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.

中文翻译：

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工程酿酒酵母作为核苷酸糖的生物合成平台

生物分子的糖基化可以极大地改变其理化特性、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。使这些底物易于使用将大大加速新糖基化反应的表征、其潜在调节机制的阐明以及糖基化分子的产生。在这项工作中，我们对酿酒酵母进行了改造，使其异源表达核苷酸糖合酶，从而从简单的起始材料（即葡萄糖和半乳糖）中获取多种尿苷二磷酸（UDP）糖。具体来说，活化的葡萄糖、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-芹菜糖(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 -Ara p) )和UDP- l-阿拉伯呋喃糖(UDP- l -Ara f )，使用植物和微生物来源的相应核苷酸糖合酶。我们还表达了编码挽救途径的基因以直接激活游离糖以实现UDP- l -Ara p和UDP -l -Ara f的生物合成。我们观察到下游产物 UDP- d -Xyl 对 UDP- d -Glc 6-脱氢酶 (UGD)具有强烈抑制作用，我们使用诱导系统 (Tet-On) 来规避这种抑制，以延迟 UDP- d -Xyl 的产生，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一项时间过程研究，以产生五种非天然 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，可以（i）对体内糖合酶进行功能表征，（ii）生物合成多种选择的UDP-糖，（iii）检查细胞内UDP-糖相互转化的调节，以及（iv）产生糖基化次生代谢物和蛋白质。