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。     

海藻糖脂的合成及应用前景

海藻糖脂是一类生物表面活性化合物,与化学表面活性剂相比,具有低毒、可生物降解、在极端条件下稳定性强、环境友好等优势,具有广泛的应用价值和前景。目前国内还没有大规模商业应用的实例,主要是生产成本高、产量低、产物成分复杂等因素导致。本文中,笔者主要介绍了海藻糖脂的制备方法,分析比较了各种方法的优势与问题,综述了海藻糖脂的性质及其在生物医学、食品、化妆品、生物修复和石油工业领域的应用前景。海藻糖脂由于其特有的性质,将在实际应用中,尤其是在生物医学工程领域方面的应用中有望得到进一步的发展。     

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。     

海藻糖合酶的分子生物学研究进展

海藻糖合酶能够将麦芽糖转化为海藻糖,在海藻糖的工业生产中具有十分重要的意义。本文从海藻糖合酶的基因克隆、基因工程应用、结构和催化机制的研究以及其在微生物体内的功能等方面讨论了海藻糖合酶的研究进展。     

海藻糖及其在微生物领域中的应用

海藻糖是生物体低湿休眠及微生物、生物大分子耐热抗干燥的稳定物质,８０年代以来受到国内外广泛关注并做了大量细致研究,本文详细介绍了这种独特双糖的理化及分子生物学特性、功能和作用机理以及它的制备和应用前景。     

海藻糖的生物学功能简介

海藻糖（Ｔｒｅｈａｌｏｓｅ,α－Ｄ－ｇｌｕｃｏｐｙｒａｎｏｓｙｌ－α－Ｄ－ｇｌｕｃｏｐｙｒａｎｏｓｉｄｅ）是一种非还原性二糖,广泛存在于海藻、酵母、霉菌、食用菌、虾、昆虫、高等植物等生物体内,是一种贮藏性碳水化合物。它具有保护生物细胞和生物活动性物质...     

微生物生产海藻糖及其应用前景

微生物生产海藻糖及其应用前景罗明典（中国北京科学院微生物研究所,北京１０００８０）在自然界有些生物能够在脱水情况下连续数年保持生命状态,一旦遇水又重新恢复生机,其重要原因在于这些生物细胞含有大量海藻糖之故,这类糖由两个葡萄糖分子聚合组成的双糖,分子式...     

Improved trehalose production from biodiesel waste using parent and osmotically sensitive mutant of Propionibacterium freudenreichii subsp. shermanii under aerobic conditions

Trehalose is an important nutraceutical of wide commercial interest in the food processing industry. Recently, crude glycerol was reported to be suitable for the production of trehalose using a food microbe, Propionibacterium freudenreichii subsp. shermanii, under static flask conditions. Similarly, enhanced trehalose yield was reported in an osmotically sensitive mutant of the same strain under anaerobic conditions. In the present study, an effort was made to achieve higher production of trehalose, propionic acid, and lactic acid using the parent and an osmotically sensitive mutant of P. freudenreichii subsp. shermanii under aeration conditions. Under aeration conditions (200 rpm in shake flasks and 30 % air saturation in a batch reactor), biomass was increased and approximately 98 % of crude glycerol was consumed. In the parent strain, a trehalose titre of 361 mg/l was achieved, whereas in the mutant strain a trehalose titre of 1.3 g/l was produced in shake flask conditions (200 rpm). In the mutant strain, propionic and lactic acid yields of 0.53 and 0.21 g/g of substrate were also achieved with crude glycerol. Similarly, in controlled batch reactor culturing conditions a final trehalose titre of approximately 1.56 g/l was achieved with the mutant strain using crude glycerol as the substrate. Enhanced production of trehalose using P. freudenreichii subsp. shermanii from waste under aeration conditions is reported here. Higher production of trehalose was not due to a higher yield of trehalose but to a higher final biomass concentration.     

Impact of heterologous expression of Escherichia coli UDP-glucose pyrophosphorylase on trehalose and glycogen synthesis in Corynebacterium glutamicum

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.     

Haemolymph sugar levels in a nectar-feeding ant: dependence on metabolic expenditure and carbohydrate deprivation

In nectar-feeding insects, sugars are an important source of fuel and energy storage. Here, we analyzed the haemolymph sugar levels in foragers of the ant Camponotus rufipes trained to collect nectar from an artificial feeder, and their dependence on the metabolic rate during feeding. The main sugar found was trehalose, followed by glucose and traces of fructose and sucrose. In foragers, trehalose level was independent of their activity and metabolic rate while feeding. Carbohydrate deprivation of the colony had a strong effect on the haemolymph sugar levels of workers, with a significant decrease in trehalose and glucose with increasing starvation. We also found a correlation between haemolymph sugar levels and behavioral states, with immobile workers having higher trehalose and fructose levels than active ones. It is suggested that under food deprivation, inside-nest workers initially stay completely immobile as a strategy to save energy, and only become active and start to search for food when the trehalose levels decrease even more. Based on a conservative estimation, well-fed ants could travel up to 500 m, or spend more than 20 h inactive at 25°C, using only the energy provided by the haemolymph trehalose, before reaching the levels found in starved nest-mates.     

