome stabilisation by progressive reduction of the Saccharomyces uvarum genome in an interspecific hybrid with Saccharomyces cerevisiae

Considerable amounts of molecular and genetic data indicate that interspecific hybridisation may not be rare among natural strains of Saccharomyces sensu stricto. Although a post-zygotic barrier operating during meiosis usually prevents the production of viable spores, stable hybrids can arise which can even evolve into distinct species. This study was aimed to analyse the genome of a fertile Saccharomyces cerevisiae x S. uvarum hybrid and monitor its changes over four filial generations of viable spores. The molecular genetic analysis demonstrated that the two species did not contribute equally to the formation and stabilisation of the hybrid genome. S. cerevisiae provided the mitochondrial DNA and the more stable part of the nuclear genome. The S. uvarum part of the hybrid nuclear genome became progressively smaller by loosing complete chromosomes and genetic markers in the course of successive meiotic divisions. Certain S. uvarum chromosomes were eliminated and/or underwent rearrangements in interactions with S. cerevisiae chromosomes. Numerous S. uvarum chromosomes acquired S. cerevisiae telomere sequences. The gradual elimination of large parts of the S. uvarum genome was associated with a progressive increase of sporulation efficiency. We hypothesise that this sort of genomic alterations may contribute to speciation in Saccharomyces sensu stricto.