In the last decade, the emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates 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 not withstanding, 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 describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked (N-linked) protein glycosylation. This technology leveraged a glyco-optimized chassis strain of Escherichia coli to source crude cell extracts that were selectively enriched with essential glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enabled a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins in just hours, requiring only the addition of DNA to enable rapid synthesis of 100% glycosylated product. The platform is flexible and modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs, including the eukaryotic trimannosyl core N-glycan (mannose3-N-acetylglucosamine2, Man3GlcNAc2) at higher conversions than those reported in vivo. Our CFGpS platform broadens the glycoengineering toolbox by providing a simplified and highly modular framework for investigating glycosylation outside the confines of a cell. As such, we anticipate that CFGpS will facilitate fundamental understanding in glycoscience and make possible new applications in on-demand biomanufacturing of glycoprotein products.

Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery

255th National Meeting (2018)

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