Mix and Match Sweets: Modular, Coordinated Cell-Free Transcription, Translation, and Glycosylation of Proteins Using Selectively Enriched Escherichia coli Cell Lysates

Glycosylation, or the attachment of glycans (sugars) to proteins, is one of the most abundant post-translational modifications in nature and plays a pivotal role in protein folding, sorting, and activity. In molecular medicine, the compositions and structures of glycans on recombinant therapeutic glycoproteins are known to impact pharmacokinetics and drug activity. However, unlike DNA, RNA, and protein synthesis, protein glycosylation is not a template-driven process. Instead, glycans are assembled by coordinated expression of glycosyltransferase enzymes and attached to proteins by oligosaccharyltransferase (OST) enzymes. The complexity of this biosynthetic process results in inherent heterogeneity of glycans produced in living organisms. The inability to precisely control protein glycosylation in eukaryotic protein expression systems (e.g., yeast and Chinese hamster ovary cells) represents a key challenge in the fields of glycoprotein synthesis and glycoprotein therapeutics. Bacterial glycoengineering, or the expression of orthogonal glycosylation machinery in Escherichia coli, is an alternative strategy for production of proteins with controllable glycosylation. However, in vivo bacterial glycoengineering is limited by i) the need to maintain cell viability during glycoprotein synthesis while simultaneously overexpressing multiple non-native, membrane-bound glycosylation enzymes and ii) lengthy design-build-test cycles for novel glycosylation pathway engineering. To address these limitations, we have developed a bacterial cell-free glycoprotein synthesis (CFGpS) system with the potential to enable controllable, user-specified protein glycosylation. Uniquely, in our CFGpS system, i) all the biosynthetic machinery for protein synthesis and glycosylation is supplied by E. coli lysates and ii) transcription, translation, and glycosylation occur in an all-in-one in vitro reaction. We engineered glycosylation chassis strains that are optimized for glycosylation and produce ~1.5 g/L protein in cell-free protein synthesis, which represents a 50% increase in potential glycoprotein yields compared to state-of-the-art E. coli glycosylation chassis and recombinant protein expression strains. We then demonstrated that E. coli cell lysates can be selectively enriched with active glycan precursors and/or the OST from Campylobacter jejuni via overexpression of orthogonal genes in the chassis strain. Next, we used these lysates individually and as a mixture to carry out one-pot synthesis of glycoproteins. By mixing and matching lysates, we also used the CFGpS system to characterize the in vitro activity of four additional OSTs with varying sequence homology to the archetypal C. jejuni OST. The CFGpS platform is modular, flexible, and has promising applications as a high-throughput prototyping platform for glycosylation pathway design and synthesis of glycoproteins of biotechnological interest. This system promises to advance our understanding of bacterial glycosylation and aid development of new glycoengineering tools.