Comparative analysis of glycogen and trehalose accumulation in methylotrophic and nonmethylotrophic yeasts

Trehalose and glycogen accumulate in certain yeast species when they are exposed to unfavorable growth conditions. Accumulations of these reserve carbohydrates in yeasts provide resistance to stress conditions. The results of this study indicate that certain Pichia species do not accumulate high levels of glycogen and trehalose under normal growth conditions. However, depending on the Pichia species, both saccharides accumulate at high levels when the Pichia cells are exposed to unfavorable or stress-inducing growth conditions. Growth in glycerol or methanol medium mostly led to trehalose accumulation in Pichia species tested in this study. It was shown that the metabolic pathways for glycogen and trehalose biosynthesis are present in Pichia species. However, it appears that the biosynthesis of trehalose and glycogen may be regulated in different manners in Pichia species than in the yeast S. cerevisiae. Published in Russian in Mikrobiologiya, 2006, Vol. 75, No. 6, pp. 737–741. The text was submitted by the author in English.     

Physiological and technological aspects of large-scale heterologous-protein production with yeasts

Commercial production of heterologous proteins by yeasts has gained considerable interest. Expression systems have been developed forSaccharomyces cerevisiae and a number of other yeasts. Generally, much attention is paid to the molecular aspects of heterologous-gene expression. The success of this approach is indicated by the high expression levels that have been obtained in shake-flask cultures. For large-scale production however, possibilities and restrictions related to host-strain physiology and fermentation technology also have to be considered. In this review, these physiological and technological aspects have been evaluated with the aid of numerical simulations. Factors that affect the choice of a carbon substrate for large-scale production involve price, purity and solubility. Since oxygen demand and heat production (which are closely linked) limit the attainable growth rate in large-scale processes, the biomass yield on oxygen is also a key parameter. Large-scale processes impose restrictions on the expression system. Many promoter systems that work well in small-scale systems cannot be implemented in industrial environments. Furthermore, large-scale fed-batch fermentations involve a substantial number of generations. Therefore, even low expression-cassette instability has a profound effect on the overall productivity of the system. Multicopy-integration systems may provide highly stable expression systems for industrial processes. Large-scale fed-batch processes are typically performed at a low growth rate. Therefore, effects of a low growth rate on the physiology and product formation rates of yeasts are of key importance. Due to the low growth rates in the industrial process, a substantial part of the substrate carbon is expended to meet maintenance-energy requirements. Factors that reduce maintenance-energy requirements will therefore have a positive effect on product yield. The relationship between specific growth rate and specific product formation rate (kg product·[kg biomass]^–1·h^–1) is the main factor influencing production levels in large-scale production processes. Expression systems characterized by a high specific rate of product formation at low specific growth rates are highly favourable for large-scale heterologous-protein production.     

Differential pattern of trehalose accumulation in wine yeast strains during the microvinification process

Trehalose accumulation in wine yeast strains growing under microvinification conditions was determined and compared to that obtained under laboratory conditions. Industrial strains accumulate 10-fold more trehalose than laboratory strains. Contrary to batch-culture growth, under microvinification conditions trehalose accumulation is not consequence of glucose exhaustion. Physiological relevance of trehalose during the process of wine making and their use for potential improvements of alcoholic fermentation are discussed.     

Incorporation of 3-deoxy-3-fluoro-D-glucose into glycogen and trehalose in fat body and flight muscle inLocusta migratoria

Flight muscle and fat body extracts fromLocusta migratoria were incubated with D-[U-¹⁴C]-glucose or D-[3-³H]-3-deoxy-3-fluoroglucose and the products were analyzed. In the case of the latter compound, radio-chromatographic analysis yielded glycogen and trehalose fractions that were shown by¹⁹F nuclear magnetic resonance to contain fluorine. Acid hydrolysis of these fractions liberated tritium labelled 3-deoxy-3-fluoro-D-glucose. In addition to the formation of fluoroglycogen and fluorotrehalose in these tissue extracts, there was an accumulation of tritium labelled fructose.     

Effect of wine yeast monoculture practice on the biodiversity of non-Saccharomyces yeasts

AIMS: The objective of this work was to study the effect of the use of Saccharomyces cerevisiae monocultures over the biodiversity of non-Saccharomyces yeasts in wine-producing areas in Chile. METHODS AND RESULTS: Microvinifications were carried out with grape musts of two areas. In one of them, the fermentation is carried out mainly in a spontaneous manner, whereas in the other the musts are inoculated with commercial yeasts. The isolated yeasts were identified by the internal transcribed (ITS)/restriction fragment length polymorphism technique. In the industrial production area less variability of yeast genera was observed as compared with the traditional area, an observation that is greatest at the end of the fermentation. Furthermore, a study of the production of extracellular enzymes was done. The majority of the yeasts showed at least one of the activities assayed with the exception of beta-glycosidase. CONCLUSION: The results suggest that in the industrialized area the diversity of yeasts is less in the traditional area. Likewise, the potentiality of the non-Saccharomyces yeasts as enzyme producers with industrial interest has been confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the negative effect of the use of monocultures over the biodiversity of yeasts in wine-producing regions.     

