New insights on trehalose: a multifunctional molecule

Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an alpha,alpha-1,1-glycosidic linkage. This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates, and lower and higher plants, where it may serve as a source of energy and carbon. In yeast and plants, it may also serve as a signaling molecule to direct or control certain metabolic pathways or even to affect growth. In addition, it has been shown that trehalose can protect proteins and cellular membranes from inactivation or denaturation caused by a variety of stress conditions, including desiccation, dehydration, heat, cold, and oxidation. Finally, in mycobacteria and corynebacteria, trehalose is an integral component of various glycolipids that are important cell wall structures. There are now at least three different pathways described for the biosynthesis of trehalose. The best known and most widely distributed pathway involves the transfer of glucose from UDP-glucose (or GDP-glucose in some cases) to glucose 6-phosphate to form trehalose-6-phosphate and UDP. This reaction is catalyzed by the trehalose-P synthase (TPS here, or OtsA in Escherichia coli ). Organisms that use this pathway usually also have a trehalose-P phosphatase (TPP here, or OtsB in E. coli) that converts the trehalose-P to free trehalose. A second pathway that has been reported in a few unusual bacteria involves the intramolecular rearrangement of maltose (glucosyl-alpha1,4-glucopyranoside) to convert the 1,4-linkage to the 1,1-bond of trehalose. This reaction is catalyzed by the enzyme called trehalose synthase and gives rise to free trehalose as the initial product. A third pathway involves several different enzymes, the first of which rearranges the glucose at the reducing end of a glycogen chain to convert the alpha1,4-linkage to an alpha,alpha1,1-bond. A second enzyme then releases the trehalose disaccharide from the reducing end of the glycogen molecule. Finally, in mushrooms there is a trehalose phosphorylase that catalyzes the phosphorolysis of trehalose to produce glucose-1-phosphate and glucose. This reaction is reversible in vitro and could theoretically give rise to trehalose from glucose-1-P and glucose. Another important enzyme in trehalose metabolism is trehalase (T), which may be involved in energy metabolism and also have a regulatory role in controlling the levels of trehalose in cells. This enzyme may be important in lowering trehalose concentrations once the stress is alleviated. Recent studies in yeast indicate that the enzymes involved in trehalose synthesis (TPS, TPP) exist together in a complex that is highly regulated at the activity level as well as at the genetic level.     

Two distinct pathways for trehalose assimilation in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast.     

Two Distinct Pathways for Trehalose Assimilation in the Yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast.     

Effect of trehalose on survival of Bradyrhizobium japonicum during desiccation

AIMS: A major reason for the ineffectiveness of legume inoculants in the field is the rapid death of rhizobia because of desiccation. The major purpose of this study was to identify conditions under which alpha,alpha-trehalose would improve survival of Bradyrhizobium japonicum during desiccation. METHODS AND RESULTS: Trehalose was added to cultures just prior to desiccation or was supplied to bacteria during the 6-day growth period. A wide variety of trehalose concentrations was tested. Trehalose added to cultures at the time of desiccation improved survival slightly, but trehalose loading during growth was much more effective in protection against desiccation. Growth of bacteria with 3 mmol l-1 trehalose increased trehalose concentration in cells by about threefold and increased survival of cells placed on soya bean [Glycine max (L.) Merr.] seeds by two- to four-fold after 2 or 24 h. Average of overall results indicate that growth of bacteria with trehalose in the medium resulted in a 294% increase in survival after 24 h of desiccation. The concentration of trehalose in cells was very highly correlated with survival of bacteria. When trehalose-loaded cells were suspended in buffer or water, 60-85% of cellular trehalose was lost in about 1 h and, in spite of these losses, survival during desiccation was not reduced. CONCLUSIONS: Accumulation of trehalose in the cytoplasm is critical to the survival of B. japonicum during desiccation. Increasing the periplasmic concentration of trehalose is also beneficial but is not so critical as the concentration of trehalose in the cytoplasm. Because B. japonicum cannot utilize trehalose as a carbon source, cells can be loaded with trehalose by providing the disaccharide during the growth period. SIGNIFICANCE AND IMPACT OF THE STUDY: Although it may not be practical to use trehalose as a carbon source in inoculant production, it may be possible to engineer greater trehalose accumulation in rhizobia. Trehalose concentration in cells should be a useful predictor of survival during desiccation.     

