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。     

海藻糖代谢途径相关基因及生物工程

海藻糖(Trehalose)是一种由两个葡萄糖分子通过α,α-1,l糖苷键连接的非还原性双糖。最早的记录是在19世纪初期作为黑麦的麦角菌的一种成分而被描述,后来发现海藻糖广泛存在于微生物、动物和植物体内,特别是在那些能抗脱水作用的生物中起着重要作用。这些特殊生物具有在脱水条件下存活多年的性质,包括所谓的“复苏植物”(Selaginella lepidophylla)、某些咸水虾、线虫及面包酵母等。当它们体内99％的水分被去掉之后,仍保持着能在获水后迅速复活的能力^[1]。研究表明,海藻糖对于生物抗逆具有重要的保护作用。海藻糖的应用研究因此得到了人们的广泛关注和重视,目前海藻糖已被用作酶、其它蛋白、生物制品甚至移植器官的保护剂。海藻糖作为生物体对抗环境胁迫的重要应激保护物质,在不同生物中存在多种合成和分解代谢途径,相关基因已相继被克隆和分析。海藻糖合成、分解及其调控是生物抗逆的重要机制,其相关基因的研究也是海藻糖生物工程的重要基础。     

海藻糖的性质及其广阔应用前景

海藻糖是一种稳定的非还原性双糖,在自然界中广泛存在。藻类植物,特别是在酵母、霉菌等真菌中,海藻糖含量可高达干重的１６％以上［１］。作为一种储存性碳水化合物,海藻糖在生物体内扮演极其特殊的角色,它能在干燥状态下保护生物体细胞内的蛋白质、脂类、糖类、核酸...     

海藻糖酶法合成途径及其酶基因的重组表达研究

在生物抗逆研究中,海藻糖合酶基因是继甘露醇、脯氨酸、甜菜碱合成酶基因之后又一个与抗逆相关的基因。海藻糖具有独特的生物学功能,能提高生物体对干旱、高温、冷冻和渗透压的抗性,发现以来就受到人们的普遍关注。随着对海藻糖化学性质、生理功能、作用机理及代谢途径等方面研究的深入,其在生物制品、食品、医药、作物育种及精细化工等领域广阔的应用前景日益显现。就海藻糖在生物体中的合成途径,以及海藻糖合成酶的基因工程研究进展进行了综述。     

昆虫海藻糖代谢及其影响因素的研究进展

海藻糖广泛存在于细菌、真菌、动物和植物中。它不仅作为能量储备物质,在外界环境胁迫或内部代谢紊乱时,也可作为保护因子,保护其生命体度过逆境。昆虫海藻糖合成酶与海藻糖酶分别是海藻糖合成与分解的关键酶,合成的海藻糖在海藻糖转运蛋白的帮助下由胞内进入胞外。胰岛素与脂动激素直接参与昆虫糖代谢,保幼激素与蜕皮激素通过和胰岛素与脂动激素通路偶联,间接参与调控昆虫海藻糖代谢。海藻糖代谢途径和昆虫生长发育密切相关,昆虫海藻糖代谢信号通路为开发害虫控制的新靶标提供理论依据。     

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。     

灰树花海藻糖合成酶基因的克隆及其在大肠杆菌中的表达

海藻糖主要作用是作为生物体的结构组分、以及保护生物膜和保护蛋白质。在灰树花中 ,海藻糖在干重中所占比例最高可达到 1 5 %～ 1 7% ,说明灰树花合成海藻糖的能力很强。将灰树花海藻糖合成酶基因克隆 ,并在大肠杆菌表达系统里表达。表达量为 1 90mg L。通过活性测定 ,证明在大肠杆菌中表达的海藻糖合成酶具有酶活性 ,结合基因工程和酶工程方法 ,为合成海藻糖的研究提供了新的方向     

海藻糖的生物合成与分解途经及其生物学功能

海藻糖是一类在干旱、低温、热击或脱水等逆境环境下具有独特抗逆保护作用的二糖,广泛分布于藻类、细菌、真菌和动植物体内。近年来,随着对海藻糖研究的深入,海藻糖已经被广泛应用到食品、医药、化妆品和分子生物学研究等领域。该文简述了海藻糖在生物体内的代谢途径、生物学功能和研究进展,并对灭蚊真菌Pythiumsp.GY1938菌株海藻糖代谢酶基因的研究前景加以展望。     

海藻糖对微生物谷氨酰氨转胺酶热稳定性研究

本实验目的是研究海藻糖对微生物谷氨酰胺转胺酶（TGase）热稳定性的作用。糖类对TGase的保护作用根据糖种类不同有所差异,海藻糖和蔗糖的保护作用优于葡萄糖对TGase的保护作用。在45℃、50℃、55℃、60℃、65℃下研究了海藻糖对TG酶的保护作用。结果表明,在50～65℃下海藻糖使谷氨酰胺转胺酶受热时的稳定性提高了约20％。海藻糖与酶复合的最合适浓度约为14％,浓度低时保护作用不明显,加入过高浓度的糖对酶的活性维持不利。50℃下处理一段时间内,海藻糖对酶的保护作用随时问变化很小。     

