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

Weston Kightlinger, Katherine E. Duncker, Ashvita Ramesh, Ariel H. Thames, Aravind Natarajan, Jessica C. Stark, Allen Yang, Liang Lin, Milan Mrksich, Matthew P. DeLisa and Michael C. Jewett ()
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Weston Kightlinger: Northwestern University
Katherine E. Duncker: Northwestern University
Ashvita Ramesh: Northwestern University
Ariel H. Thames: Northwestern University
Aravind Natarajan: Cornell University
Jessica C. Stark: Northwestern University
Allen Yang: Northwestern University
Liang Lin: Northwestern University
Milan Mrksich: Northwestern University
Matthew P. DeLisa: Cornell University
Michael C. Jewett: Northwestern University

Nature Communications, 2019, vol. 10, issue 1, 1-13

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-12024-9

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