Glycans and glycosylated biomolecules are directly involved in almost every biological process as well as the etiology of most major diseases. Hence, glycoscience knowledge is essential to efforts aimed at addressing fundamental challenges in understanding and improving human health, protecting the environment and enhancing energy security, and developing renewable and sustainable resources that can serve as the source of next-generation materials. While much progress has been made, there remains an urgent need for new tools that can overexpress structurally uniform glycans and glycoconjugates in the quantities needed for characterization and that can be used to mechanistically dissect the enzymatic reactions and multi-enzyme assembly lines that promote their construction. To address this technology gap, cell-free synthetic glycobiology has emerged as a simplified and highly modular framework to investigate, prototype, and engineer pathways for glycan biosynthesis and biomolecule glycosylation outside the confines of living cells. From nucleotide sugars to complex glycoproteins, we summarize here recent efforts that harness the power of cell-free approaches to design, build, test, and utilize glyco-enzyme reaction networks that produce desired glycomolecules in a predictable and controllable manner. We also highlight novel cell-free methods for shedding light on poorly understood aspects of diverse glycosylation processes and engineering these processes toward desired outcomes. Taken together, cell-free synthetic glycobiology represents a promising set of tools and techniques for accelerating basic glycoscience research (e.g., deciphering the “glycan code”) and its application (e.g., biomanufacturing high-value glycomolecules on demand).

聚糖和糖基化生物分子直接参与几乎所有生物过程以及大多数主要疾病的病因学。因此，糖科学知识对于解决理解和改善人类健康、保护环境和加强能源安全以及开发可作为下一代材料来源的可再生和可持续资源方面的基本挑战的努力至关重要。尽管已经取得了很大进展，但仍然迫切需要新的工具，这些工具可以以表征所需的数量过度表达结构均一的聚糖和糖缀合物，并可用于机械地剖析酶促反应和促进其构建的多酶装配线。为了解决这一技术差距，无细胞合成糖生物学已经成为一种简化且高度模块化的框架，用于研究、原型设计和设计活细胞范围外的聚糖生物合成和生物分子糖基化途径。从核苷酸糖到复杂的糖蛋白，我们在此总结了最近的努力，这些努力利用无细胞方法的力量来设计、构建、测试和利用糖酶反应网络，以可预测和可控的方式产生所需的糖分子。我们还重点介绍了新颖的无细胞方法，以阐明各种糖基化过程中人们知之甚少的方面，并设计这些过程以获得所需的结果。总而言之，无细胞合成糖生物学代表了一套有前途的工具和技术，可用于加速基础糖科学研究（例如，破译“聚糖代码”）及其应用（例如，按需生物制造高价值糖分子）。