New insights into trehalose metabolism by Saccharomyces cerevisiae: NTH2 encodes a functional cytosolic trehalase, and deletion of TPS1 reveals Ath1p-dependent trehalose mobilization

In the yeast Saccharomyces cerevisiae, the synthesis of endogenous trehalose is catalyzed by a trehalose synthase complex, TPS, and its hydrolysis relies on a cytosolic/neutral trehalase encoded by NTH1. In this work, we showed that NTH2, a paralog of NTH1, encodes a functional trehalase that is implicated in trehalose mobilization. Yeast is also endowed with an acid trehalase encoded by ATH1 and an H+/trehalose transporter encoded by AGT1, which can together sustain assimilation of exogenous trehalose. We showed that a tps1 mutant defective in the TPS catalytic subunit cultivated on trehalose, or on a dual source of carbon made of galactose and trehalose, accumulated high levels of intracellular trehalose by its Agt1p-mediated transport. The accumulated disaccharide was mobilized as soon as cells entered the stationary phase by a process requiring a coupling between its export and immediate extracellular hydrolysis by Ath1p. Compared to what is seen for classical growth conditions on glucose, this mobilization was rather unique, since it took place prior to that of glycogen, which was postponed until the late stationary phase. However, when the Ath1p-dependent mobilization of trehalose identified in this study was impaired, glycogen was mobilized earlier and faster, indicating a fine-tuning control in carbon storage management during periods of carbon and energy restriction.     

海藻糖介导的信号转导与植物抗逆性

海藻糖是一种非还原性二糖,它广泛存在于细菌、真菌、酵母、昆虫、无脊椎动物和植物等生物体内。海藻糖不仅作为碳水化合物的储备,而且还是一个多功能分子。海藻糖作为一种信号分子,启动信号转导级联反应,改变基因表达和酶的活性,与激素也有一定的关系。采用基因工程和通过外源施加的方法增加海藻糖在植物体内的积累可以提高植物的抗逆性,这为提高农作物的抗逆性提供了新的策略。     

Comparison of electroporation and Chariot? for delivery of ��-galactosidase into mammalian cells: strategies to use trehalose in cell preservation

There are many compounds that can and have been used as cryoprotectants including disaccharides such as trehalose. Many organisms in nature use trehalose to help protect themselves at colder temperatures. Trehalose has also been used to a limited extent for the preservation of mammalian cells and tissues, but mainly as a supplement to other cryoprotectants like dimethyl sulfoxide. Recently, the use of trehalose as the primary cryoprotectant has gained much interest because of its low-potential cytotoxicity. Trehalose does not readily pass through mammalian cells membranes and research has shown that it is most effective when present on both sides of the cell membrane prior to preservation. Different strategies for introducing disaccharide sugars into cells have been investigated with limited success. In this study, two separate strategies are investigated for the introduction of disaccharide sugars into cells. Electroporation using an electric pulse to create temporary holes in the membrane so that molecules could pass through and a transport peptide (Chariot?) that covalently binds to the molecule of interest and then moves it across the membrane. Both strategies have the potential to load disaccharide sugars into cells at concentrations that would provide ample protection during preservation. In preparation for cryopreservation studies, smooth muscle cells that are difficult to cryopreserve using conventional preservation protocols were used to evaluate and compare the translocation potential of these two strategies using β-galactosidase. Assessment of each loading strategy was done by measuring viability and the presence of β-galactosidase inside the cells. The results indicate that both methods appear feasible as potential delivery systems and that treatment cytotoxicity can be minimized. The next step is definition of the best loading strategy to introduce trehalose into cells followed by preservation by freezing.     