Trehalose accumulation in a high-trehalose-accumulating mutant of Saccharomycopsis fibuligera sdu does not respond to stress treatments

The isolation of high-trehalose-accumulating mutant A11 from Saccharomycopsis fibuligera sdu has been previously described. In this paper, accumulation of trehalose under various stress conditions in S. fibuligera A11 was investigated. Neither activation of trehalose-6-phosphate synthase (SfTps1) nor change in trehalose content was observed under stress exposure of S. fibuligera A11 cells. A fragment of the Sftps1 gene in this strain was also cloned by degenerate PCR using the CoDeHOP strategy and multiply-aligned Tps1 sequences. This sequence allowed us to investigate the expression of the Sftps1 gene, which was also kept constant under the various stress conditions. Altogether, these results indicate that trehalose metabolism in S. fibuligera A11 in response to stress conditions clearly differs from that of Saccharomyces cerevisiae and most other fungi. The expression of the Sftps1 gene was not responsive to different stress treatments.     

Stress effect of ethanol on fermentation kinetics by stationary-phase cells of Saccharomyces cerevisiae

When 4% (v/v) ethanol was added progressively to two strains exhibiting different fermentative abilities, K1 (a commercial wine strain) and V5 (a strain derived of a wine yeast), the fermentation rate correlated directly to the ethanol concentration for both strains. In contrast, the effect of sudden addition of 2%, 4% or 6% (v/v) ethanol was different depending on the strain. While the same effect was observed for K1 whatever the way of ethanol addition, V5 required an adaptation period after the shock addition of ethanol.     

Biochemical aspects of stuck and sluggish fermentation in grape must

Recently a number of studies have focused on the factors responsible for the occurrence of stuck and sluggish fermentations. Results from these studies indicate that together with nutritional deficiencies and inhibitory substances, technological practices could lead to such situations. This review explains, from a biochemical point of view, the influence of nutritional deficiencies, inhibitory substances and technological practices on yeast cell development and physiology and the fermentation process. Received 07 February 1997/ Accepted in revised form 01 July 1997     

Aquaporins in Saccharomyces cerevisiae wine yeast

AQY1 and AQY2 were sequenced from five commercial and five native wine yeasts. Of these, two AQY1 alleles from UCD 522 and UCD 932 were identified that encoded three or four amino-acid changes, respectively, compared with the Sigma1278b sequence. Oocytes expressing these AQY1 alleles individually exhibited increased water permeability vs. water-injected oocytes, whereas oocytes expressing the AQY2 allele from UCD 932 did not show an increase, as expected, owing to an 11 bp deletion. Wine strains lacking Aqy1p did not show a decrease in spore fitness or enological aptitude under stressful conditions, limited nitrogen, or increased temperature. The exact role of aquaporins in wine yeasts remains unclear.     

Intraspecific diversity of Oenococcus oeni isolated during red wine-making in Japan

Using molecular and chemotaxonomic techniques, we studied the intraspecific diversity of Oenococcus oeni, a lactic acid bacterium isolated during red wine-making in Japan. The results confirmed high values of DNA-DNA relatedness and strong similarity among 16S rDNA sequences of the isolates with the O. oeni-type strain. Pulsed-field gel electrophoresis (PFGE) by NotI identified four patterns among the strains. Three different patterns of lactate dehydrogenase mobility were seen and there was a strong correlation between PFGE pattern and mobility. The present results suggest that the different strains of O. oeni comprise one species, and that variations in the genomic profiles of the different strains of O. oeni, including Japanese isolates are well correlated.     

Mutagen Formation in the Nitrite—Piperic Acid Reaction

A morphological mutant of Neurospora crassa, which showed great changes in cell wall β-glucan structures, was obtained. The mutant lacked spore-forming ability. Chemical analysis indicated that the mutant cell walls had more carbohydrates and less proteins than the wild type. In the structural polymers of cell walls, heteroglycan and chitin were not apparently changed in their sugar composition and structures. On the other hand, the alkali-soluble β-glucan of this mutant showed significant changes in the chemical structure, particularly, the number and length of branches. The mutant glucan had about 2.5 times as many branches as that from wild type and the number of 1,3-linked glucose residues was greatly reduced.     

The Role of Trehalose and Glycogen in the Survival of Aging Saccharomyces cerevisiae Cells

The role of the storage carbohydrates trehalose and glycogen in the survival of aging Saccharomyces cerevisiae cells was studied. Culture aging for one week did not reduce cell viability. During this period, the cells accumulated the storage carbohydrates and showed increased activity of the glycolytic enzymes hexokinase and phosphofructokinase. However, further aging led to a drastic drop in cell viability and to a decrease in the cellular content of trehalose and glycogen and in the activity of hexokinase and phosphofructokinase. The possible reasons for these changes are discussed.     

