Bacterial Cell-Free Systems for Biosynthesis of Complex Glycoproteins and Glycoconjugates

The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycoprotein on demand, and to evolve enzymes, pathways, and host organisms that catalyze prescribed glycosylation reactions. Bacteria equipped with recombinant glycosylation pathways have the potential to both improve our fundamental understanding of the glycosylation process and generate structurally-defined glycoproteins for use as vaccines and therapeutics. These developments notwithstanding, cell-based production of homogeneous glycoproteins remains a significant challenge due to the complexity of this process in vivo, cell viability constraints, and the inability to control glycosylation components at precise ratios. To address these challenges, we develop a novel cell-free glycoprotein synthesis (CFGpS) system that seamlessly integrates protein biosynthesis with asparagine-linked (N-linked) protein glycosylation. This technology leveraged a glyco-optimized Escherichia coli strain to source crude extracts that were selectively enriched with glycosylation components, including oligosaccharyltransferases and lipid-linked oligosaccharides. The resulting extracts enabled a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The platform is flexible and modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs to synthesize therapeutically-relevant glycoproteins including biologically-active human erythropoietin. Building upon CFGpS, we further develop technology for in vitro bioconjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic E. coli. Upon rehydration, iVAX reactions synthesize clinically-relevant doses of bioconjugate vaccines against diverse bacterial pathogens including Franciscella tularensis subsp. tularensis (type A) strain Schu S4. Notably, cell-free derived glycoconjugate elicited F. tularensis polysaccharide-specific antibodies in mice at significantly higher levels compared to vaccines produced in bacteria. Our CFGpS and iVAX platforms broaden the glycoengineering toolbox by providing a simplified and highly modular framework for investigating glycosylation outside the confined cell. As such, we anticipate that our cell-free glycosylation systems will facilitate fundamental understanding in glycoscience and make possible new applications in on-demand biomanufacturing of glycoprotein and glycoconjugate products.