作物淀粉生物合成与转基因修饰研究进展

淀粉是高等植物中碳水化合物的主要贮藏形式 ,也是粮食作物产品的最主要成分。淀粉虽然都由直链淀粉和枝链淀粉组成 ,但在不同作物中两者的比例和枝链淀粉结构的存在很大差异。现已明确 ,直链淀粉是在颗粒结合淀粉合成酶 (granule boundstarchsynthase,GBSS)催化下合成的 ,而枝链淀粉是四种酶共同作用的结果 ,它们分别是腺嘌呤 -葡萄糖焦磷酸化酶 (ADP glucosepyrophosphorylase ,AGP) ,可溶性淀粉合成酶 (solublestarchsynthase ,SSS) ,淀粉分枝酶 (starchbranchingenzyme ,SBE)和脱分枝酶 (starchdebranchingenzyme ,DBE)。一方面 ,在不同作物中 ,这些酶本身存在多种形式 ,如在玉米胚乳中 ,AGP有大亚基和小亚基之分 ,SBE又可分BE1,BEIIa ,BEIIb 3种 ,SSS也可分为SSI和SSIII(或SSIIa)两种 ,而DBE也有异淀粉酶 (isoamylase)和限制性糊精酶 (pullu lanase)两种。另一方面 ,控制特定酶的基因 ,在不同作物甚至在同一种作物的不同品种中也可能存在不同的复等位基因 ,如籼稻和粳稻的GBSS分别由蜡质基因Wxa 和Wxb 控制 ,两者编码的GBSS活性差异显著。此外 ,环境条件也可通过影响基因的转录使酶的含量或催化性能发生变化。迄今 ,国内外已获得多种马铃薯和水稻的转基因材料 ,对淀粉合成进行修饰 ,试图培育优质品     

基因工程改良淀粉品质

淀粉对人类生活十分重要,它不仅是人们的能量和营养来源,而且还是重要的工业原材料。对于淀粉合成过程及淀粉的加工、使用一直是淀粉研究的重点内容。淀粉的合成在最后阶段涉及到３个关键性的酶是：ＡＤＰＧ焦磷酸化酶、淀粉合成酸以及淀粉分支酶。它们分别催化ＡＤＰ－葡萄糖的形成、葡聚糖链的延伸以及分支链的形成。另外淀粉去分支酶对淀粉最终结构的形成也起到重要作用。本文将介绍上述４个酶近年来的生物化学和分子生物学研究     

Towards a more versatile alpha-glucan biosynthesis in plants

Starch is an important storage polysaccharide in many plants. It is composed of densely packed alpha-glucans, consisting of 1,4- and 1,4,6-linked glucose residues. The starch polymers are used in many industrial applications. The biosynthetic machinery for assembling the granule has been manipulated in many different ways to gain insight into the process of starch biosynthesis and to engineer starches with improved functionalities. With respect to the latter, two generic technologies with great potential have been developed: (i) introduction of new linkage types in starch polymers (1,3- and 1,6-linkages), and (ii) engineering granule-boundness. The toolbox to engineer this new generation of starch polymers is discussed.     

植物淀粉合成的调控酶

淀粉是植物中最普通的碳水化合物,是人类最主要的食品来源与重要的工业原料。植物淀粉的生物合成主要涉及了4种酶—ADPG焦磷酸化酶、淀粉合成酶、淀粉分支酶和淀粉去分支酶,它们在淀粉的生物合成中发挥着不同作用。近年来,随着基因工程技术的迅速发展及与这些酶有关的众多突变体的发现,使人们对这些酶的结构、特性、功能及表达调控等方面的研究取得了重要进展。并且,人们已开始利用基因工程技术调控植物淀粉的数量与特性,取得了一定成效。在此,文章介绍了调控植物淀粉合成关键酶的生化特性、基因调控及利用基因工程改良植物淀粉等方面所取得进展。     