氯化钙浸种对玉米幼苗抗逆性的影响及其与谷胱甘肽还原酶的关系

两个抗逆性不同的玉米品种经过CaCl2 浸种处理后 ,能显著提高其幼苗在低温、高温、干旱和盐胁迫下的存活率 ;相反 ,Ca2 螯合剂EGTA浸种处理会降低玉米幼苗在上述逆境胁迫下的存活率 ,表明外源Ca2 处理能增强玉米幼苗对低温、高温、干旱和盐胁迫的多重抗逆性。此外 ,Ca2 浸种处理能使玉米幼苗在多种逆境胁迫下保持相对较高的谷胱甘肽还原酶 (GR)活性 ,而EGTA处理正好相反 ,表明GR可能参与了Ca2 提高的玉米幼苗多重抗逆性的调控     

金针菇菌种中短期保藏因素的正交分析

【背景】金针菇(Flammulina velutipes)是我国一种重要的栽培食用菌,年产量超过250万t,规模已跃居世界首位。菌种保藏技术是金针菇栽培和新品种研发的基础,但相关研究十分薄弱,已成为制约我国金针菇产业进一步发展的瓶颈问题。【目的】探索不同保藏因素对金针菇优良菌种中短期保藏的影响,为建立高效、低成本、易操作的保藏方法奠定基础。【方法】以温度、甘油、海藻糖、甘露醇以及保护剂体积5个因素进行正交试验。经12个月保藏,考察金针菇菌种在木屑培养基中的菌丝生长速度,通过极差分析和回归分析解析保藏因素的效应。【结果】温度、海藻糖、甘油和甘露醇对金针菇菌种的中短期保藏有极显著的影响,保护剂体积的影响不显著。温度是最重要的影响因子,与其它4个因素的互作效应均达到极显著水平。20°C是较好的短期保藏温度,-80°C为理想的中期保藏温度。渗透型与非渗透型保护剂间的互作效应对金针菇菌种的中短期保藏有极显著影响,海藻糖和甘露醇间的互作效应不显著。高浓度的海藻糖、甘油及甘露醇均不利于金针菇菌种的中短期保藏。保藏效果较佳的保护剂为10%甘油和0.3 mol/L甘露醇混合液。【结论】建立的菌种中短期保藏方法填补了金针菇产业发展的空白,研究结果可为其它大型真菌的中短期保藏提供重要参考。     

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.     

Regulation of expression of trehalose-6-phosphate synthase during cold shock in Arthrobacter strain A3

Trehalose is a chemical chaperone known to protect a variety of organisms against cold stress. Members of the genus Arthrobacter, which belongs to the Actinomycetales group, exhibit strong resistance to stress conditions, but exactly how trehalose synthesis is regulated in conditions of cold stress is still unknown. Here, we report that Arthrobacter strain A3, which was isolated from the alpine permafrost, has only two trehalose synthesis pathways (OtsA/B and TreS), while other Arthrobacter spp. have three. Mutants and immunoblot analyses indicate that trehalose is mainly synthesized via OtsA at low temperatures in Arthrobacter strain A3. Therefore, we have focused on the regulation of OtsA expression during cold shock. The results indicated that both low temperature and accumulation of trehalose can inhibit OtsA expression. The elongation factor Tu, which binds to OtsA, stabilizes the expression of OtsA in the cold.     

Engineering trehalose synthesis in Lactococcus lactis for improved stress tolerance

Trehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose in Propionibacterium freudenreichii in response to acid stress, a result that led to the idea that endowing Lactococcus lactis with the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway of L. lactis, i.e., trePP and pgmB, encoding trehalose 6-phosphate phosphorylase and β-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given that L. lactis lacks trehalose 6-phosphate phosphatase, the respective gene, otsB, from the food-grade organism P. freudenreichii was used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery.     

Fractionation process for the protective isolation of ergosterol and trehalose from microbial biomass

A new process is described for the two phase extraction of ergosterol and trehalose from microbial biomass. Baker’s yeast was used as a model organism to develop the method, which was then applied for extracting 13 oleaginous microbes. Major findings of the study were that the ergosterol content was not dependent on intracellular oil content and that 1-butanol and alkaline pH were needed to protect ergosterol. Saponification for 3–4 h at 85–100 °C followed by extraction of the reaction mixture with toluene gave the maximal ergosterol yield. Trehalose was stable at this temperature and remained in water solution, but the maximal yield was obtained after a shorter reaction time at lower alkalinity. Although trehalose alone is stable at alkaline pH, extraction yields of trehalose from yeast decreased with increasing alkalinity. This finding led us to propose a two-step process in which trehalose is separated in the first step and ergosterol in the second. The possibility to apply this method to fractionate oleaginous microbes in process scale is discussed from technical viewpoints.     

