Stimulation of Yeast Sporulation by Glycerol

S ummary : Glycerol stimulated sporulation of Saccharomyces cerevisiae Hanson, especially when the cells were precultured in a complex growth medium instead of a chemically defined medium. Optimum spore yields occurred with 1–4% of glycerol but some were produced in 16% glycerol. Sporulation in glycerol was much less sensitive to ammonium sulphate inhibition than it was in acetate. Growth occurred with glycerol as sole carbon source and glutamic acid as sole nitrogen source, but not with ammonium sulphate as the sole nitrogen source.     

葡萄酒中酵母菌高产甘油的研究进展

甘油是酵母菌酒精发酵过程中的副产物,甘油作为非挥发性物质,对葡萄酒的香气没有贡献,但是其黏性和甜味,可使葡萄酒具有圆润、柔滑、甘甜、肥硕、更易入口的特性,也可用于平衡酒中的酸感,增加口感复杂性,是高品质果酒的重要成分。近年来,国内外对高产甘油酵母的研究日益增多,着重于提高葡萄酒中酵母发酵生产甘油的能力。     

酵母细胞渗透压调节与甘油代谢

酵母甘油代谢与调控的信息主要来自于酿酒酵母和酿酒酵母细胞对高渗应答的研究。本文综述了酵母细胞非协迫条件下的甘油合成与分解代谢特征;甘油在酵母细胞渗透压调节过程中的作用与酵母耐高渗机理;增强甘油合成的外环境及其甘油合成的途径工程;以及酵母感受胞外高渗信息及控制在高渗协迫条件下甘油合成的高渗甘油应答途径。     

酵母细胞对高渗环境的适应与胞内甘油累积

甘油是包括酿酒酵母在内的许多种酵母细胞中的主要相容性溶质。为适应在高渗环境下的生存,酵母细胞将在胞内累积甘油。胞内甘油累积的增加可由甘油合成的增强,甘油利用的减弱,细胞膜通透性下降导致的胞内甘油流失的减少以及从环境中吸取更多的甘油而产生。本文综述了酵母细胞对环境渗透压变化的信号传导,高渗诱导的基因表达,环境渗透压升高时酵母细胞内甘油的累积以及甘油合成的限速步骤。     

Pilot Plant Glycerol Production with a Slow-Feed Osmophilic Yeast Fermentation

A slow feed batch fermentation is described for the production of glycerol from sugar. The conversion efficiency was approximately 1 mole of glycerol produced per mole of glucose utilized after the cell growth phase. The glycerol production phase was extended several-fold by periodic glucose addition. The yeast cell count remained constant during this time as limited by phosphate, a deficiency required for an efficient glycerol fermentation. A small amount of phosphate was supplied during the extended fermentation, maintaining an active culture, by the normal autolysis of spent cells. Interfering or inhibitory by-products did not accumulate, and the osmophilic yeasts are tolerant of high glycerol concentrations. These factors combined to allow a particularly efficient fermentation well suited to product enrichment by supplying large quantities of substrate over an extended period.     

Identification of Positive Regulators of the Yeast Fps1 Glycerol Channel

The yeast Fps1 protein is an aquaglyceroporin that functions as the major facilitator of glycerol transport in response to changes in extracellular osmolarity. Although the High Osmolarity Glycerol pathway is thought to have a function in at least basal control of Fps1 activity, its mode of regulation is not understood. We describe the identification of a pair of positive regulators of the Fps1 glycerol channel, Rgc1 (Ypr115w) and Rgc2 (Ask10). An rgc1/2Δ mutant experiences cell wall stress that results from osmotic pressure associated with hyper-accumulation of glycerol. Accumulation of glycerol in the rgc1/2Δ mutant results from a defect in Fps1 activity as evidenced by suppression of the defect through Fps1 overexpression, failure to release glycerol upon hypo-osmotic shock, and resistance to arsenite, a toxic metalloid that enters the cell through Fps1. Regulation of Fps1 by Rgc1/2 appears to be indirect; however, evidence is presented supporting the view that Rgc1/2 regulate Fps1 channel activity, rather than its expression, folding, or localization. Rgc2 was phosphorylated in response to stresses that lead to regulation of Fps1. This stress-induced phosphorylation was partially dependent on the Hog1 MAPK. Hog1 was also required for basal phosphorylation of Rgc2, suggesting a mechanism by which Hog1 may regulate Fps1 indirectly.     

