Investigation on Oligosaccharides as Antimicrobial and Prebiotics

低聚糖作为抗菌剂和益生元的研究

• 批准号: 8514952
• 负责人: Peng George Wang
• 金额: $ 29.32万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8514952
• 关键词: Adhesions; Anti-Infective Agents; Antigens; Bacteria; Bacterial Adhesion; Bacterial Infections; Biological; Breast Feeding; Carbohydrates; Cell-Free System; Cells; Characteristics; Child; Cloning; Collaborations; Development; Dietary Fiber; Dose; Enteral; Enzyme Gene; Enzymes; Epithelial; Escherichia coli; Exhibits; Exposure to; Fermentation; Fucose; Galactose; Gene Cluster; Genitourinary system; Glucosamine; Glucose; Glycoconjugates; Human; Human Milk; Individual; Infant; Infection; Intestines; Investigation; Kilogram; Laboratories; Large Intestine; Link; Lipids; Metabolism; Monosaccharides; Nutritional; Oligosaccharides; Pathway interactions; Pharmacologic Substance; Phase; Play; Population; Process; Production; Property; Reagent; Recombinants; Recycling; Reporting; Research; Role; Safety; Series; Sialic Acids; Small Intestines; Solid; Staging; Surface; Synthetic Genes; System; Technology; Virus Diseases; Yeasts; antimicrobial; efficacy trial; gastrointestinal; glycosyltransferase; high risk; inhibitor/antagonist; large scale production; microbial; microbiome; pathogen; pre-clinical; prebiotics; preclinical study; prevent; programs; research clinical testing; research study; respiratory; safety study; safety testing; sugar; sugar nucleotide

DESCRIPTION (provided by applicant): Human milk oligosaccharides (HMOs) have been thought to play a role in the development of specific intestinal flora in breast-fed infants for many years. Nowadays it is known that they are also potent inhibitors of bacterial adhesion to epithelial surfaces (initial stage of the infection process). Oligosaccharides are not hydrolyzed in the upper small intestine and reach the large intestine intact, where they serve as substrates for bacterial metabolism. Thus, HMOs are considered as the ''dietary fiber'' of human milk. Another characteristic of oligosaccharides is their proposed ''anti-infective effect''. This role is achieved thanks to their capacity to inhibit the adhesion of bacteria to the epithelial surfaces, thereby playing an important protective role against infection in the gastrointestinal, respiratory and urogenital tracts by direct and indirect mechanisms. Therefore, HMOs have antimicrobial activity and may be useful in treating and/or preventing specific enteric bacterial and viral infections. However, the road to convert HMOs into pharmaceuticals or nutritional substances has been blocked by the lack of pure, single component oligosaccharides from human milk in quantities large enough for scientific investigation, as well as preclinical tolerance and safety studies and for safety and clinical testing in populations that are exposed to gastrointestinal pathogens. Therefore, this proposed research program aims to develop practical processes to produce HMOs on multi-gram to kilo-gram scales. Since it was repeatedly reported that 2-linked fucosyloligosaccharides exhibited more antimicrobial activity than non-2-linked fucosyloligosaccharides, we will choose five 2-linked fucosyloligosaccharides such as 2'-FL, LNF-I, 2H-antigen, LDFH-I and Ley as our main targets. Moreover, non-2-linked fucosyloligosaccharides LNF-II and LNF-III will provide us the opportunity to confirm the observation of higher antimicrobial activity for 2-linked fucosyloligosaccharides. In addition, the non-fucosylated oligosaccharide LNT and LNnT will provide control experiments to evaluate the effect of fucose in oligosaccharides. Over the past 14 years, the Wang lab has been developing enzymatic oligosaccharides synthesis. We have invented and further developed the "superbeads" and "superbug" technology for large scale oligosaccharide production. The most efficient approach for oligosaccharide synthesis is to follow the natural carbohydrate biosynthetic pathway where oligosaccharides are assembled together by specific glycosyltransferases using individual sugar nucleotides as building blocks. These building blocks are themselves biosynthesized and recycled from individual monosaccharides through a series of biosynthetic enzymes. For small to medium scale synthesis of oligosaccharides, Wang has developed simple solid phase synthetic systems by immobilizing all the necessary biosynthetic enzymes onto a so-called "superbeads". These beads function as stable and versatile synthetic reagents, which can be used to synthesize a variety of glycoconjugates in cell-free systems. For large-scale production, Wang "superbug" essentially transfers the entire natural biosynthetic pathway into an E. coli strain. The approach includes cloning each enzyme along the biosynthetic pathway and connecting the genes of these enzymes together to produce an artificial gene cluster. A recombinant E. coli transformed with such a gene cluster is then used to produce the oligosaccharide through fermentation and purification. Thus, the "superbeads" and "superbug" approaches will be used in this program to produce the 9 oligosaccharides. Specifically, there are four aims: Aim I: Production of HMOs by immobilizing multiple enzymes (superbeads). This involves investigation on the necessary microbial glycosyltransferases, development of superbeads for UDP-GlcNAc, UDP-Gal and GDP-Fuc production, and combination of the glycosyltransferases with sugar nucleotide production to produce oligosaccharides. Aim II: Production of HMOs by recombinant E. coli (Superbug), which involves combination of the biosynthetic pathways of these HMOs into one or several recombinant E. coli strains. Aim III: Production of HMOs by GRAS (Generally Recognized as Safe) yeast cells. This new system will provide safer production system for HMOs synthesis. Aim IV: Characterization of the oligosaccharides through systematic biomedical and microbiome approaches in collaboration with other specialized laboratories, also in our own lab, with the advantage of multi-gram or kilo-gram scale neutral human milk oligosaccharides produced from this project. It is expected that the biosynthetic technology developed in Aim I - III will be transferred to biotech company(s) (such as the biotech startup Carbogene USA LLC which specializes in large scale oligosaccharide production) and GMP processes will be developed to produce the oligosaccharides in quantities large enough for preclinical studies of tolerance and safety, as well as for safety, dose-ranging, and efficacy trials in infants and children who are at high risk of exposure to gastrointestinal pathogens.

