Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies

Abstract

Glycosylation of proteins profoundly impacts their physical and biological properties. Yet our ability to engineer novel glycoprotein structures remains limited. Established bacterial glycoengineering platforms require secretion of the acceptor protein to the periplasmic space and preassembly of the oligosaccharide substrate as a lipid-linked precursor, limiting access to protein and glycan substrates respectively. Here, we circumvent these bottlenecks by developing a facile glycoengineering platform that operates in the bacterial cytoplasm. The Glycoli platform leverages a recently discovered site-specific polypeptide glycosyltransferase together with variable glycosyltransferase modules to synthesize defined glycans, of bacterial or mammalian origin, directly onto recombinant proteins in the E. coli cytoplasm. We exploit the cytoplasmic localization of this glycoengineering platform to generate a variety of multivalent glycostructures, including self-assembling nanomaterials bearing hundreds of copies of the glycan epitope. This work establishes cytoplasmic glycoengineering as a powerful platform for producing glycoprotein structures with diverse future biomedical applications.

An enzyme-based protocol for cell-free synthesis of nature-identical capsular oligosaccharides from Actinobacillus pleuropneumoniae serotype 1

“…The resulting oligosaccharides can be readily coupled to the carrier protein and the risk of undesired chemical modification of the final antigen, which can occur during linker introduction and activation, is reduced. As an alternative to in vitro enzymatic synthesis, assembling entire glycoconjugates in engineered E. coli strains has emerged as a promising and potentially cost effective alternative for vaccine provision (46)(47)(48). However, the bacterial enzymatic machinery has to be altered and adapted to each serotype and it remains to be seen if these systems will be available for a broad variety of pathogens.…”

An Engineered Pathway for Production of Terminally Sialylated N-glycoproteins in the Periplasm of Escherichia coli

“…Keys and co-workers reported a biosynthetic pathway, based on NGTase, for sitespecific polysialylation of recombinant proteins with α-2,8-linked polysialic acid (polySia) chains in the E. coli cytoplasm, albeit with only approximately 20% of target molecules modified with polySia (Keys et al, 2017). Other workers have achieved approximately 62% glycosylation of N-glycoproteins with an α-2,3-linked, terminally sialylated N-glycan trisaccharide (Neu5Acα-2,3-Gal-β-1,4-GlcNAc-) (Tytgat et al, 2019). Scale-up of either approach is limited by the need to supply the expensive and unstable donor Neu5Ac, however.…”

Genetically Encoded, Multivalent Liquid Glycan Array (LiGA)

“…The biosynthesis of a DNA-encoded bona fide multivalent display of diverse glycan structures on phage remains an interesting bioengineering challenge. Recent advances in this area include biosynthesis of multivalent display of glycoproteins on mammalian cell surface 59 and multivalent display of biosynthesized glycans on DNA-free virus-like particles 60 . Unlike these and other display systems that employ the biosynthesis of glycans, LiGA decouples DNA encoding and glycan display from biosynthesis.…”