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).

Ribosome display is a powerful in vitro method for selection and directed evolution of proteins expressed from combinatorial libraries. However, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this gap, we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (N-linked) glycoproteins in conformations amenable to downstream functional and glycostructural interrogation. The ability to generate glycosylated ribosome− nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free Nglycoprotein synthesis. This integration enabled a first-in-kind method for ribosome stalling of target proteins modified efficiently and site-specifically with different N-glycan structures. Moreover, the observation that encoding mRNAs remained stably attached to ribosomes provides evidence of a genotype−glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N-glycoproteins with advantageous biological and biophysical properties.

Ribosome display is a powerful in vitro method for the selection and directed evolution of proteins expressed from combinatorial libraries. However, because ribosome display is typically performed with standard in vitro translation reagents, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this technological gap, here we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (N-linked) glycoproteins in conformations amenable to downstream functional and glyco-structural interrogation. The ability to generate glycosylated ribosome-nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free synthesis of (N-glycoproteins. This integration yielded a novel capability for translating and displaying target proteins modified efficiently and site-specifically with different (N-glycan structures. Moreover, the encoding mRNAs remained stably attached to stalled ribosomes both before and after biopanning, thereby providing the genotype-glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of (N-linked glycoproteins with advantageous biological and biophysical properties.