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: 8322023
• 负责人: Peng George Wang
• 金额: $ 30.9万
• 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/8322023
• 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，母乳低聚糖（HMO）一直被认为在母乳喂养的婴儿特定肠道菌群的发育中发挥着作用。如今，人们知道它们也是细菌粘附到上皮表面（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，在那里它们充当细菌代谢的底物。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其所提出的“抗感染作用”。这一作用的实现得益于它们能够抑制细菌与上皮表面的粘附，从而通过直接和间接机制对胃肠道、呼吸道和泌尿生殖道感染发挥重要的保护作用。因此，HMO 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，由于缺乏来自母乳的纯单组分低聚糖，其数量不足以进行科学研究、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测试，因此将 HMO 转化为药物或营养物质的道路受到阻碍。因此，该研究计划旨在开发生产数克至公斤级 HMO 的实用工艺。由于多次报道2-连接岩藻糖基低聚糖比非2-连接岩藻糖基低聚糖表现出更强的抗菌活性，因此我们将选择2'-FL、LNF-I、2H-抗原、LDFH-I和Ley等5种2-连接岩藻糖基低聚糖作为我们的主要目标。此外，非2-连接岩藻糖基低聚糖LNF-II和LNF-III将为我们提供机会来确认观察到2-连接岩藻糖基低聚糖具有更高的抗菌活性。此外，非岩藻糖基化低聚糖LNT和LNnT将提供对照实验来评估低聚糖中岩藻糖的作用。在过去的14年里，王实验室一直致力于酶促寡糖合成的开发。我们发明并进一步开发了用于大规模寡糖生产的“超级珠”和“超级细菌”技术。寡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，其中寡糖通过特定的糖基转移酶使用单个糖核苷酸作为构建块组装在一起。这些结构单元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 通过将所有必需的生物合成酶固定在所谓的“超级珠子”上，开发了简单的固相合成系统。这些珠子用作稳定且多功能的合成试剂，可用于在无细胞系统中合成各种糖缀合物。为了大规模生产，王的“超级细菌”本质上将整个天然生物合成途径转移到大肠杆菌菌株中。该方法包括沿着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。然后用这样的基因簇转化的重组大肠杆菌通过发酵和纯化来生产寡糖。因此，该计划将使用“超级珠子”和“超级细菌”方法来生产 9 种寡糖。具体来说，有四个目标： 目标 I：通过固定化多种酶（超级珠）生产 HMO。这涉及对必要的微生物糖基转移酶的研究，开发用于 UDP-GlcNAc、UDP-Gal 和 GDP-Fuc 生产的超级珠，以及将糖基转移酶与糖核苷酸生产相结合以生产寡糖。目标 II：通过重组大肠杆菌（Superbug）生产 HMO，其中涉及将这些 HMO 的生物合成途径组合到一种或多种重组大肠杆菌菌株中。目标 III：通过 GRAS（公认安全）酵母细胞生产 HMO。该新系统将为HMO合成提供更安全的生产系统。目标四：与其他专业实验室（也在我们自己的实验室）合作，通过系统的生物医学和微生物组方法表征寡糖，利用该项目生产的多克或公斤级中性母乳寡糖的优势。预计 Aim I - III 中开发的生物合成技术将转移给生物技术公司（例如专门从事大规模寡糖生产的生物技术初创公司 Carbogene USA LLC），并且将开发 GMP 工艺来生产足够数量的寡糖，用于临床前耐受性和安全性研究，以及婴儿的安全性、剂量范围和功效试验 以及接触胃肠道病原体高风险的儿童。