Advances in flavonoid glycosyltransferase research: integrating recent findings with long-term citrus studies

Flavonoid glycosides are required for a number of crucial roles in planta and have the potential for development in a variety of agricultural, medicinal, and biotechnological applications. A number of recent advancements have been made in characterizing glycosyltransferases, the enzymes that are responsible for the synthesis of these important molecules. In this review, glycosyltransferases are considered with regard to biochemical properties, expression patterns, levels of enzyme activity during development, and structure/function relationships. This is presented with historical context to highlight critical findings, particularly with regard to the innovative work that has come from research on citrus species. The plant glycosyltransferase crystal structures that have been solved over the past decade, either alone or in complex with sugar donor and/or acceptor molecules, are discussed. The application of results from these structures to inform current structure/function work as well as implications and goals for future crystallography and tertiary modeling studies are considered. A thorough understanding of the properties of glycosyltransferases will be a critical step in any future biotechnological application of these enzymes in areas such as crop improvement and custom design of enzymes to produce desired compounds for nutritional and/or medicinal usage.     

Synthesis of oligosaccharides by bacterial enzymes

Many human pathogens initiate disease by utilizing their microbial adhesin proteins to attach to glycoconjugates on host cell mucosal surfaces. Soluble oligosaccharides of identical or similar structure to these naturally occurring ligands can both prevent bacterial attachment as well as mediate the release of attached bacteria. Since it has not been possible to isolate large quantities of these compounds, we have developed enzyme-based technologies to synthesize several relevant human oligosaccharides. Using cloned bacterial glycosyltransferases, we can synthesize several hundred grams of these oligosaccharides at a time. The availability of these large quantities will allow these compounds to be tested as anti-adhesive pharmaceutical agents as well as lead to expanded practical applications.     

Comparative aspects of glycosyltransferases

Glycosyltransferases, the enzymes that build oligosaccharides and glycoconjugates, have received much interest in recent years owing to their biological functions and their potential uses in biotechnology. Despite the fact that many glycosyltransferases recognize similar donor or acceptor substrates, there is surprisingly limited sequence identity between different classes. On the one hand, the glycosyltransferases are found in a large number of families, by sequence-based classification. On the other hand, only two structural folds have been identified among the fewer than one dozen glycosyltransferases that have been crystallized at present. Detection of conserved motifs that have a direct role in the functional aspects of glycosyltransferases is one approach for identifying remote similarity. With the availability of more crystal structures, the use of the fold-recognition approach is also very promising.     

Molecular cloning, expression, and uses of mammalian glycosyltransferases.

Lineage specific expression patterns of mammalian oligosaccharides are regulated largely by glycosyltransferases. Several distinct glycosyltransferase genes have recently been isolated by gene transfer and traditional cloning approaches. These each maintain a type II transmembrane topology, that yields a COOH-terminal, Golgi-resident catalytic domain. However, significant primary sequence similarities between cloned glycosyltransferases have been found only between enzymes with substantial functional similarity. These cloned DNA segments, and their cognate enzymes, have been used to determine glycosyltransferase expression patterns, characterize the molecular basis for mutant glycosyltransferase alleles, and identify oligosaccharide ligands for members of the Selectin family of cell adhesion molecules. They have also shown promise for use in the preparative synthesis of defined oligosaccharide molecules, and for transgenic animal approaches designed to explore the function(s) of oligosaccharides in mammalian organisms.     

Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides

Mammalian cell surfaces are all covered with bioactive oligosaccharides which play an important role in molecular recognition events such as immune recognition, cell-cell communication and initiation of microbial pathogenesis. Consequently, bioactive oligosaccharides have been recognized as a medicinally relevant class of biomolecules for which the interest is growing. For the preparation of complex and highly pure oligosaccharides, methods based on the application of glycosyltransferases are currently recognized as being the most effective. The present paper reviews the potential of glycosyltransferases as synthetic tools in oligosaccharide synthesis. Reaction mechanisms and selected characteristics of these enzymes are described in relation to the stereochemistry of the transfer reaction and the requirements of sugar nucleotide donors. For the application of glycosyltransferases, accepted substrate profiles are summarized and the whole-cell approach versus isolated enzyme methodology is compared. Sialyltransferase-catalyzed syntheses of gangliosides and other sialylated oligosaccharides are described in more detail in view of the prominent role of these compounds in biological recognition.     