Thermal and rheological properties of nixtamalized maize starch

The effect of nixtamalization process on thermal and rheological characteristics of corn starch was studied. Starch of raw sample had higher gelatinization temperature than its raw counterpart, because, the Ca(2+) ions stabilize starch structure of nixtamalized sample; however, the enthalpy values were not different in both samples. The temperature of the phase transition of the retrograded starches (raw and nixtamalized) were not different at the storage times assessed, but the enthalpy values of the above mentioned transition was different, indicating a lower reorganization of the starch structure in the nixtamalized sample. The viscoamylographic profile showed differences between both starches, since raw starch had higher peak viscosity than the nixtamalized sample due to partial gelatinization of some granules during this heat treatment. Rheological test showed that at low temperature (25 degrees C) the raw and nixtamalized starches presented different behaviour; however, the elastic characteristic was more important in the starch gel structure. The nixtamalization process produced changes in thermal and rheological characteristics becoming important in those products elaborated from nixtamalized maize.     

The complexities of starch biosynthesis in cereal endosperms

Starch serves not only as an energy source for plants, animals, and humans but also as an environmentally friendly alternative for fossil fuels. Here, we describe recent findings concerning the synthesis of this important molecule in the cereal endosperm. Results from six separate transgenic reports point to the importance of adenosine diphosphate glucose pyrophosphorylase in controlling the amount of starch synthesized. The unexpected cause underlying the contrast in sequence divergence of its two subunits is also described. A major unresolved question concerning the synthesis of starch is the origin of nonrandom or clustered alpha-1,6 branch-points within the major component of starch, amylopectin. Developing evidence that several of the starch biosynthetic enzymes involved in amylopectin synthesis occur in complexes is reviewed. These complexes may provide the specificity for the formation of nonrandom branch-points.     

Manipulation of starch granule size distribution in potato tubers by modulation of plastid division

Starch granule size is an important parameter for starch applications in industry. Starch granules are formed in amyloplasts, which are, like chloroplasts, derived from proplastids. Division processes and associated machinery are likely to be similar for all plastids. Essential roles for FtsZ proteins in plastid division in land plants have been revealed. FtsZ forms the so-called Z ring which, together with inner and outer plastid division rings, brings about constriction of the plastid. It has been shown that modulation of the expression level of FtsZ may result in altered chloroplast size and number. To test whether FtsZ is also involved in amyloplast division and whether this, in turn, may affect the starch granule size in crop plants, FtsZ protein levels were either reduced or increased in potato. As shown previously in other plant species, decreased StFtsZ1 protein levels in leaves resulted in a decrease in the number of chloroplasts in guard cells. More interestingly, plants with increased StFtsZ1 protein levels in tubers resulted in less, but larger, starch granules. This suggests that the stoichiometry between StFtsZ1 and other components of the plastid division machinery is important for its function. Starch from these tubers also had altered pasting properties and phosphate content. The importance of our results for the starch industry is discussed.     

Enzymic starch utilization and genetic engineering

Starch is widely used in the food and beverage, paper and textile industries. Genetic engineering will allow optimization of starch conversion technologies both by creating novel enzymes/micro organisms of the desired efficiency, stability and activity to fit any process, and by ensuring they are produced in commercial quantities. Plants could also be genetically engineered to produce starches of the desired amylopectin: amylose ratio. To ensure a continuous supply of starch, starch-producing plants could be genetically engineered to be disease- and insect-resistant, high yielding, and able to grow under any climatic and soil conditions. However, screening for novel microorganisms should not be neglected and should be used to complement genetic engineering.     

Properties and applications of starch-converting enzymes of the alpha-amylase family.

Starch is a major storage product of many economically important crops such as wheat, rice, maize, tapioca, and potato. A large-scale starch processing industry has emerged in the last century. In the past decades, we have seen a shift from the acid hydrolysis of starch to the use of starch-converting enzymes in the production of maltodextrin, modified starches, or glucose and fructose syrups. Currently, these enzymes comprise about 30% of the world's enzyme production. Besides the use in starch hydrolysis, starch-converting enzymes are also used in a number of other industrial applications, such as laundry and porcelain detergents or as anti-staling agents in baking. A number of these starch-converting enzymes belong to a single family: the alpha-amylase family or family13 glycosyl hydrolases. This group of enzymes share a number of common characteristics such as a (beta/alpha)(8) barrel structure, the hydrolysis or formation of glycosidic bonds in the alpha conformation, and a number of conserved amino acid residues in the active site. As many as 21 different reaction and product specificities are found in this family. Currently, 25 three-dimensional (3D) structures of a few members of the alpha-amylase family have been determined using protein crystallization and X-ray crystallography. These data in combination with site-directed mutagenesis studies have helped to better understand the interactions between the substrate or product molecule and the different amino acids found in and around the active site. This review illustrates the reaction and product diversity found within the alpha-amylase family, the mechanistic principles deduced from structure-function relationship structures, and the use of the enzymes of this family in industrial applications.     

