Investigation on Oligosaccharides as Antimicrobial and Prebiotics

描述(由申请人提供)：多年来，人类乳寡糖(HMOS)一直被认为在母乳喂养婴儿的特定肠道菌群发育中发挥作用。现已知，它们也是细菌黏附上皮细胞表面(感染过程的初始阶段)的有效抑制物。低聚糖不会在较高的小肠中被水解，而是完整地到达大肠，在那里它们作为细菌新陈代谢的底物。因此，HMO被认为是人类乳汁中的“膳食纤维”。低聚糖的另一个特点是它们被认为具有“抗感染作用”。这一作用的实现要归功于它们能够抑制细菌与上皮表面的黏附，从而通过直接和间接机制发挥重要的保护作用，防止胃肠道、呼吸道和泌尿生殖道的感染。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，由于缺乏数量足以用于科学研究、临床前耐受性和安全性研究以及暴露于胃肠道病原体的人群的安全性和临床测试的纯、单组分低聚糖，将HMO转化为药物或营养物质的道路受阻。因此，这项拟议的研究计划旨在开发生产多克到千克规模的HMO的实用工艺。鉴于2‘-FL、LNF-I、2H-抗原、LDFH-I和LEY等5种2’-连接岩藻糖低聚糖的抗菌活性不断被报道，因此我们选择2‘-FL、LNF-I、2H-抗原、LDFH-I和LEY等5种2-连接岩藻糖低聚糖作为研究的主要靶点。此外，非2-连接岩藻糖低聚糖LNF-II和LNF-III将为我们提供机会来证实2-连接岩藻糖低聚糖具有更高的抗菌活性。此外，非岩藻糖化低聚糖LNT和LNnT将提供对照实验，以评估岩藻糖在低聚糖中的作用。在过去的14年里，王的实验室一直在开发酶促低聚糖的合成。我们发明并进一步开发了规模化低聚糖生产的“超级珠子”和“超级细菌”技术。低聚糖合成的最有效方法是遵循天然的碳水化合物生物合成途径，即寡糖通过特定的糖基转移酶以单个糖核苷酸为基础组装在一起。这些构件本身就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，王开发了简单的固相合成系统，通过将所有必要的生物合成酶固定在所谓的“超级珠子”上。这些小球是一种稳定的、多功能的合成试剂，可用于在无细胞体系中合成各种糖偶联物。在大规模生产中，王的“超级细菌”基本上将整个自然生物合成途径转移到了大肠杆菌菌株中。该方法包括克隆生物合成途径中的每一种酶，并将这些酶的基因连接在一起，产生一个人造基因簇。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生产低聚糖。因此，在这个项目中将使用“超级珠子”和“超级细菌”的方法来生产这9种寡糖。具体地说，有四个目标：目标一：通过固定化多种酶(超级珠)生产HMO。这包括对必需的微生物糖基转移酶的研究，开发用于UDP-GlcNAc、UDP-Gal和GDP-Fuc生产的超球，以及将糖基转移酶与糖核苷酸生产相结合来生产寡糖。目的II：利用重组大肠杆菌(Superbug)生产HMOS，它涉及将这些HMOS的生物合成途径组合成一个或几个重组大肠杆菌菌株。目的III：用GRAS(公认为安全的)酵母细胞生产HMOS。这一新系统将为HMOS的合成提供更安全的生产系统。目的四：与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，利用本项目生产的多克或千克规模的中性人乳低聚糖，对低聚糖进行表征。预计在AIM I-III中开发的生物合成技术将转移到生物技术公司(S)(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)，并将开发转基因低聚糖工艺，以生产足够数量的寡糖，用于耐受性和安全性的临床前研究，以及用于高风险暴露于胃肠道病原体的婴儿和儿童的安全性、剂量范围和疗效试验。

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.