ENGINEERING A SYNTHETIC GLYCOBIOLOGY TOOLKIT FOR CUSTOMIZABLE CELL-FREE GLYCOSYLATION PATHWAYS
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Glycobiology is the study of glycans and glycan-conjugated molecules that regulate diverse biological processes and serve as essential biomarkers and therapeutic targets. Despite recent advances in synthetic glycobiology, such as the development of glycovaccines and glycotherapeutics in E. coli, significant technical bottleneck still remains largely due to the complex and unpredictable nature of glycosylation pathways. In particular, intracellular glycan biosynthesis is difficult to control due to enzyme competition and the limited availability of functional glycosyltransferases, many of which are membrane-bound and poorly expressed in the recombinant systems.To address these challenges, we engineered a modular, cell-free synthetic glycobiology toolkit to enable customizable glycan assembly and conjugation. Central to this effort was the application of the SIMPLEx strategy (solubilization of Integral Membrane Proteins with high-levels of expression) to render difficult-to-express glycosyltransferases (GTs) water-soluble and functionally active. We successfully solubilized ~98 GTs, predominantly of human origin, and demonstrated in vitro reconstitution of glycan synthesis using these enzymes as efficient biocatalysts. We next applied this strategy to solubilize the oligosaccharyltransferase (OST), a GT-C fold enzyme with 13 transmembrane helices that catalyzes en bloc N-linked protein glycosylation. By combining an optimized SIMPLEx architecture with AI-driven de novo protein design strategy called WRAP (Water-soluble RFdiffused Amphipathic Protein), we created two functional, water-soluble OST variants. These approaches not only simplify OST preparation but also significantly reduce associated time and costs. Finally, we are leveraging these solubilized components to engineer a fully cytoplasmic N-linked glycosylation pathway in E. coli, bypassing the native periplasmic bottlenecks. Together, these efforts lay the foundation for a versatile synthetic glycotoolkit that enables the rational design of glycosylation pathways for both research and therapeutic development.