乳酸菌食品级nisin控制的基因表达系统NICE

乳酸菌安全应用于人们的生产和生活已有上千年的历史,是一种食品级的微生物。在过去二十年里,其生理及遗传学特性已被彻底研究。由于其遗传可行且操作简单,乳酸菌除了其传统应用外已被广泛用于表达异源基因,在食品、农业及医药工程领域具有重要的应用前景。人们已开发了一系列乳酸菌食品级基因表达系统。本文主要介绍了乳酸菌,重点是其模式菌Lactococcus lactis最常见的食品级诱导表达系统--nisin控制的基因表达系统NIC E及其食品级诱导物nisin、食品级的宿主及表达载体系统,以及NICE系统在表达异源基因方面的应用。     

Cell wall structure and function in lactic acid bacteria

The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.     

Glycosyltransferases:key players involved in the modification of plant secondary metabolites

Glycosyltransferases are members of the multigene superfamily in plants that can transfer single or multiple activated sugars to a range of plant molecules,resulting in the glycosylation of plant compounds.Although the activities of many glycosyltransferases and their products have been recognized for a long time,only in recent years were some glycosyltransferase genes identified and a few functionally characterized in detail.Glycosylation is thought to be one of the most important modification reactions towards plant secondary metabolites,and plays a key role in maintaining cell homeostasis,thus likely participating in the regulation of plant growth,development and in defense responses to stress environments.With advances in plant genome projects and the development of novel technologies in analyzing gene function,significant progress could be made in gaining new insights into the properties and precise biological roles of plant secondary product glycosyltransferases,and the new knowledge will have extensive application prospects in the catalytic synthesis of glycoconjugates and metabolic engineering of crops.In this review,we summarize the current research,highlighting the possible biological roles,of plant secondary metabolite glycosyltransferases and discuss their potential applications as well as aspects to be further studied in the near future.     

Glycosyltransferases: key players involved in the modification of plant secondary metabolites

Glycosyltransferases are members of the multigene superfamily in plants that can transfer single or multiple activated sugars to a range of plant molecules, resulting in the glycosylation of plant compounds. Although the activities of many glycosyltransferases and their products have been recognized for a long time, only in recent years were some glycosyltransferase genes identified and a few functionally characterized in detail. Glycosylation is thought to be one of the most important modification reactions towards plant secondary metabolites, and plays a key role in maintaining cell homeostasis, thus likely participating in the regulation of plant growth, development and in defense responses to stress environments. With advances in plant genome projects and the development of novel technologies in analyzing gene function, significant progress could be made in gaining new insights into the properties and precise biological roles of plant secondary product glycosyltransferases, and the new knowledge will have extensive application prospects in the catalytic synthesis of glycoconjugates and metabolic engineering of crops. In this review, we summarize the current research, highlighting the possible biological roles, of plant secondary metabolite glycosyltransferases and discuss their potential applications as well as aspects to be further studied in the near future.     

Synthetic glycobiology: Exploits in the Golgi compartment

The challenge of engineering glycosylation has been confronted by synthetic chemists, biochemists and cell biologists, each with the primary goal of optimizing glycoconjugates for therapeutic applications. In nature, glycans are constructed by glycosyltransferases that are organized in an assembly line in the endoplasmic reticulum and Golgi compartment. Recent insights into the domain architecture, localization and regulation of glycosyltransferases have provided a platform for engineering their position within the secretory pathway and access to substrates. Using this knowledge, glycosyltransferase assembly lines have been redesigned for the production of specific glycan structures using protein engineering and chemical approaches. These efforts epitomize the emerging field of 'synthetic glycobiology'.     