描述（由申请人提供）：人们认为，人乳寡糖（HMO）在母乳喂养婴儿的特定肠道菌群的发展中发挥了作用多年。如今，众所周知，它们也是对上皮表面（感染过程的初始阶段）细菌粘附的有效抑制剂。寡糖不会在小肠上部水解并完整到达大肠，它们是细菌代谢的底物。因此，HMO被视为人牛奶的“饮食纤维”。寡糖的另一个特征是他们提出的“抗感染效应”。由于它们能够抑制细菌对上皮表面的粘附的能力，因此可以通过直接和间接机制抑制重要的保护作用，从而发挥了重要的保护作用，从而发挥了重要的保护作用，从而发挥了重要的保护作用。因此，HMO具有抗菌活性，可能有助于治疗和/或预防特定的肠细菌和病毒感染。但是，将HMOS转化为药物或营养物质的道路已被人牛奶中的纯少量寡糖所阻断，足以进行科学研究，以及临床前耐受性和安全性研究，以及用于胃肠道病原体的人群中的安全性和临床测试。因此，该拟议的研究计划旨在开发实用过程，以在多克到千克量表上生产HMO。由于反复报道，与非2连接的烟糖基醇相比，两种连接的葡萄糖基醇表现出更多的抗菌活性，因此我们将选择五种五种2链接的岩藻糖基醇，例如2'-FL，例如2'-FL，LNF-I，LNF-I，LNF-I，2h- antigen，ldfh-i和我们的主要目标。此外，非2连接的五氯乙糖LNF-II和LNF-III将为我们提供确认观察到较高抗菌活性的2-链接岩藻糖基醇的较高活性。此外，非关糖基化的寡糖LNT和LNNT将提供控制实验，以评估岩藻糖在寡糖中的影响。在过去的14年中，王实验室一直在开发酶促寡糖的合成。我们已经发明并进一步开发了用于大规模寡糖生产的“超级小球”和“ Superbug”技术。寡糖合成的最有效方法是遵循天然碳水化合物生物合成途径，在这些途径中，使用单个糖核苷酸作为基础，通过特定的糖基转移酶将寡糖组装在一起。这些构件本身是通过一系列生物合成酶从单个单糖中进行生物合成和回收的。对于寡糖的中小型合成，Wang通过将所有必要的生物合成酶固定在所谓的“ SuperBeads”上，从而开发了简单的固相合成系统。这些珠子起着稳定且多功能的合成试剂的作用，可用于合成无细胞系统中的各种糖缀合物。对于大规模生产，Wang“ Superbug”基本上将整个天然生物合成途径转移到大肠杆菌菌株中。该方法包括将每个酶沿生物合成途径克隆，并将这些酶的基因连接在一起以产生人工基因簇。然后，使用这种基因簇转化的重组大肠杆菌通过发酵和纯化来产生寡糖。因此，将在该程序中使用“超级小球”和“超级咬合”方法来生产9种寡糖。具体来说，有四个目标：目标I：通过固定多种酶（SuperBeads）固定HMO的生产。这涉及研究必要的微生物糖基转移酶，开发UDP-GLCNAC的超级蛋白酶，UDP-GAL和GDP-FUC的产生，以及将糖基转移酶与糖核苷酸产生的糖基转移酶的组合结合在一起，以产生寡糖。 AIM II：由重组大肠杆菌（Superbug）生产HMO，涉及将这些HMO的生物合成途径组合为一个或几个重组大肠杆菌菌株。 AIM III：GRA（通常被认为是安全的）酵母细胞生产HMO。这个新系统将为HMOS合成提供更安全的生产系统。 AIM IV：通过系统的生物医学和微生物组方法与其他专业实验室合作（也是在我们自己的实验室中）通过系统的生物医学和微生物组方法来表征寡糖，并且在该项目中产生的多克或千克量表中性人牛奶寡糖的优势。 It is expected that the biosynthetic technology developed in Aim I - III will be transferred to biotech company(s) (such as the biotech startup Carbogene USA LLC which specializes in large scale oligosaccharide production) and GMP processes will be developed to produce the oligosaccharides in quantities large enough for preclinical studies of tolerance and safety, as well as for safety, dose-ranging, and efficacy trials in infants以及暴露于胃肠道病原体的高风险的孩子。