Microbial production of fucosylated human milk oligosaccharides

项目摘要 微生物法生产岩藻糖基化人乳低聚糖 本项目旨在建立高效、特异的人乳微生物生产工艺 低聚糖（HMO）。HMO是调节新生儿健康的有效生物活性化合物， 作为治疗成人疾病的潜在药物开发的兴趣。健康维护组织有200多人 在初乳中存在20-23 g/L的化合物，在成熟乳中存在12-14 g/L的化合物。不像它们的共同前身 乳糖、HMO不能被人类婴儿消化，而是通过有效地 抗微生物剂和抗病毒药、益生元和炎症免疫细胞反应级联的调节剂。这些 和其他潜在的好处，使他们有吸引力的研究目标，预防或治疗疾病， 儿童和成人都有。β1−3-连接半乳糖苷Galβ3GlcNAcβOR，称为1型聚糖， 在100多种健康维护组织中发现的主要健康维护组织成分。在20个HMO核心结构中， 鉴定出11个含有至少一个1型聚糖末端分支。乳糖-N-四糖（LNT，Galβ3GlcNAcβ3Lac） 是最简单的1型聚糖HMO。LNT及其岩藻糖基化衍生物是最丰富的HMO之一。 虽然1型聚糖结构在人乳中占主导地位，但它们的丰度较低（并且有时 在其他哺乳动物的乳汁中完全不存在。研究1型糖尿病个体的生物学功能 含聚糖的HMO及其作为益生元和抗菌剂的潜在应用需要获得 足够量的这些结构确定的化合物。这些分子的潜力， 从自然来源获取，以及难以大规模分离单个HMO进行研究和应用 推动了新生产方法的发展。化学和体外酶促合成 基于当前方法的HMO对于工业规模合成而言预期是昂贵的。全细胞生物催化剂 正在成为替代性的自我调节生产平台，具有显著的潜力， HMO的生产成本。短链，线性和小的单岩藻糖基化HMO已全部产生 细胞生物催化剂，但尚未探索具有更高复杂性的结构。在这个项目中，我们将 建立用于生产岩藻糖基化HMO的策略，包括乳糖-N-岩藻五糖II（LNFP II）、乳糖-N- 在活工程菌中从乳糖和L-岩藻糖中分离岩藻五糖I（LNFP I）和乳糖-N-二岩藻糖基己糖I（LNDFH I） 大肠杆菌细胞。值得注意的是，我们将开发一种创新的方法来控制秩序和网站的 在全细胞系统中进行糖基化，为将来微生物生产其他复杂的HMO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.