Microbial production of fucosylated human milk oligosaccharides

项目摘要 微生物法生产岩藻糖化人乳低聚糖 这项拟议的项目旨在建立高效和特定的母乳微生物生产工艺。 低聚糖(HMOS)。HMOS是一种有效的生物活性化合物，可调节新生儿健康，并具有 有兴趣开发为治疗成人疾病的潜在药物。HMO是一个超过200人的班级 化合物在初乳中含量为20-23g/L，在成熟乳中含量为12-14g/L。与它们共同的前身不同 乳糖，HMOS是人类婴儿不能消化的，相反，它通过有效地改善新生儿的健康 抗菌素和抗病毒药物、益生元和炎症免疫细胞反应的调节剂级联反应。这些 和其他HMO的潜在好处使它们成为预防或治疗疾病的有吸引力的研究目标 无论是儿童还是成人。β1−3-连接半乳糖苷Galβ3GlcNAcβOR，称为1型多糖，是 在100多个HMO中发现了主要的HMO成分。在20个HMO核心结构中， 经鉴定，11个含有至少一个类型1糖链末端支链。N-内酯-四糖(LnT，Galβ3GlcNAcβ3Lac) 是最简单的1型糖链HMO。LNT及其岩藻糖化衍生物是最丰富的HMO之一。 虽然1型糖链结构在母乳中占主导地位，但它们的含量较少(有时 完全不存在的)在其他哺乳动物的乳汁中。研究个体1型的生物学功能 含有葡聚糖的HMO及其作为益生素和抗菌剂的潜在应用需要获得 足够数量的这些结构定义的化合物。这些分子的潜力，它们的有限 从自然来源获取，难以大规模分离单个卫生保健组织进行研究和应用 推动了新生产方式的发展。化学合成和体外酶促合成 基于目前方法的HMO对于工业规模的合成来说预计将是昂贵的。全细胞生物催化剂 正在成为替代的自律生产平台，具有极大的潜力来减少 HMO的生产成本。已整体生产了短链、线形和小分子单糖HMOS。 细胞生物催化剂，但具有更高复杂性的结构尚未被探索。在这个拟议的项目中，我们将 建立了一种生产岩藻糖化HMO的策略，包括LNFP II、LNFP II和LNFP II 由乳糖和L岩藻糖合成的岩藻五糖I(LNFP I)和N-二岩藻糖基六糖I(LNDFH I) 大肠埃希菌细胞。值得注意的是，我们将开发一种创新的方法来控制 在整个细胞系统中进行糖基化，为未来微生物生产其他复杂的HMOS奠定基础。

Project Summary Microbial production of fucosylated human milk oligosaccharides This proposed project aims to establish efficient and specific microbial production processes for human milk oligosaccharides (HMOs). HMOs are potent bioactive compounds that modulate neonatal health and are of interest for development as potential drug treatments for adult diseases. HMOs are a class of over 200 compounds present at 20-23 g/L in colostrum and 12-14 g/L in mature milk. Unlike their common precursor lactose, HMOs are indigestible by human infants and instead improve neonatal health by serving as effective antimicrobials and antivirals, prebiotics, and regulators of inflammatory immune cell-response cascades. These and other potential benefits of HMOs make them attractive targets of study for preventing or treating diseases in both children and adults. β1−3-Linked galactosides Galβ3GlcNAcβOR, which are called Type 1 glycans, are major HMO components found in more than 100 HMOs. Among the 20 HMO core structures that have been identified, 11 contain at least one Type 1 glycan-terminated branch. Lacto-N-tetraose (LNT, Galβ3GlcNAcβ3Lac) is the simplest Type 1 glycan HMO. LNT and its fucosylated derivatives are among the most abundant HMOs. While Type 1 glycan structures are predominant in human milk, they are less abundant (and sometimes completely absent) in the milk of other mammals. Investigating the biological functions of individual Type 1 glycan-containing HMOs and their potential applications as prebiotics and antimicrobials requires access to sufficient quantities of these structurally defined compounds. The potential of these molecules, their limited access from natural sources, and difficulty in large-scale isolation of individual HMOs for studies and applications have motivated the development of novel production methods. Chemical and in vitro enzymatic syntheses of HMOs based on current methods are expected to be costly for industrial-scale synthesis. Whole cell biocatalysts are emerging as alternative self-regulating production platforms that have significant potential to reduce the production cost of HMOs. Short-chain, linear and small monofucosylated HMOs have been produced in whole cell biocatalysts, but structures with higher complexity have not been explored. In this proposed project, we will establish a strategy for producing fucosylated HMOs including lacto-N-fucopentaose II (LNFP II), lacto-N- fucopentaose I (LNFP I) and lacto-N-difucosylhexaose I (LNDFH I) from lactose and L-fucose in live engineered Escherichia coli cells. Notably, we will develop an innovative method to control the order and the site of glycosylation in whole cell systems to lay the groundwork for future microbial production of other complex HMOs.