优良菌种发酵葡萄酒优化条件的探讨

以诱变耐低温果酒酵母菌种YU2.28和产香酵母S15.3为发酵菌株,进行了葡萄酒发酵条件优化的试验研究.探讨了菌种生长温度、通氧量等因素,通过对菌种的生长情况和发酵醪液中总酯含量的变化分析,确定了自选酵母酿制葡萄酒的最佳技术参数,并对优化条件下发酵得到的葡萄酒进行GC/MS分析.结果显示：YU2.28和S15.3以1：3比例的混合发酵,接种量3%,调节醪液pH值为4.0,SO2添加量40 mg/L,发酵温度20℃,主发酵6 d内控制以230r/min的摇床转速进行摇瓶发酵,并进行9 h（每天1.5 h）供氧处理,后发酵30 d,酿造出的葡萄酒品质较佳,具有酒体丰盈,酒液澄清透亮,香气醇和的特征.成品酒香气成分共检测出醇类9种,酯类8种,酸类6种和少量的醛类、酮类等成分.     

Levels of trehalose and glycogen inArthrobacter globiformis under conditions of nutrient starvation and osmotic stress

Cells ofArthrobacter globiformis grown in carbohydrate-rich media were found to contain large quantities of low-M_r carbohydrates (800 g/mg protein) and only small amounts of amino acids, in addition to high amounts of glycogen (2 mg/mg protein). At increasing osmotic values of the medium, low-M_r carbohydrate levels increased to 1300 g/mg protein. Low-M_r pools were extracted from the cells with hot 75% ethanol, and subjected to thin layer, gel and gas-liquid chromatography. They turned out to consist mainly of ,-trehalose. Levels of trehalose inArthrobacter cells have the tendency to remain constant, both during nutrient exhaustion (resulting in glycogen consumption), and on addition of excess of carbon source to the medium (resulting in an increased glycogen content of the cells). The stress-tolerant properties ofArthrobacter (resistance to nutrient starvation, desiccation and high salt concentration) are discussed with respect to the high glycogen and trehalose contents of the cells.     

Intracellular accumulation of trehalose and glycogen in an extreme oligotroph,Rhodococcus erythropolis N9T-4

An extreme oligotroph, Rhodococcus erythropolis N9T-4, showed intracellular accumulation of trehalose and glycogen under oligotrophic conditions. No trehalose accumulation was observed in cells grown on the rich medium. Deletion of the polyphosphate kinase genes enhanced the trehalose accumulation and decreases the intracellular glycogen contents, suggesting an oligotrophic relationship between among the metabolic pathways of trehalose, glycogen, and inorganic polyphosphate biosyntheses.     

Analysis of the expression of some stress induced genes in several commercial wine yeast strains at the beginning of vinification

AIMS: During fermentation yeast cells should cope with stress conditions. We pursue a better understanding of the stress response in wine yeasts at the beginning of vinification. METHODS AND RESULTS: We analyse by means of quantitative PCR the expression of several stress induced genes in 24 efficient commercial wine yeast strains at the beginning of vinifications performed under standard conditions or with small variations in pH and temperature. In all cases, high levels (with differences among strains) of GPD1 mRNA but quite low expression of other stress genes (TRX2, HSP104 and SSA3) were found. For all these genes, mRNA levels increase as temperature decreases or pH increases. CONCLUSIONS: Important levels of expression of GPD1 (but not of other stress genes) are required during the first hours of vinification, because of the need for glycerol production to counteract the hyperosmotic stress at this point. The differences among strains suggest that certain level of expression is enough to ensure the continuity of the process. Variations in the pH and temperature of the vinification can affect gene expression. SIGNIFICANCE AND IMPACT OF THE STUDY: A common pattern of stress response between efficient wine strains exists, which could be used as a criterion for selection. Studies of this kind can allow the establishment of connections between gene expression and physiological traits.     

大斑芫菁滞育幼虫在滞育不同阶段体内糖类和醇类含量的变化

在以卵滞育的昆虫中昆虫滞育时的生理代谢特点已经得到了大量研究。本文对以末龄幼虫（5龄）滞育的大斑芫菁Mylabris phalerate（Pallas）在不同滞育阶段体内糖类和醇类代谢的特征进行了研究。结果表明： 滞育个体血淋巴中的海藻糖含量高于非滞育个体,且随滞育时间的加大逐渐升高,滞育5个月时达到最大值,为5.61 μmol/mL。糖原的含量随滞育的进程逐渐减少,滞育初期（0.5个月）为0.72 mg/mL,到滞育末期（5个月）时仅为0.1 mg/mL。滞育个体脂肪体中的海藻糖含量都高于非滞育个体,滞育1个月时为非滞育个体的3倍,至滞育末期时达非滞育个体的5倍,为2.5 μmol/g脂肪体。糖原含量总体变化趋势是随滞育时间的加大逐渐减少,滞育早期和中期都高于非滞育个体。在滞育过程中血淋巴积累的小分子多元醇主要为甘油,其次是山梨醇;而在脂肪体中主要为甘油,其次是甘露醇,少量积累山梨醇：表明大斑芫菁滞育幼虫主要积累的是海藻糖和一些小分子多元醇。滞育幼虫在准备滞育时储备了大量糖原,这些糖原可能为滞育期间海藻糖、山梨醇和甘油的代谢提供了原料。