Significant differences in the activities of alpha-amylases in the absence and presence of polyethylene glycol assayed on eight starches solubilized by two methods

Starch is a reserve chemical source of the energy of the sun found in plants as a water-insoluble granule that differs in their chemical and physical properties, depending on the source. The granules can be solubilized by heating in water or by treatment with various reagents, such as 1M NaOH. alpha-Amylases are widely distributed enzymes that initiate the hydrolysis of starch into low molecular weight maltodextrins. We recently found that the activities of a single alpha-amylase on two different starches were significantly different. We then determined the activities of Bacillus amyloliquefaciens and porcine pancreas alpha-amylases, using eight different starches, solubilized by two methods: autoclaving at 121 degrees C and 1M NaOH at 20 degrees C. There were significant differences in the activities of both of the amylases on all eight of the starches. Previously, it had been found that polyethylene glycol (PEG) stabilized and activated the activities of both enzymes, using a soluble amylose as the substrate. Addition of PEG to the enzymes greatly increased the activities on the eight starches, but the activities still differed significantly. The different activities with the starches were hypothesized as differences in the amounts of secondary and tertiary structures that are partially retained when the different starches are solubilized; the activities on addition of PEG is hypothesized as the formation of highly active species from a series of less active forms.     

Fermentation of native and processed starches by the porcine caecal anaerobe Clostridium butyricum (NCIMB 7423)

Starch fermentation by the porcine caecal anaerobe Clostridium butyricum was examined using gas and volatile fatty acid production as determinants of activity. Potato starch and amylopectin were studied in their native form as well as after retrogradation, which should render them resistant to pancreatic α-amylase digestion. Fermentation of both substrates was enhanced by pancreatin digestion of the native material, possibly due to the removal or disruption of part of the structure of the starch by the pancreatic enzymes. However, pancreatic digestion of retrograded potato starch apparently reduced the amount available for bacterial fermentation, whereas no significant effect was observed with amylopectin. The data suggests that starches which are high in amylopectin would be more likely to influence fermentation in the large intestine in monogastric animals, and that the presence of residual pancreatic enzymes in the lower gut could potentially enhance starch fermentation by this micro-organism.     

The phosphoglucan phosphatase like sex Four2 dephosphorylates starch at the C3-position in Arabidopsis

Starch contains phosphate covalently bound to the C6-position (70 to 80% of total bound phosphate) and the C3-position (20 to 30%) of the glucosyl residues of the amylopectin fraction. In plants, the transient phosphorylation of starch renders the granule surface more accessible to glucan hydrolyzing enzymes and is required for proper starch degradation. Phosphate also confers desired properties to starch-derived pastes for industrial applications. In Arabidopsis thaliana, the removal of phosphate by the glucan phosphatase Starch Excess4 (SEX4) is essential for starch breakdown. We identified a homolog of SEX4, LSF2 (Like Sex Four2), as a novel enzyme involved in starch metabolism in Arabidopsis chloroplasts. Unlike SEX4, LSF2 does not have a carbohydrate binding module. Nevertheless, it binds to starch and specifically hydrolyzes phosphate from the C3-position. As a consequence, lsf2 mutant starch has elevated levels of C3-bound phosphate. SEX4 can release phosphate from both the C6- and the C3-positions, resulting in partial functional overlap with LSF2. However, compared with sex4 single mutants, the lsf2 sex4 double mutants have a more severe starch-excess phenotype, impaired growth, and a further change in the proportion of C3- and C6-bound phosphate. These findings significantly advance our understanding of the metabolism of phosphate in starch and provide innovative options for tailoring novel starches with improved functionality for industry.     

