GlycoSNAP: A High-Throughput Screening Methodology for Engineering Designer Glycosylation Enzymes.

Area of Science:

• Microbiology
• Biochemistry
• Synthetic Biology

Background:

• The Campylobacter jejuni protein glycosylation locus (pgl) is a key system for bacterial N-linked glycosylation.
• Transferring this pathway to E. coli allows for efficient glycosylation of proteins with the C. jejuni heptasaccharide.
• Engineering glycosylation enzymes is crucial for expanding glycan diversity and target protein compatibility.

Purpose of the Study:

• To develop a genetic assay, glycoSNAP, for engineering bacterial glycosylation enzymes in E. coli.
• To facilitate the screening of enzyme variants with altered functions, such as substrate specificity and acceptor site recognition.

Main Methods:

• Development of the glycoSNAP (glycosylation of secreted N-linked acceptor proteins) genetic assay.
• Application of glycoSNAP to screen combinatorial libraries of bacterial oligosaccharyltransferases (OSTs).
• Utilizing glycoSNAP to screen acceptor site libraries for identifying natural and engineered sequons.

Main Results:

• The glycoSNAP assay enables direct engineering of glycosylation enzymes within E. coli.
• Successfully screened OST libraries for relaxed substrate specificity.
• Identified natural and engineered sequons recognized by OSTs using acceptor site libraries.

Conclusions:

• glycoSNAP is an effective tool for engineering bacterial N-linked glycosylation systems.
• This assay accelerates the development of novel glycosylation capabilities in E. coli.
• Facilitates the expansion of bacterial glycosylation for diverse glycan and protein targets.