Osmosensing and osmoregulatory compatible solute accumulation by bacteria

Bacteria inhabit natural and artificial environments with diverse and fluctuating osmolalities, salinities and temperatures. Many maintain cytoplasmic hydration, growth and survival most effectively by accumulating kosmotropic organic solutes (compatible solutes) when medium osmolality is high or temperature is low (above freezing). They release these solutes into their environment when the medium osmolality drops. Solutes accumulate either by synthesis or by transport from the extracellular medium. Responses to growth in high osmolality medium, including biosynthetic accumulation of trehalose, also protect Salmonella typhimurium from heat shock. Osmotically regulated transporters and mechanosensitive channels modulate cytoplasmic solute levels in Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Lactobacillus plantarum, Lactococcus lactis, Listeria monocytogenes and Salmonella typhimurium. Each organism harbours multiple osmoregulatory transporters with overlapping substrate specificities. Membrane proteins that can act as both osmosensors and osmoregulatory transporters have been identified (secondary transporters ProP of E. coli and BetP of C. glutamicum as well as ABC transporter OpuA of L. lactis). The molecular bases for the modulation of gene expression and transport activity by temperature and medium osmolality are under intensive investigation with emphasis on the role of the membrane as an antenna for osmo- and/or thermosensors.     

Influence of trehalose on the molecular chaperone activity of p26, a small heat shock/alpha-crystallin protein

Encysted embryos of the primitive crustacean Artemia franciscana are among the most resistant of all multicellular eukaryotes to environmental stress, in part due to massive amounts of a small heat shock/alpha-crystallin protein (p26) that acts as a molecular chaperone. These embryos also contain very large amounts of the disaccharide trehalose, well known for its ability to protect macromolecules and membranes against damage due to water removal and temperature extremes. Therefore, we looked for potential interactions between trehalose and p26 in the protection of a model substrate, citrate synthase (CS), against heat denaturation and aggregation and in the restoration of activity after heating in vitro. Both trehalose and p26 decreased the aggregation and irreversible inactivation of CS at 43 degrees C. At approximate physiological concentrations (0.4 M), trehalose did not interfere with the ability of p26 to assist in the reactivation of CS after heating, but higher concentrations (0.8 M) were inhibitory. We also showed that CS and p26 interact physically during heating and that trehalose interferes with complex formation and disrupts CS-p26 complexes that form at high temperatures. We suggest from these results that trehalose may act as a "release factor," freeing folding intermediates of CS that p26 can chaperone to the native state. Trehalose and p26 can act synergistically in vitro, during and after thermal stress, suggesting that these interactions also occur in vivo.     

Inhibition of yeast glutathione reductase by trehalose: possible implications in yeast survival and recovery from stress

Accumulation of trehalose has been implicated in the tolerance of yeast cells to several forms of stress, including heat-shock and high ethanol levels. However, yeast lacking trehalase, the enzyme that degrades trehalose, exhibit poor survival after exposure to stress conditions. This suggests that optimal cell viability also depends on the capacity to rapidly degrade the high levels of trehalose that build up under stress. Here, we initially examined the effects of trehalose on the activity of an important antioxidant enzyme, glutathione reductase (GR), from Saccharomyces cerevisiae. At 25 degrees C, GR was inhibited by trehalose in a dose-dependent manner, with 70% inhibition at 1.5M trehalose. The inhibition was practically abolished at 40 degrees C, a temperature that induces a physiological response of trehalose accumulation in yeast. The inhibition of GR by trehalose was additive to the inhibition caused by ethanol, indicating that enzyme function is drastically affected upon ethanol-induced stress. Moreover, two other yeast enzymes, cytosolic pyrophosphatase and glucose 6-phosphate dehydrogenase, showed temperature dependences on inhibition by trehalose that were similar to the temperature dependence of GR inhibition. These results are discussed in terms of the apparent paradox represented by the induction of enzymes involved in both synthesis and degradation of trehalose under stress, and suggest that the persistence of high levels of trehalose after recovery from stress could lead to the inactivation of important yeast enzymes.     

Effect of sugars and growth media on the dehydration of Lactobacillus delbrueckii ssp. bulgaricus

AIMS: The efficiency of trehalose, sucrose and maltose to protect Lactobacillus bulgaricus during drying has been evaluated in bacteria grown at low water activity. METHODS AND RESULTS: Bacteria were grown in MRS (control), and in MRS supplemented with sucrose (MRS-sucrose) or with polyethyleneglycol (PEG) (MRS-PEG) as low water activity media. The growth in low water activity media (MRS-sucrose and MRS-PEG) prior to drying enhanced the effectiveness of trehalose as thermoprotectant during drying. The efficiency of sucrose was improved when bacteria were grown in MRS-sucrose. On the other hand, the growth in both low water activity media did not affect the efficiency of maltose. The damage produced during dehydration has been evaluated by means of growth kinetics in milk. The preservation of bacteria dehydrated with sucrose, after growing them in MRS-sucrose, appears to be as efficient as the dehydration with trehalose. CONCLUSIONS: The growth of L. bulgaricus in low water activity media enhances the protective action of trehalose and sucrose. SIGNIFICANCE AND IMPACT OF THE THE STUDY: These results may aid the dairy industry to improve the recovery of the starters at low costs after preservation processes.