Advances in bacterial glycoprotein engineering: A critical review of current technologies, emerging challenges, and future directions.

Protein glycosylation, which involves the addition of carbohydrate chains to amino acid side chains, imparts essential properties to proteins, offering immense potential in synthetic biology applications. Despite its importance, natural glycosylation pathways present several limitations, highlighting the need for new tools to better understand glycan structures, recognition, metabolism, and biosynthesis, and to facilitate the production of biologically relevant glycoproteins. The field of bacterial glycoengineering has gained significant attention due to the ongoing discovery and study of bacterial glycosylation systems. By utilizing protein glycan coupling technology, a wide range of valuable glycoproteins for clinical and diagnostic purposes have been successfully engineered. This review outlines the recent advances in bacterial protein glycosylation from the perspective of synthetic biology and metabolic engineering, focusing on the development of new glycoprotein therapeutics and vaccines. We provide an overview of the production of high-value, customized glycoproteins using prokaryotic glycosylation platforms, with particular emphasis on four key elements: (i) glycosyltransferases, (ii) carrier proteins, (iii) glycosyl donors, and (iv) host bacteria. Optimization of these elements enables precise control over glycosylation patterns, thus enhancing the potential of the resulting products. Finally, we discuss the challenges and future prospects of leveraging synthetic biology technologies to develop microbial glyco-factories and cell-free systems for efficient glycoprotein production.