Genetically modified probiotics in foods

Probiotics have many potential therapeutic uses, but have not been universally accepted because of a lack of understanding of their action. Lactic acid bacteria (LAB) have been modified by traditional and genetic engineering methods to produce new varieties. Modern techniques of molecular biology have facilitated the identification of probiotic LAB strains, but only a few LAB have been modified by recombinant-DNA technology because of consumer resistance to their introduction to markets, especially in Europe.     

Towards lactic acid bacteria-based biorefineries

Lactic acid bacteria (LAB) have long been used in industrial applications mainly as starters for food fermentation or as biocontrol agents or as probiotics. However, LAB possess several characteristics that render them among the most promising candidates for use in future biorefineries in converting plant-derived biomass—either from dedicated crops or from municipal/industrial solid wastes—into biofuels and high value-added products. Lactic acid, their main fermentation product, is an attractive building block extensively used by the chemical industry, owing to the potential for production of polylactides as biodegradable and biocompatible plastic alternative to polymers derived from petrochemicals. LA is but one of many high-value compounds which can be produced by LAB fermentation, which also include biofuels such as ethanol and butanol, biodegradable plastic polymers, exopolysaccharides, antimicrobial agents, health-promoting substances and nutraceuticals. Furthermore, several LAB strains have ascertained probiotic properties, and their biomass can be considered a high-value product. The present contribution aims to provide an extensive overview of the main industrial applications of LAB and future perspectives concerning their utilization in biorefineries. Strategies will be described in detail for developing LAB strains with broader substrate metabolic capacity for fermentation of cheaper biomass.     

A new pathway for the synthesis of oligosaccharides by the use of non-Leloir glycosyltransferases

The growing recognition of the roles of carbohydrates in fundamental biological processes and their potential application as functional foods and new therapeutics have generated a need for larger amounts of different carbohydrate structures. Leloir glycosyltransferases catalyze the synthesis of complex oligosaccharides. However they are difficult or expensive to obtain, and require expensive nucleotide activated sugars. In contrast non-Leloir pathway enzymes use sucrose, which is known to be a high energy donor of d-glucose for glucosyltransferases like dextransucrase, or a donor of d-fructose for fructosyltransferases like inulin- and levansucrases for the synthesis of polysaccharides. Here we present the synthesis and kinetic studies of oligosaccharides using non-Leloir glycosyltransferases and sucrose analogues as new substrates, like β-d-fructofuranosyl-α-d-galactopyranoside (Gal-Fru) by a fructosyltransferase (FTF) from B. subtilis NCIMB 11871. The sucrose analogues carry a high binding energy in the glycosidic bond similar to that of sucrose. Thus, β-d-Fructofuranosyl-α-d-galactopyranoside (Gal-Fru) and β-d-Fructofuranosyl-α-d-fucopyranoside (d-Fuc-Fru) have been shown to be substrates for fructosyltransferases, which produce oligo- or polysaccharides, also in the presence of acceptors.     

Rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors

We describe the chemoenzymatic synthesis of a variety of monodisperse hyaluronan (beta 4-glucuronic acid-beta 3-N-acetylglucosamine (HA)) oligosaccharides. Potential medical applications for HA oligosaccharides (approximately 10-20 sugars in length) include killing cancerous tumors and enhancing wound vascularization. Previously, the lack of defined oligosaccharides has limited the exploration of these sugars as components of new therapeutics. The Pasteurella multocida HA synthase, pmHAS, a polymerizing enzyme that normally elongates HA chains rapidly (approximately 1-100 sugars/s), was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid transferase and N-acetylglucosamine transferase). The two resulting enzymes were purified and immobilized individually onto solid supports. The two types of enzyme reactors were used in an alternating fashion to produce extremely pure sugar polymers of a single length (up to HA20) in a controlled, stepwise fashion without purification of the intermediates. These molecules are the longest, non-block, monodisperse synthetic oligosaccharides hitherto reported. This technology platform is also amenable to the synthesis of medicant-tagged or radioactive oligosaccharides for biomedical testing. Furthermore, these experiments with immobilized mutant enzymes prove both that pmHAS-catalyzed polymerization is non-processive and that a monomer of enzyme is the functional catalytic unit.     

