A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

TLDR

Glycosylation is essential for cellular function and therapeutic protein efficacy, yet engineering precise glycan structures remains a bottleneck. The authors present GlycoPRIME, a modular, cell‑free platform that assembles glycosylation pathways by rapid in‑vitro mixing and expression. GlycoPRIME constructs pathways by combining cell‑free synthesized glycosyltransferases that extend a glucose primer on proteins via an N‑glycosyltransferase. Using GlycoPRIME, 37 pathways were built to generate 23 unique glycan motifs—18 of which are novel—and the platform produced a vaccine candidate with an α‑galactose adjuvant and human antibody constant regions with minimal sialic acid motifs, underscoring its promise for glycoengineering.

Abstract

Glycosylation plays important roles in cellular function and endows protein therapeutic 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.