The Simultaneous Abolition of Three Starch Hydrolases Blocks Transient Starch Breakdown in Arabidopsis

In this study, we investigated which enzymes are involved in debranching amylopectin during transient starch degradation. Previous studies identified two debranching enzymes, isoamylase 3 (ISA3) and limit dextrinase (LDA), involved in this process. However, plants lacking both enzymes still degrade substantial amounts of starch. Thus, other enzymes/mechanisms must contribute to starch breakdown. We show that the chloroplastic α-amylase 3 (AMY3) also participates in starch degradation and provide evidence that all three enzymes can act directly at the starch granule surface. The isa3 mutant has a starch excess phenotype, reflecting impaired starch breakdown. In contrast, removal of AMY3, LDA, or both enzymes together has no impact on starch degradation. However, removal of AMY3 or LDA in addition to ISA3 enhances the starch excess phenotype. In plants lacking all three enzymes, starch breakdown is effectively blocked, and starch accumulates to the highest levels observed so far. This provides indirect evidence that the heteromultimeric debranching enzyme ISA1-ISA2 is not involved in starch breakdown. However, we illustrate that ISA1-ISA2 can hydrolyze small soluble branched glucans that accumulate when ISA3 and LDA are missing, albeit at a slow rate. Starch accumulation in the mutants correlates inversely with plant growth.     

Approaches to influence starch quantity and starch quality in transgenic plants

Starch plays a major role as a transitory and long-term storage compound in higher plants, and therefore is of prime importance for plant growth and development. Additionally, starch serves as a widely used material for a variety of industrial uses. The formation of starch can arbitrarily be divided into three types of event: (I) those leading to the supply of glucose-1-phosphate in the plastids; (II) the conversion of glucose-1-phosphate to ADP-glucose catalysed by the enzyme ADP-glucose pyrophosphorylase; and (III) the enzymatic reactions converting ADP-glucose to long-chain glucans (amylopectin, amylose). In recent years, numerous cDNA and genomic sequences encoding enzymes involved in starch metabolism have been identified. Some of these have been used to down-regulate enzyme activities via the antisense RNA technique. Additionally, bacterial genes have been ectopically expressed in transgenic plants in order to increase corresponding enzyme activities. By modulating the activity of ADP-glucose pyrophosphorylase in plastids, it was possible to decrease and increase, respectively, the starch content in source and sink organs of transgenic plants. In addition, down-regulation of granule-bound starch synthase (isoform I) resulted in the production of starch that was almost completely free of amylose. Further experiments aimed to modulate starch structure are currently underway and will briefly be discussed.     

Kinetic Analysis of Aspergillus oryzae Cultivations on Starch

Cultivation of Aspergillus oryzae on starch is described as a combination of two rate processes: Starch hydrolysis and the cellular activities of the fungi including growth, enzyme production and maintenance. Kinetic models are presented to describe growth, enzyme production, starch hydrolysis and uptake of the hydrolysis products. Numerical values of the model parameters indicated that the rate controlling step of A. oryzae growth on starch was not starch hydrolysis, but the substrate uptake process. Glucose was one of the starch hydrolysis products. About 35% of the substrate consumed for biomass synthesis was glucose. Its accumulation in the medium did not cause repression of the starch hydrolysing enzymes. Steady state starch hydrolysis rates increased with initial starch concentration in the medium. Starch hydrolysing enzymes of A. oryzae have extensive industrial uses. This study may help in a more detailed understanding of the kinetic aspects of the production of these enzymes.     

