A Cell-Free Biosynthesis Platform for Modular Construction of Protein Glycosylation Pathways

Cell-free systems are emerging as a robust technology to characterize, design, and optimize proteins and biosynthetic pathways with unprecedented control and throughput. However, the production and testing proteins with post-translational-modifications (PTMs) in vitro remains difficult. Protein glycosylation (the attachment of oligosaccharides to amino acid sidechains by glycosyltransferase enzymes) is among the most abundant PTMs, is present in 70% of protein therapeutics, and is known to endow protein therapeutics with beneficial properties¹. Despite their importance, the membrane-bound and complex nature of most glycosylation systems has impeded the development of biosynthetic pathways to produce glycoproteins outside of their native hosts, particularly in vitro.

Here we take a modular, unit-operations approach to develop a versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, crude Escherichia coli lysates are selectively enriched with individual glycosyltransferases by cell-free protein synthesis and then mixed-and-matched to construct multienzyme glycosylation pathways. Importantly, GlycoPRIME uses a soluble bacterial N-glycosyltransferase to install a glucose primer that is elaborated to therapeutically-relevant glycosylation motifs. This soluble system enables the first entirely in vitro glycoprotein production method that does require the synthesis of membrane-associated glycosylation components in living cells, obviating the need to make new genetic constructs and strain lines for each enzyme combination. We demonstrate GlycoPRIME by screening 37 putative protein glycosylation pathways in vitro. These pathways provide new biosynthetic routes to 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We used selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant glycan in a one-pot cell-free system and human antibody constant regions with minimal sialic acid glycan in glycoengineered E. coli. We anticipate that these methods and pathways will make possible new applications in cell-free synthesis and glycoengineering.

¹Sethurman & Stadheim, Current Opinion in Biotechnology, 2006