酵母细胞甘油代谢与生理功能研究进展

甘油是酵母细胞生长代谢过程中常见的多元醇物质。尽管甘油的结构简单,代谢途径并不复杂,但是其在细胞内的生理功能十分重要。甘油代谢过程主要参与细胞的高渗透压生理调节和厌氧条件下的胞内氧化还原平衡调节。近年来许多学者在酵母细胞的甘油代谢及生理功能方面开展了深入的研究。在扼要介绍甘油生理代谢的基础上,重点阐述甘油代谢参与细胞高渗压甘油应答信号途径和氧化还原平衡调节的生理机制,同时就酵母细胞甘油合成的代谢工程进行归纳和评述。     

Glycerol Production by Fermenting Yeast Cells Is Essential for Optimal Bread Dough Fermentation

Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO₂ production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts.     

Roles of Sugar Alcohols in Osmotic Stress Adaptation. Replacement of Glycerol by Mannitol and Sorbitol in Yeast

For many organisms there is a correlation between increases of metabolites and osmotic stress tolerance, but the mechanisms that cause this protection are not clear. To understand the role of polyols, genes for bacterial mannitol-1-P dehydrogenase and apple sorbitol-6-P dehydrogenase were introduced into a Saccharomyces cerevisiae mutant deficient in glycerol synthesis. Sorbitol and mannitol provided some protection, but less than that generated by a similar concentration of glycerol generated by glycerol-3-P dehydrogenase (GPD1). Reduced protection by polyols suggested that glycerol had specific functions for which mannitol and sorbitol could not substitute, and that the absolute amount of the accumulating osmoticum might not be crucial. The retention of glycerol and mannitol/sorbitol, respectively, was a major difference. During salt stress, cells retained more of the six-carbon polyols than glycerol. We suggest that the loss of >98% of the glycerol synthesized could provide a safety valve that dissipates reducing power, while a similar high intracellular concentration of retained polyols would be less protective. To understand the role of glycerol in salt tolerance, salt-tolerant suppressor mutants were isolated from the glycerol-deficient strain. One mutant, sr13, partially suppressed the salt-sensitive phenotype of the glycerol-deficient line, probably due to a doubling of [K(+)] accumulating during stress. We compare these results to the "osmotic adjustment" concept typically applied to accumulating metabolites in plants. The accumulation of polyols may have dual functions: facilitating osmotic adjustment and supporting redox control.     

Determination of Growth and Glycerol Production Kinetics of a Wine Yeast Strain Saccharomyces cerevisiae Kalecik 1 in Different Substrate Media

Summary Glycerol has been known as an important by-product of wine fermentations improving the sensory quality of wine. This study was carried out with an endogenic wine yeast strain Saccharomyces cerevisiae Kalecik 1. The kinetics of growth and glycerol biosynthesis were analysed at various initial concentrations of glucose, fructose, and sucrose in a batch system. Depending on the determined values of Monod constants, glucose (K_s = 28.09 g/l) was found as the most suitable substrate for the yeast growth. Initial glucose, fructose and sucrose concentrations necessary for maximum specific yeast growth rate were determined as 175 g, 100 l, and 200 g/l, respectively. The yeast produced glycerol at very high concentrations in fructose medium. Fructose was determined as the most suitable substrate for glycerol production while the strain showed low tendency to use it for growth. S. cerevisiae Kalecik 1 could not produce glycerol below 200 g/l initial sucrose concentration. When natural white grape juice was used as fermentation medium, maximum glycerol concentration and dry weight of the yeast were determined as 9.3 g/l and 11.8 g/l, respectively.     