Acid trehalase in yeasts and filamentous fungi: localization, regulation and physiological function

Yeasts and filamentous fungi are endowed with two different trehalose-hydrolysing activities, termed acid and neutral trehalases according to their optimal pH for enzymatic activity. A wealth of information already exists on fungal neutral trehalases, while data on localization, regulation and function of fungal acid trehalases have remained elusive. The gene encoding the latter enzyme has now been isolated from two yeast species and two filamentous fungi, and sequences encoding putative acid trehalase can be retrieved from available public sequences. Despite weak similarities between amino acids sequences, this type of trehalase potentially harbours either a transmembrane segment or a signal peptide at the N-terminal sequence, as deduced from domain prediction algorithms. This feature, together with the demonstration that acid trehalase from yeasts and filamentous fungi is localized at the cell surface, is consistent with its main role in the utilisation of exogenous trehalose as a carbon source. The growth on this disaccharide is in fact pretty effective in most fungi except in Saccharomyces cerevisiae. This yeast species actually exhibits a "Kluyver effect" on trehalose. Moreover, an oscillatory behaviour reminiscent of what is observed in aerobic glucose-limited continuous cultures at low dilution rate is also observed in batch growth on trehalose. Finally, the S. cerevisiae acid trehalase may also participate in the catabolism of endogenous trehalose by a mechanism that likely requires the export of the disaccharide, its extracellular hydrolysis, and the subsequent uptake of the glucose released. Based on these recent findings, we suggest to rename "acid" and "neutral" trehalases as "extracellular" and "cytosolic" trehalases, which is more adequate to describe their localization and function in the fungal cell.     

Trehalose: A Key Organic Osmolyte Effectively Involved in Plant Abiotic Stress Tolerance

Trehalose is a natural non-reducing sugar that is found in the vast majority of organisms such as bacteria, yeasts, invertebrates and even in plants. Regarding its features, it is considered as a unique compound. It plays a key role as a carbon source in lower organisms and as an osmoprotectant or a stabilizing molecule in higher animals and plants. Although in plants it is present in a minor quantity, its levels rise upon exposure to abiotic stresses. Trehalose is believed to play a protective role against different abiotic stressful cues such as temperature extremes, salinity, desiccation. Moreover, it regulates water use efficiency and stomatal movement in most plants. Detectable endogenous trehalose levels are vital for sustaining growth under stressful cues. Exogenously applied trehalose in low amounts mitigates physiological and biochemical disorders induced by various abiotic stresses, delays leaf abscission and stimulates flowering in crops. External application of trehalose also up-regulates the stress responsive genes in plants exposed to environmental cues. The genetically modified plants with trehalose biosynthesis genes exhibit improved tolerance against stressful conditions. An increased level of trehalose has been observed in transgenic plants over-expressing genes of microbial trehalose biosynthesis. However, these transgenic plants display enhanced tolerance to heat, cold, salinity, and drought tolerance. Due to multiple bio-functions of this sugar, it has gained considerable ground in various fields. However, exogenous use of this bio-safe sugar would only be possible under field conditions upon adopting strategies of low-cost production of trehalose. In short, trehalose is a unique chemical that preserves vitality of plant life under harsh ecological conditions. Certainly, the new findings of this disaccharide will revolutionize a wide array of new avenues.

     

The nature of stability of yeast cells to drying]

The residual water and dry matter condition in the lyophilized biomass of the yeast Saccharomyces cerevisiae was studied by NMR-relaxation technique. It was shown that the slow component of the transverse magnetization NMR signal spectrum corresponding to the so-called "isolated mobile water" was caused in fact by the interaction of the disaccharide trehalose with the cell biopolymers. The big amount of hydrogen bonds formed by trehalose and their three-dimensional orientation closed to the orientation in water clusters assure the valuable functioning of this disaccharide during the process of removing water out of cells. When stationary phase yeast biomass containing a lot of trehalose was dried the cell organelles condition remained practically unchanged what led to the high resistance of such cells to dehydration.     

