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）：多年来，人们一直认为人乳寡糖（HMOs）在母乳喂养婴儿特定肠道菌群的发育中起作用。如今，已知它们也是细菌粘附上皮表面（感染过程的初始阶段）的有效抑制剂。低聚糖在小肠上部不被水解，并完整地到达大肠，在那里它们作为细菌代谢的底物。因此，hmo被认为是母乳中的“膳食纤维”。低聚糖的另一个特点是它们的“抗感染作用”。这一作用是由于它们能够抑制细菌对上皮表面的粘附，从而通过直接和间接的机制在胃肠道、呼吸道和泌尿生殖道感染中发挥重要的保护作用。因此，HMOs具有抗菌活性，可用于治疗和/或预防特定的肠道细菌和病毒感染。然而，将HMOs转化为药物或营养物质的道路一直受到阻碍，因为缺乏从人乳中提取的纯单组分低聚糖，其数量足以进行科学调查，以及临床前耐受性和安全性研究，以及在暴露于胃肠道病原体的人群中进行安全性和临床试验。因此，这个拟议的研究计划旨在开发实用的工艺，以生产数克到公斤级的hmo。由于已有文献多次报道，2-linked focusyloligosaccharides比非2-linked focusyloligosaccharides表现出更强的抗菌活性，因此我们将选择5种2-linked focusyloligosaccharides，如2'-FL, LNF-I, 2H-antigen， LDFH-I和Ley作为我们的主要靶点。此外，非2链聚酰基低聚糖LNF-II和LNF-III将为我们提供机会证实2链聚酰基低聚糖具有更高的抗菌活性。此外，非聚焦低聚糖LNT和LNnT将提供对照实验来评估低聚糖的聚焦效果。在过去的14年里，王实验室一直致力于酶促低聚糖合成。我们发明并进一步发展了大规模生产低聚糖的“超级微珠”和“超级细菌”技术。低聚糖合成最有效的方法是遵循天然碳水化合物生物合成途径，通过特定的糖基转移酶使用单个糖核苷酸作为构建块将低聚糖组装在一起。这些构建模块本身是通过一系列生物合成酶从单个单糖中生物合成和回收的。对于中小规模的低聚糖合成，Wang通过将所有必要的生物合成酶固定在所谓的“超级微珠”上，开发了简单的固相合成系统。这些小珠作为稳定和通用的合成试剂，可用于在无细胞系统中合成各种糖缀合物。对于大规模生产，Wang的“超级细菌”基本上将整个天然生物合成途径转移到大肠杆菌菌株中。该方法包括沿生物合成途径克隆每种酶，并将这些酶的基因连接在一起以产生人工基因簇。用这种基因簇转化的重组大肠杆菌然后通过发酵和纯化来生产低聚糖。因此，“超级微珠”和“超级细菌”的方法将在这个程序中使用，以产生9低聚糖。具体来说，有四个目标：目标1：通过固定化多种酶（超珠）来生产HMOs。这包括研究必要的微生物糖基转移酶，开发用于生产UDP-GlcNAc， UDP-Gal和GDP-Fuc的超珠，以及将糖基转移酶与糖核苷酸生产相结合以生产低聚糖。目的II：重组大肠杆菌（超级细菌）生产HMOs，包括将这些HMOs的生物合成途径结合到一个或几个重组大肠杆菌菌株中。目的三：用GRAS（公认安全）酵母细胞生产HMOs。该系统将为HMOs合成提供更安全的生产系统。目标四：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，也在我们自己的实验室中，利用本项目生产的多克或公斤级中性人乳低聚糖的优势，对低聚糖进行表征。预计Aim I - III开发的生物合成技术将转让给生物技术公司（如专门从事大规模低聚糖生产的生物技术初创公司Carbogene USA LLC），并开发GMP流程以生产足够数量的低聚糖，用于耐受性和安全性的临床前研究，以及安全性，剂量范围，对暴露于胃肠道病原体高风险的婴儿和儿童进行疗效试验。