海藻糖及其在生物工程方面的应用

自海藻糖发现以来对其化学性质、生理功能、作用机理、代谢途径等已进行了较为深入的研究,其分子生物学的研究也渐渐兴起。研究表明海藻糖能提高生物体对干旱、低温、高温、pH、盐渍等逆境条件下的抗性。离体试验表明海藻糖能保护生物膜、蛋白质的结构并能保持逆境下的酶活性,同时,外源海藻糖同样对生物体有保护作用。由于海藻糖具有这些独特的生物学功能,它已在许多方面得以应用,可作为食品工业的一种添加剂和甜味剂,使干燥食品在得水后保持原有的色、香、味;也可作为医药工业的非特异性生物制品和生化药品保护剂,使其在常温下保存,从而降低运输与储存费用;另外,在农业研究中可利用现代分子生物技术培育表达海藻糖的转基因作物,提高农作物的抗旱、抗冻等抗逆性能。     

GUSTATORY REACTION TIME AND TIME INTENSITY MEASUREMENTS OF TREHALOSE AND SUCROSE SOLUTIONS AND THEIR MIXTURES

Dynamic sweetness perception of commercial food grade trehalose, sucrose solutions and their mixtures were studied for a wide range of concentrations. For gustatory reaction time (GRT), concentrations ranged from 2.3 to 13.8% for sucrose and up to 23.0% for trehalose. For time intensity (T-I) sucrose or trehalose solutions (concentration range 2.3–36.8%) and their combinations (23.0 and 36.8% total solids) were analyzed. Trehalose had bigger GRT along the studied range. Both sugars presented similar values for persistence and times of plateau and to maximum intensity, while a significant difference was observed in intensity and GRT at equal concentrations. Trehalose had longer persistence than sucrose in equi -sweet solutions. Overall sweetness profile of some sucrose solutions (i.e., 29.9% sucrose solution and 0.6 sucrose/trehalose ratio mixture at 36.8% total solids) were perceived as similar to mixtures of sucrose/trehalose or single trehalose solutions, which suggests the possibility of sugar replacement without completely modifying sweetness perception.

PRACTICAL APPLICATIONS

It has been suggested that trehalose may be a potential substitute for sucrose and other sugars used in food formulation because, although its chemical structure is very similar to that of sucrose, it is more stable at low pH and high temperatures. It is not involved in caramelization and does not participate in Maillard reaction with amino acids/proteins. In order to fully establish the potential of trehalose as a functional replacement of sucrose we have determined the sweetness dynamic profile (gustatory reaction time and time-intensity curves) of trehalose solutions and sucrose/trehalose solutions; this aspect is needed for adequately replacing (partially or totally) sucrose in food systems.     

Heterologous expression of Escherichia coli ppsA (phosphoenolpyruvate synthetase) and galU (UDP-glucose pyrophosphorylase) genes in Corynebacterium glutamicum, and its impact on trehalose synthesis

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on a Corynebacterium glutamicum strain expressing the Escherichia coli enzyme UDP-glucose pyrophosphorylase (GalU). Biochemical network analysis suggest a further bottleneck for trehalose synthesis resulting from the coupling of phosphotransferase (PTS) mediated glucose uptake, and glucose catabolism in C. glutamicum. To overcome this coupling, we propose the expression of E. coli phosphoenolpyruvate synthetase (PpsA), in addition to GalU expression, in C. glutamicum. Although GalU expression improved trehalose synthesis in C. glutamicum, the simultaneous expression of GalU and PpsA did not result in a further increase in trehalose yield, but resulted in an increased catabolic rate of glucose, which could be ascribed to the operation of a futile cycle between phosphoenolpyruvate and pyruvate. The impact of GalU and PpsA expression on polysaccharide content, side product excretion and metabolic fluxes is discussed, as well as alternative ways to decouple glucose uptake and catabolism, in order to increase trehalose yield.     

Impact of Heterologous Expression of Escherichia coli UDP-Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.     

Metabolic engineering of Corynebacterium glutamicum for trehalose overproduction: role of the TreYZ trehalose biosynthetic pathway

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.     

Novel enzymatic production of trehalose from sucrose using amylosucrase and maltooligosyltrehalose synthase-trehalohydrolase

Trehalose (α-d-glucopyranosyl α-d-glucopyranoside) is an important non-reducing disaccharide used in the food industry due to its mild sweetness (45% that of sucrose), low cariogenicity, high glass transition temperature, low hygroscopicity, and protein protection properties. In this study, we accomplished the production of trehalose from sucrose as a sole substrate using a novel dual-enzyme system, in which amylosucrase (ASase) and maltooligosyltrehalose synthase-trehalohydrolase (MTSH) fusion enzyme were employed. The biotransformation of sucrose to trehalose was confirmed by high-performance anion-exchange chromatography (HPAEC) analysis. Trehalose was successfully produced by both simultaneous and sequential methods by using ASase and MTSH. A higher trehalose production yield (3.15 ± 0.83 mM trehalose/20 mM sucrose) was observed in the sequential method than the simultaneous method (1.43 ± 0.14 mM trehalose/20 mM sucrose), indicating that the production of maltooligosaccharides from sucrose by ASase was an important step in the biosynthesis of trehalose.     

Metabolic Engineering of Corynebacterium glutamicum for Trehalose Overproduction: Role of the TreYZ Trehalose Biosynthetic Pathway

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.