Department of Chemical & Biomolecular Engineering, University of California, Berkeley, California 94720, United States.
California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, California 94720, United States.
ACS Synth Biol. 2024 Apr 19;13(4):1215-1224. doi: 10.1021/acssynbio.3c00666. Epub 2024 Mar 11.
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 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-Ara), and UDP-l-arabinofuranose (UDP-l-Ara) 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-Ara and UDP-l-Ara. 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 , (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.
生物分子的糖基化可以极大地改变它们的物理化学性质、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。使这些底物易于获得将极大地加速新糖基化反应的表征、阐明其潜在的调控机制以及糖基化分子的生产。在这项工作中，我们设计了 来异源表达核苷酸糖合酶，以便从简单的起始材料（即葡萄糖和半乳糖）中获取各种尿苷二磷酸 (UDP)-糖。具体来说，激活的葡萄糖、尿苷二磷酸 d-葡萄糖 (UDP-d-Glc) 可以转化为尿苷二磷酸 d-葡萄糖醛酸 (UDP-d-GlcA)、尿苷二磷酸 d-木糖 (UDP-d-Xyl)、尿苷二磷酸 d-阿洛酮糖 (UDP-d-Api)、尿苷二磷酸 d-岩藻糖 (UDP-d-Fuc)、尿苷二磷酸 l-鼠李糖 (UDP-l-Rha)、尿苷二磷酸 l-阿拉伯吡喃糖 (UDP-l-Ara) 和尿苷二磷酸 l-阿拉伯呋喃糖 (UDP-l-Ara) 使用来自植物和微生物来源的相应核苷酸糖合酶。我们还表达了编码补救途径的基因，以直接激活游离糖来实现 UDP-l-Ara 和 UDP-l-Ara 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，我们通过诱导系统 (Tet-On) 来避免这种情况，该系统延迟 UDP-d-Xyl 的产生以维持上游 UDP-糖库。最后，我们使用含有生物合成途径的菌株进行了时间过程研究，以生产五种非天然 UDP-糖，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) 功能表征糖合酶，(ii) 生物合成各种选择的 UDP-糖，(iii) 研究细胞内 UDP-糖相互转化的调节，以及 (iv) 生产糖基化的次生代谢物和蛋白质。