玉米淀粉生物合成及其遗传操纵

淀粉是许多植物重要的储藏物质。淀粉突变体以及转基因植物中淀粉变异的特点使我们对淀粉生物合成的过程有了较深入的了解,许多研究的结果揭示了玉米淀粉的生物合成涉及4类酶--ADPG焦磷酸化酶、淀粉合成酶、淀粉分支酶和去分支酶。随着编码这些酶的基因的克隆,利用转基因技术对淀粉合成过程进行遗传操纵业已成为可能,并且在提高淀粉产量以及不同特性淀粉品质的种质资源创新等方面展示出巨大的潜力。 Abstract:Starch is the most important source of calories and a vital storage component in plants.The characterization and production of starch variants from mutation and with transgenic technology has improved our understanding of the synthesis of starch granule.In starch biosynthesis in plants,four enzymes,including ADP-glucose pyrophosphorylase,starch synthase,starch branching enzyme and starch debranching enzyme,are widely accepted from an enormous amount of research aimed primarily at enzyme characterization.As many genes encoding the enzymes and their multiple isoforms in starch biosynthesis pathway have been isolated,genetic manipulation of the starch biosynthesis pathway shows to be a practical way by which starch quantity is increased and starch with novel properties can be created.     

植物体内淀粉磷酸化的生物学研究进展

植物中淀粉是主要储存碳水化合物形式,是食品和工业应用中最重要的植物原材料之一。而植物淀粉中惟一的取代是磷酸化作用,更体现了淀粉的独特性能。淀粉的品质和理化性质影响其应用。因此对淀粉的研究是有重要意义的。主要介绍了淀粉磷酸化作用机制,概述GWD功能与磷酸化作用和淀粉代谢的关系的生物学研究进展,并在此基础上讨论利用基因工程改良淀粉品质的可能途径以及今后的研究任务。     

Composition, structure, functionality, and chemical modification of legume starches: a review

The major carbohydrate of the legume seed is starch, which represents up to 45% of the total seed weight. In recent years, substantial progress has been made in understanding the relationship between starch structure and functionality. However, these studies have been mainly on cereal and tuber starches. The present status of knowledge on the composition, structure, functionality, digestibility, and chemical modification of legume starches is reviewed. In addition present concepts of granule structure, gelatinization, retrogradation, and rheology are also reviewed. Future research needs in the area of legume starch chemistry are discussed.     

Potato starch synthases: Functions and relationships

Starch, a very compact form of glucose units, is the most abundant form of storage polyglucan in nature. The starch synthesis pathway is among the central biochemical pathways, however, our understanding of this important pathway regarding genetic elements controlling this pathway, is still insufficient. Starch biosynthesis requires the action of several enzymes. Soluble starch synthases (SSs) are a group of key players in starch biosynthesis which have proven their impact on different aspects of the starch biosynthesis and functionalities. These enzymes have been studied in different plant species and organs in detail, however, there seem to be key differences among species regarding their contributions to the starch synthesis. In this review, we consider an update on various SSs with an emphasis on potato SSs as a model for storage organs. The genetics and regulatory mechanisms of potato starch synthases will be highlighted. Different aspects of various isoforms of SSs are also discussed.     

The diurnal metabolism of leaf starch

Starch is a primary product of photosynthesis in leaves. In most plants, a large fraction of the carbon assimilated during the day is stored transiently in the chloroplast as starch for use during the subsequent night. Photosynthetic partitioning into starch is finely regulated, and the amount of carbohydrate stored is dependent on the environmental conditions, particularly day length. This regulation is applied at several levels to control the flux of carbon from the Calvin cycle into starch biosynthesis. Starch is composed primarily of branched glucans with an architecture that allows the formation of a semi-crystalline insoluble granule. Biosynthesis has been most intensively studied in non-photosynthetic starch-storing organs, such as developing seeds and tubers. Biosynthesis in leaves has received less attention, but recent reverse-genetic studies of Arabidopsis (thale cress) have produced data generally consistent with what is known for storage tissues. The pathway involves starch synthases, which elongate the glucan chains, and branching enzymes. Remarkably, enzymes that partially debranch glucans are also required for normal amylopectin synthesis. In the last decade, our understanding of starch breakdown in leaves has advanced considerably. Starch is hydrolysed to maltose and glucose at night via a pathway that requires recently discovered proteins in addition to well-known enzymes. These sugars are exported from the plastid to support sucrose synthesis, respiration and growth. In the present review we provide an overview of starch biosynthesis, starch structure and starch degradation in the leaves of plants. We focus on recent advances in each area and highlight outstanding questions.