Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in bacteria

Belen Sotomayor; Thomas C Donahue; Sai Pooja Mahajan; May N Taw; Sophia W Hulbert; Erik J Bidstrup; D Natasha Owitipana; Alexandra Pang; Xu Yang; Souvik Ghosal; Christopher A Alabi; Parastoo Azadi; Jeffrey J Gray; Michael C Jewett; Lai-Xi Wang; Matthew P DeLisa

doi:10.1038/s41467-025-61440-7

Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in bacteria

Nat Commun. 2025 Jul 4;16(1):6152. doi: 10.1038/s41467-025-61440-7.

Authors

Belen Sotomayor¹, Thomas C Donahue^{1

2}, Sai Pooja Mahajan³, May N Taw⁴, Sophia W Hulbert⁵, Erik J Bidstrup¹, D Natasha Owitipana², Alexandra Pang¹, Xu Yang⁶, Souvik Ghosal⁷, Christopher A Alabi^{1

7}, Parastoo Azadi⁶, Jeffrey J Gray³, Michael C Jewett⁸, Lai-Xi Wang², Matthew P DeLisa^{9

10

11

12}

Affiliations

¹ Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA.
² Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, 20742, USA.
³ Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA.
⁴ Department of Microbiology, Cornell University, Ithaca, NY, 14853, USA.
⁵ Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY, 14853, USA.
⁶ Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA, 30602-4712, USA.
⁷ Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853, USA.
⁸ Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.
⁹ Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA. md255@cornell.edu.
¹⁰ Department of Microbiology, Cornell University, Ithaca, NY, 14853, USA. md255@cornell.edu.
¹¹ Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY, 14853, USA. md255@cornell.edu.
¹² Cornell Institute of Biotechnology, Cornell University, 130 Biotechnology Building, Ithaca, NY, 14853, USA. md255@cornell.edu.

Abstract

Human immunoglobulin G (IgG) antibodies are a major class of biotherapeutics and undergo N-linked glycosylation in their Fc domain, which is critical for immune functions and therapeutic activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. Previous attempts to engineer Escherichia coli for this purpose have met limited success due in part to the lack of oligosaccharyltransferase (OST) enzymes that can install N-glycans at the conserved N297 site in the Fc region. Here, we identify a single-subunit OST from Desulfovibrio marinus with relaxed substrate specificity that catalyzes glycosylation of native Fc acceptor sites. By chemoenzymatic remodeling the attached bacterial glycans to homogeneous, asialo complex-type G2 N-glycans, the E. coli-derived Fc binds human FcγRIIIa/CD16a, a key receptor for antibody-dependent cellular cytotoxicity (ADCC). Overall, the discovery of D. marinus OST provides previously unavailable biocatalytic capabilities and sets the stage for using E. coli to produce fully human antibodies.

MeSH terms

Bacterial Proteins* / genetics
Bacterial Proteins* / metabolism
Escherichia coli* / genetics
Escherichia coli* / metabolism
Glycosylation
Hexosyltransferases* / chemistry
Hexosyltransferases* / genetics
Hexosyltransferases* / metabolism
Humans
Immunoglobulin Fc Fragments / metabolism
Immunoglobulin G* / genetics
Immunoglobulin G* / metabolism
Membrane Proteins* / chemistry
Membrane Proteins* / genetics
Membrane Proteins* / metabolism
Polysaccharides / metabolism
Receptors, IgG / metabolism
Substrate Specificity

Substances

Immunoglobulin G
Hexosyltransferases
dolichyl-diphosphooligosaccharide - protein glycotransferase
Membrane Proteins
Polysaccharides
Receptors, IgG
Immunoglobulin Fc Fragments
Bacterial Proteins

Abstract

MeSH terms

Substances

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