Current status and application of lactic acid bacteria in animal production systems with a focus on bacteria from honey bee colonies

Lactic acid bacteria (LAB) are widely distributed in nature and, due to their beneficial effects on the host, are used as probiotics. This review describes the applications of LAB in animal production systems such as beekeeping, poultry, swine and bovine production, particularly as probiotics used to improve health, enhance growth and reproductive performance. Given the importance of honeybees in nature and the beekeeping industry as a producer of healthy food worldwide, the focus of this review is on the coexistence of LAB with honeybees, their food and environment. The main LAB species isolated from the beehive and their potential technological use are described. Evidence is provided that 43 LAB bacteria species have been isolated from beehives, of which 20 showed inhibition against 28 species of human and animal pathogens, some of which are resistant to antibiotics. Additionally, the presence of LAB in the beehive and their relationship with antibacterial properties of honey and pollen is discussed. Finally, we describe the use of lactic bacteria from bee colonies and their antimicrobial effect against foodborne pathogens and human health . This review broadens knowledge by highlighting the importance of honeybee colonies as suppliers of LAB and functional food.     

A review of food-grade vectors in lactic acid bacteria: from the laboratory to their application

Lactic acid bacteria (LAB) have a long history of use in fermented foods and as probiotics. Genetic manipulation of these microorganisms has great potential for new applications in food safety, as well as in the development of improved food products and in health. While genetic engineering of LAB could have a major positive impact on the food and pharmaceutical industries, progress could be prevented by legal issues related to the controversy surrounding this technology. The safe use of genetically modified LAB requires the development of food-grade cloning systems containing only the DNA from homologous hosts or generally considered as safe organisms, and not dependent antibiotic markers. The rationale for the development of cloning vectors derived from cryptic LAB plasmids is the need for new genetic engineering tools, therefore a vision from cryptic plasmids to applications in food-grade vectors for LAB plasmids is shown in this review. Replicative and integrative vectors for the construction of food-grade vectors, and the relationship between resistance mechanism and expression systems, will be treated in depth in this paper. Finally, we will discuss the limited use of these vectors, and the problems arising from their use.     

Expression of human glycosyltransferase genes in yeast as a tool for enzymatic synthesis of sugar chain

We planned the production of human glycosyltransferases in yeast for the enzymatic synthesis of various sugar chains. More than 160 genes encoding various glycosyltransferases were prepared as N-terminal transmembrane region truncated forms by PCR and were inserted into the entry vector of Invitrogen Ltd's Gateway system. About fifty glycosyltransferases were chosen for the synthesis of human type oligosaccharides, and expressed as two different forms in yeast. One is a soluble form, which is secreted into the culture medium by methylotrophic yeast, and the other is an immobilized form, which is displayed at the budding yeast cell wall as a fusion protein with Pir protein. To date, in both systems, some sialyltranferases and fucosyltransferases have been produced as active forms, indicating the potential usefulness of these systems for the enzymatic synthesis of various types of human sugar chains attached to proteins and lipids.     

Biocatalytic synthesis of oligosaccharides

Tremendous advances in biocatalytic approaches to oligosaccharide synthesis have taken place in the past two years. The use of isolated enzymes, both glycosyltransferases and glycosidases, or engineered whole cells allows the preparation of natural oligosaccharides and analogs required for glycobiology research.     

A bacterial glycosyltransferase gene toolbox: Generation and applications

The bioactivity of many natural products produced by microorganisms can be attributed to their sugar substituents. These substituents are transferred as nucleotide-activated sugars to an aglycon by glycosyltransferases. Engineering these enzymes can broaden their substrate specificity and can therefore have an impact on the bioactivity of the secondary metabolites.In this review we present the generation of a glycosyltransferase gene toolbox which contains more than 70 bacterial glycosyltransferases to date. Investigations of the function, specificity and structure of these glycosyltransferases help to understand the great potential of these enzymes for natural product biosynthesis.