Reduction of Ethanol Yield and Improvement of Glycerol Formation by Adaptive Evolution of the Wine Yeast Saccharomyces cerevisiae under Hyperosmotic Conditions

There is a strong demand from the wine industry for methodologies to reduce the alcohol content of wine without compromising wine''s sensory characteristics. We assessed the potential of adaptive laboratory evolution strategies under hyperosmotic stress for generation of Saccharomyces cerevisiae wine yeast strains with enhanced glycerol and reduced ethanol yields. Experimental evolution on KCl resulted, after 200 generations, in strains that had higher glycerol and lower ethanol production than the ancestral strain. This major metabolic shift was accompanied by reduced fermentative capacities, suggesting a trade-off between high glycerol production and fermentation rate. Several evolved strains retaining good fermentation performance were selected. These strains produced more succinate and 2,3-butanediol than the ancestral strain and did not accumulate undesirable organoleptic compounds, such as acetate, acetaldehyde, or acetoin. They survived better under osmotic stress and glucose starvation conditions than the ancestral strain, suggesting that the forces that drove the redirection of carbon fluxes involved a combination of osmotic and salt stresses and carbon limitation. To further decrease the ethanol yield, a breeding strategy was used, generating intrastrain hybrids that produced more glycerol than the evolved strain. Pilot-scale fermentation on Syrah using evolved and hybrid strains produced wine with 0.6% (vol/vol) and 1.3% (vol/vol) less ethanol, more glycerol and 2,3-butanediol, and less acetate than the ancestral strain. This work demonstrates that the combination of adaptive evolution and breeding is a valuable alternative to rational design for remodeling the yeast metabolic network.     

Heterologous Expression of Glycerol 3-Phosphate Dehydrogenase Gene [DhGPD1] from the Osmotolerant Yeast Debaryomyces hansenii in Saccharomyces cerevisiae

The role for the gene encoding glycerol 3-phosphate dehydrogenase (DhGPD1) from the osmotolerant yeast Debaryomyces hansenii, in glycerol production and halotolerance, was studied through its heterologous expression in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Δ). The expression of the DhGPD1 gene in the gpd1Δ background restored glycerol production and halotolerance to wild type levels, corroborating its role in the salt-induced production of glycerol. Although the gene was functional in S. cerevisiae, its heterologous expression was not efficient, suggesting that the regulatory mechanism may not be shared by these two yeasts.     

Long-Chain Glycerol Diether and Polyol Dialkyl Glycerol Triether Lipids of Sulfolobus acidocaldarius

Cells of Sulfolobus acidocaldarius contain about 2.5% total lipid on a dry-weight basis. Total lipid was found to contain 10.5% neutral lipid, 67.6% glycolipid, and 21.7% polar lipid. The lipids contained C(40)H(80) isopranol glycerol diethers. Almost no fatty acids were present. The glycolipids were composed of about equal amounts of the glycerol diether analogue of glucosyl galactosyl diglyceride and a glucosyl polyol glycerol diether. The latter compound contained an unidentified polyol attached by an ether bond to the glycerol diether. The polar lipids contained a small amount of sulfolipid, which appeared to be the monosulfate derivative of glucosyl polyol glycerol diether. About 40% of the lipid phosphorus was found in the diether analogue of phosphatidyl inositol. The remaining lipid phosphorus was accounted for by approximately equal amounts of two inositol monophosphate-containing phosphoglycolipids, inositolphosphoryl glucosyl galactosyl glycerol diether and inositolphosphoryl glucosyl polyol glycerol diether.     

Glycerol Transport in Mitochondria

Biophysics - The entry of glycerol molecules in unphosphorylated form into mitochondria was studied. The effective binding of glycerol with membranes of rat liver mitochondria was demonstrated...     

Glycerol metabolism in Rhizobium.

Four strains of Rhizobium japonicum and one strain of R. trifolii were grown on glycerol and found to contain a soluble ATP-glycerol kinase and a particulate glycerolphosphate dehydrogenase. Both enzymes are induced by glycerol. The presence of NAD+-or NADP+-glycerol dehydrogenase was not detected in any of the strains. No significant differences were found in the glycerol metabolic pathway between fast-and slow-growing rhizobia.