Oligosaccharides to Prevent Infectious Diarrhea

描述（由申请人提供）：病原体附着在宿主细胞上是感染的第一步，通常由特定的分子相互作用[1][2]介导。致病性弯曲杆菌、霍乱弧菌、肠致病性大肠杆菌（EPEC）、肠出血性大肠杆菌（EHEC）和致病性诺瓦克病毒菌株是人类感染性腹泻[3]的主要细菌和病毒病因，它们通过结合1（1,2）聚焦的细胞受体[4][5]粘附在肠道上皮表面。人母乳[6][7]中含有丰富的1（1,2）聚焦聚糖，在体外和体内均已被证明能有效地阻止这些病原体[4][5]的结合和感染。因此，这些分子代表了一类具有预防传染性腹泻潜力的新型药物，这种疾病每年在全球造成200多万人死亡。然而，生产足够数量的1（1,2）聚焦聚糖作为抗感染药物来影响全球腹泻发病率仍然是一个重大挑战。化学合成是可能的，但受到立体特异性问题、产品杂质和高总成本的限制。体外酶合成也是可能的，但由于需要昂贵的核苷酸-糖前体而受到限制。糖syn公司的主要目标是开发通过微生物发酵廉价批量生产1（1,2）浓缩聚糖的方法，并设想了三类潜在的抗感染产品：1)纯化的1（1,2）浓缩寡糖，2)在细胞表面表达1（1,2）浓缩聚糖的酵母菌株，以及3)纯化的1（1,2）浓缩糖蛋白。[[]本申请中概述的研究目标是在乳酵母中生产这些产品类别中的第一个例子，即纯化的1（1,2）聚焦点低聚糖，22-聚焦点乳糖（22- fl），其量足以在体外和体内感染模型中测试该分子作为单一药物的功效。通过内源基因操作和引入编码所需活性的外源基因，对克鲁维酵母菌的聚糖合成途径进行了工程设计。具体来说，K.lactis将被改造成合成关键的前体糖，GDP- focus，随后在细胞质中产生22- focus乳糖。随后的目标是提高从K.lactis中回收的2<-岩藻糖基乳糖的产量，使其接近商业化所需的水平。为了实现这一目标，将通过控制细胞合成酶和前体池的水平来增加2<聚焦乳糖的产量，平衡2；-生物反应器条件下的fl产量与总体细胞活力和生长性能。

DESCRIPTION (provided by applicant): Adherence of pathogens to their host cells is the obligatory first step of infection and is frequently mediated by specific molecular interactions [1][2]. Virulent Campylobacter species, Vibrio cholerae, enteropathogenic E.coli (EPEC), enterohemorrhagic E.coli (EHEC) and pathogenic strains of Norwalk virus, the leading bacterial and viral causes of human infectious diarrhea [3], adhere to gut epithelial surfaces through binding to 1(1,2) fucosylated cellular receptors[4][5]. 1(1,2) fucosylated glycans, which are abundant in human breast milk[6][7], have been shown both in vitro and in vivo effectively to prevent binding and infection by these pathogens[4][5]. These molecules therefore represent a new class of agent with potential to prevent infectious diarrhea, a condition that is the cause annually of over 2 million deaths worldwide [8]. However the production of 1(1,2) fucosylated glycans as anti-infective agents in sufficient quantities to impact global diarrhea incidence remains a significant challenge. Chemical syntheses are possible, but are limited by stereo-specificity issues, product impurities, and high overall cost[9][10][11]. In vitro enzymatic syntheses are also possible but are limited by a requirement for expensive nucleotide-sugar precursors. Glycosyn Inc.<s broad goal is to develop ways to manufacture 1(1,2) fucosylated glycans cheaply and in bulk through microbial fermentation, and three classes of potential anti-infective products are envisaged: 1) purified 1(1,2) fucosylated oligosaccharides, 2) yeast strains expressing 1(1,2) fucosylated glycans on their cell surface, and 3) purified 1(1,2) fucosylated glycoproteins. [[[The goal of the studies outlined in this application are to produce in the dairy yeast Kluyveromyces lactis an example of the first of these product classes, namely a purified 1(1,2) fucosylated oligosaccharide, 22-fucosyllactose (22-FL), in sufficient amounts to test this molecule<s efficacy as a single agent in in vitro and in vivo infection models. Glycan synthetic pathways in Kluyveromyces lactis will be engineered through a combination of endogenous gene manipulation and the introduction of heterologous genes encoding desired activities. Specifically, K.lactis will be engineered to synthesize the key precursor sugar, GDP-fucose, and subsequently to make 22-fucosyllactose in the cell cytoplasm. A subsequent goal is to increase the yield of 2<-fucolsyllactose recovered from K.lactis to approach levels that will be required for commercialization. To achieve this the production of 2<fucosyllactose will be increased by manipulating cellular levels of synthetic enzymes and precursor pools, balancing 2;-FL production with overall cell viability and growth performance under bioreactor conditions.