Accepted 17 Jun. 2008Received 16 Mar. 2008

Trehalose is a non-reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose-6-phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In.silico expression profiling of all Arabidopsis trehalose-6-phosphate synthases (TPSs) and trehalose-6-phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

Accepted 17 Jun. 2008Received 16 Mar. 2008

Trehalose is a non-reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose-6-phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In.silico expression profiling of all Arabidopsis trehalose-6-phosphate synthases (TPSs) and trehalose-6-phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

The transmembrane domain of acid trehalase mediates ubiquitin-independent multivesicular body pathway sorting

Trehalose serves as a storage source of carbon and plays important roles under various stress conditions. For example, in many organisms trehalose has a critical function in preserving membrane structure and fluidity during dehydration/rehydration. In the yeast Saccharomyces cerevisiae, trehalose accumulates in the cell when the nutrient supply is limited but is rapidly degraded when the supply of nutrients is renewed. Hydrolysis of trehalose in yeast depends on neutral trehalase and acid trehalase (Ath1). Ath1 resides and functions in the vacuole; however, it appears to catalyze the hydrolysis of extracellular trehalose. Little is known about the transport route of Ath1 to the vacuole or how it encounters its substrate. Here, through the use of various trafficking mutants we showed that this hydrolase reaches its final destination through the multivesicular body (MVB) pathway. In contrast to the vast majority of proteins sorted into this pathway, Ath1 does not require ubiquitination for proper localization. Mutagenesis analyses aimed at identifying the unknown targeting signal revealed that the transmembrane domain of Ath1 contains the information sufficient for its selective sequestration into MVB internal vesicles.     

Trehalose Biosynthesis in Response to Abiotic Stresses

Trehalose is a non‐reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose‐6‐phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In‐silico expression profiling of all Arabidopsis trehalose‐6‐phosphate synthases (TPSs) and trehalose‐6‐phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

海藻糖的生产制备及其应用前景

海藻糖是一种广泛分布于细菌、真菌和动植物体内的双糖。在生物体内 ,它不仅作为结构成分和能量物质存在 ,而且在热击和脱水等协迫条件下 ,对生物体和生物大分子起着良好的非特异性保护作用。由于其独特的生物学功能 ,它在食品、分子生物学、医药、化妆品、农业等方面具有广阔的应用前景。简述海藻糖的生产制备、应用研究及其前景展望。     

Engineered Trehalose Permeable to Mammalian Cells

Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.     

酵母内源性海藻糖代谢调控研究进展

作为一种天然稳定剂的双糖,海藻糖(Trehalose)在逆境下对生物体活性的保护功能既吸引了广泛的研究兴趣,也使其具有良好的应用价值和潜力。本文聚焦重要模式微生物和工业应用微生物酵母,结合组学研究最新进展,从海藻糖代谢途径、应激条件下的海藻糖代谢和转录特征以及提高胞内海藻糖含量策略等方面,对内源性海藻糖研究新进展进行了综述。     

Construction of Saccharomyces cerevisiae strains that accumulate relatively low concentrations of trehalose, and their application in testing the contribution of the disaccharide to stress tolerance

Genetically related diploid strains of Saccharomyces cerevisiae that accumulate varied amounts of trehalose during starvation for nitrogen have been constructed. Strains that produced greater than 5% trehalose (dry cell weight) were more tolerant of thermal, or freeze-thaw stresses than strains that produced less than 4% trehalose. Thus trehalose appears to play a role in stress tolerance of yeast. The significance of these results is that, for the first time, a series of related, unmutated strains have been used to test the effect of trehalose on thermotolerance. Previous studies employed either heat shock treatment, or mutated strains to provide trehalose variations, and as such the contribution of the disaccharide to stress tolerance could not necessarily be separated from other factors such as heat shock proteins.