生物分子的糖基化可以极大地改变其物理化学性质,细胞识别,亚细胞定位,和免疫原性。糖基化反应依赖于使用核苷酸二磷酸(NDP)-糖逐步添加糖。使这些底物容易获得将大大加速新的糖基化反应的表征,阐明其潜在的调节机制,和糖基化分子的产生。在这项工作中,我们设计了酿酒酵母异源表达核苷酸糖合酶,以从简单的起始材料获得各种各样的尿苷二磷酸(UDP)-糖(即,葡萄糖和半乳糖)。具体来说,激活的葡萄糖,尿苷二磷酸d-葡萄糖(UDP-d-Glc),可以转化为UDP-d-葡萄糖醛酸(UDP-d-GlcA),UDP-d-木糖(UDP-d-Xyl),UDP-d-apiose(UDP-d-Api),UDP-d-岩藻糖(UDP-d-Fuc),UDP-l-鼠李糖(UDP-l-Rha),UDP-l-阿拉伯吡喃糖(UDP-l-Arap),和使用植物和微生物来源的相应核苷酸糖合酶的UDP-1-阿拉伯呋喃糖(UDP-1-Araf)。我们还表达了编码补救途径的基因,以直接激活游离糖,从而实现UDP-1-Arap和UDP-1-Araf的生物合成。我们观察到下游产物UDP-d-Xyl对UDP-d-Glc6-脱氢酶(UGD)的强烈抑制作用,我们使用诱导系统(Tet-On)来延迟UDP-d-Xyl的生产以维持上游UDP-糖池。最后,我们使用含有生物合成途径的菌株进行了时程研究,以产生5种非天然UDP-糖,以阐明它们的时间依赖性相互转化以及UDP-d-Xyl在调节UDP-糖代谢中的作用.这些工程酵母菌株是一个强大的平台,(i)在功能上表征体内糖合酶,(ii)生物合成UDP糖的多样化选择,(iii)检查细胞内UDP-糖相互转化的调节,和(iv)产生糖基化的次级代谢产物和蛋白质。
Glycosylation of biomolecules can greatly alter their physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Glycosylation reactions rely on the stepwise addition of sugars using nucleotide diphosphate (NDP)-sugars. Making these substrates readily available will greatly accelerate the characterization of new glycosylation reactions, elucidation of their underlying regulation mechanisms, and production of glycosylated molecules. In this work, we engineered Saccharomyces cerevisiae to heterologously express nucleotide sugar synthases to access a wide variety of uridine diphosphate (UDP)-sugars from simple starting materials (i.e., glucose and galactose). Specifically, activated glucose, uridine diphosphate d-glucose (UDP-d-Glc), can be converted to UDP-d-glucuronic acid (UDP-d-GlcA), UDP-d-xylose (UDP-d-Xyl), UDP-d-apiose (UDP-d-Api), UDP-d-fucose (UDP-d-Fuc), UDP-l-rhamnose (UDP-l-Rha), UDP-l-arabinopyranose (UDP-l-Arap), and UDP-l-arabinofuranose (UDP-l-Araf) using the corresponding nucleotide sugar synthases of plant and microbial origins. We also expressed genes encoding the salvage pathway to directly activate free sugars to achieve the biosynthesis of UDP-l-Arap and UDP-l-Araf. We observed strong inhibition of UDP-d-Glc 6-dehydrogenase (UGD) by the downstream product UDP-d-Xyl, which we circumvented using an induction system (Tet-On) to delay the production of UDP-d-Xyl to maintain the upstream UDP-sugar pool. Finally, we performed a time-course study using strains containing the biosynthetic pathways to produce five non-native UDP-sugars to elucidate their time-dependent interconversion and the role of UDP-d-Xyl in regulating UDP-sugar metabolism. These engineered yeast strains are a robust platform to (i) functionally characterize sugar synthases in vivo, (ii) biosynthesize a diverse selection of UDP-sugars, (iii) examine the regulation of intracellular UDP-sugar interconversions, and (iv) produce glycosylated secondary metabolites and proteins.