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Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria
Aravind Natarajan1, Thapakorn Jaroentomeechai2, Marielisa Cabrera-Sánchez1
1Department of Microbiology, Cornell University, Ithaca, NY, USA.
Nature Chemical Biology
|July 29, 2020
Summary
Scientists engineered bacteria to create cancer-associated O-glycans, like Tn and T, on proteins. This synthetic biology approach enables efficient production of specific O-glycoforms for research and therapeutic uses.
Area of Science:
- Synthetic biology
- Glycobiology
- Biotechnology
Background:
- Cellular glycosylation pathways are crucial for protein function.
- Re-engineering or constructing novel glycosylation pathways is a key goal in synthetic glycobiology.
- Understanding and manipulating O-linked protein glycosylation is important for various biological processes.
Purpose of the Study:
- To develop orthogonal pathways for eukaryotic O-linked protein glycosylation in Escherichia coli.
- To install cancer-associated mucin-type glycans (Tn, T, sialyl-Tn, sialyl-T) onto proteins.
- To enable cell-free synthesis of O-glycoproteins and generate specific glycoforms for therapeutic applications.
Main Methods:
- Engineering orthogonal glycosylation pathways in Escherichia coli.
- Utilizing glycoengineered bacteria to produce glycosylation machinery.
- Performing one-pot cell-free synthesis of O-glycoproteins.
- Generating antigenically authentic Tn-MUC1 glycoform.
Main Results:
- Successfully installed Tn, T, sialyl-Tn, and sialyl-T glycans onto serine residues of human O-glycoprotein motifs in E. coli.
- Developed a cell-free system for O-glycoprotein construction using bacterial extracts.
- Generated a Tn-MUC1 glycoform recognized by the cancer-specific antibody 5E5.
- Demonstrated the ability to produce structurally diverse O-glycoforms.
Conclusions:
- Orthogonal glycoprotein biosynthesis pathways were established in E. coli.
- These pathways facilitate the production of specific O-glycoforms, including cancer-associated ones.
- The developed methods offer facile access to diverse O-glycoforms for scientific and therapeutic applications.
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