Stepwise Assembly of the Bacteroides fragilis Capsular Polysaccharide A Repeating Unit in Escherichia coli

Bacterial surface polysaccharides are versatile structures that provide specificity to the behavior and interactions of a given bacterial strain. One surface polysaccharide displayed on the organism Bacteroides fragilis, Capsular Polysaccharide A, has been implicated as a potential therapeutic for autoimmune disorders. This polymer is composed of repeating units of the tetrasaccharide 2-acetamido-4-amino-2,4,6-trideoxygalactopyranose (AATGal), 4,6-O-pyruvate-galactopyranose (PyrGal), N-acetylgalactosamine (GalNAc), and galactofuranose (Galf). While this and other bacterial surface polysaccharides are attractive to study and apply to biomedicine, it can be difficult to acquire quick, inexpensive access to these pure materials. In this work, we developed a recombinant expression system in Escherichia coli for the stepwise production of the CPSA polymer. A series of sequential plasmids were prepared, each incorporating successive genes required for CPSA biosynthesis. Using these iterative plasmids, we were able to observe production of the CPSA repeating unit and precursors by liquid chromatography mass spectrometry (LC-MS) analysis of cell lysates. We found that it was critical to include the CPSA polymerase but not the flippase, indicating that a native E. coli flippase could support polymer production. We also provide evidence that the CPSA polymer produced by E. coli can be ligated to LPS by the E. coli WaaL ligase, and deletion of this gene led to the formation of a water-soluble polymer. Overall, this work describes the first recombinant system for CPSA production and outlines a key strategy for the production of complex glycopolymers.