Enhancing O-linking oligosaccharyltransferase functionality through directed evolution.

Area of Science:

• Biotechnology
• Vaccine Development
• Enzymology

Background:

• Polysaccharide protein conjugate vaccines are crucial for preventing bacterial infections.
• Oligosaccharyltransferases catalyze the linkage of polysaccharides to carrier proteins for vaccine construction.
• The O-linking enzyme PglS offers broad substrate specificity but variable efficiency.

Purpose of the Study:

• To enhance the glycosylation efficiency of the PglS enzyme for improved bioconjugate vaccine production.
• To identify mutations in PglS that increase the transfer of specific polysaccharides to carrier proteins.
• To optimize bioconjugation processes for scalable vaccine manufacturing.

Main Methods:

• Directed evolution was used to identify beneficial mutations in the PglS enzyme.
• The group B Streptococcal serotype V capsular polysaccharide was used as a model substrate.
• Carrier proteins with engineered sequons and combinatorial mutations were utilized.
• Enzyme-linked immunosorbent assay and mass spectrometry (MS) were employed for analysis.

Main Results:

• Single amino acid substitutions in PglS significantly improved the transfer of the target polysaccharide.
• Combinatorial mutations and multiple sequons further enhanced bioconjugate production and quality.
• Engineered PglS variants unexpectedly glycosylated two serine residues within the sequon, boosting activity.
• The ratio of polysaccharide to carrier protein was increased by the engineered enzymes.

Conclusions:

• Engineered PglS variants represent a promising tool for improving the efficiency of bioconjugate vaccine production.
• Enhanced glycosylation activity through enzyme engineering facilitates scalable manufacturing of next-generation vaccines.
• The discovery of dual serine glycosylation expands understanding of PglS enzymatic mechanisms.