Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria

Abstract

A major objective of synthetic glycobiology is to re-engineer existing cellular glycosylation pathways from the top-down or construct non-natural ones from the bottom-up for new and useful purposes. Here, we developed a set of orthogonal pathways for eukaryotic O-linked protein glycosylation in Escherichia coli that installed the cancer-associated mucin-type glycans Tn, T, sialyl-Tn and sialyl-T onto serine residues in acceptor motifs derived from different human Oglycoproteins. These same glycoengineered bacteria were used to supply crude cell extracts enriched with glycosylation machinery that permitted cell-free construction of O-glycoproteins in a one-pot reaction. In addition, O-glycosylation-competent bacteria were able to generate an antigenically authentic Tn-MUC1 glycoform that exhibited reactivity with antibody 5E5, which specifically recognizes cancer-associated glycoforms of MUC1. We anticipate that the orthogonal glycoprotein biosynthesis pathways developed here will provide facile access to structurally diverse O-glycoforms for a range of important scientific and therapeutic applications.

Shotgun scanning glycomutagenesis: a simple and efficient strategy for constructing and characterizing neoglycoproteins

“…The notion that discrete glycan structures attached to the same site in a protein can have disparate effects is not unprecedented, having been documented for other glycoproteins (53) (54). Thus, in the future, it will of interest to extend SSGM for use with alternative glycan structures, including for example Man 3 GlcNAc 2 or other human N- and O- linked glycans that have been engineered in E. coli (32, 55, 56), so that the consequences of varying glycan structures at discrete locations can be systematically investigated.…”

“…Proteins samples (∼2 µg) were separated by SDS-PAGE gel and bands corresponding to glycosylated YebF-Im7 DQNAT were excised and subjected to in-gel digestion by trypsin followed by extraction of tryptic peptides essentially as described 50 . Gel slices were washed and then destained by treatment with a 1:1 mixture of acetonitrile (Fisher Chemical) and 50 mM aqueous NH 4 HCO 3 followed by treatment with 100% acetonitrile.…”

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells

“…Owing to its lack of any native protein glycosylation systems, E. coli offers a blank canvas on which prescribed, orthogonal glycosylation pathways can be assembled without concern over interference from endogenous glycoenzymes. Combined with its fast growth, ease of genetic manipulation, and the ability to express a wide range of recombinant proteins, E. coli cells equipped with glycosylation machinery are capable of biosynthesizing designer glycoproteins bearing various therapeutically-important glycan epitopes such as the eukaryotic core N-glycan Man 3 GlcNAc 2 (Valderrama-Rincon et al, 2012;Glasscock et al, 2018), bacterial O-polysaccharide (O-PS) antigen structures (Feldman et al, 2005), human blood group antigens (Hug et al, 2011;Shang et al, 2016), authentic human O-glycans (Du et al, 2018;Natarajan et al, 2020), and polysialic acid-containing glycans (Keys et al, 2017;Tytgat et al, 2019). Taken together, efforts in cellular glycoengineering have yielded a variety of expression platforms, both prokaryotic, and eukaryotic, for producing glycoproteins with chemically-defined carbohydrate structures.…”

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

“…With a long-term interest in synthesizing diverse glycoproteins in cell-free systems, we next ported an O-linked glycosylation system, known to have broad glycan specificity, into the CFGpS platform (60,67,68). We selected the O-OST PglO from Neisseria gonorrhoeae which accepts the C. jejuni heptasaccharide LLO as a donor but differs from PglB in acceptor sequence preferences (69).…”

“…We recently described cellfree glycoprotein synthesis (CFGpS), a platform for one-pot biomanufacturing of defined glycoproteins in extracts enriched with heterologous, membrane-bound glycosylation machinery (47). To date, CFGpS has been used to produce model glycoproteins, human glycoproteins, and conjugate vaccines (38,47,(58)(59)(60). Importantly, CFGpS reactions can be freeze-dried for shelfstability and rehydrated at the point of care to make effective vaccines (38).…”