Abstract
Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.
Highlights
Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties
We demonstrate that pathways identified using GlycoPRIME can be transferred to cell-free and cellular biosynthesis systems by producing (i) a protein vaccine candidate with an adjuvanting α1-3-linked galactose (αGal) glycan[6,7,33] in a one-pot cell-free protein synthesis driven glycoprotein synthesis (CFPS-GpS) platform and (ii) the constant region (Fc) of the human immunoglobulin (IgG1) antibody in the E. coli cytoplasm with minimal sialic acid glycans known to improve in vivo pharmacokinetics[5,34]
By removing the need for linked oligosaccharides (LLOs) production in living cells, GlycoPRIME is the first system to enable the biosynthesis of a glycosylation target, GTs, and glycoproteins entirely in vitro
Summary
Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications. Essential biosynthetic pathways in eukaryotic organisms limit the diversity of glycan structures that can be engineered in those systems[9,13] Bacterial glycoengineering addresses these limitations by expressing heterologous glycosylation pathways in laboratory Escherichia coli strains that lack endogenous glycosylation enzymes[13,14]. E. coli-based cell-free protein synthesis (CFPS) systems can produce gram per liter titers of complex proteins in hours[26], enabling the rapid discovery, prototyping, and optimization of metabolic pathways without reengineering an organism for each pathway iteration[23,24,25]
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