**Abstract**

Capsular Polysaccharide A (CPSA) is a polymer of a tetrasaccharide unit found on the surface of the symbiotic gut bacteria *Bacteroides fragilis*. CPSA has been suggested to be important for maintaining a natural equilibrium between Th1 and Th2 cell levels in the normal immune system of mammals. If this equilibrium is disrupted, the human body can develop different autoimmune disorders. The gene locus responsible for CPSA biosynthesis has been previously identified. The locus was proposed to encode one glycosyl-1-phosphate transferase (WcfS) and three glycosyltransferases (WcfN, -P and -Q ), three sugar modifying enzymes (WcfM, WcfR and WcfO), a flippase (Wzx) and a polysaccharide polymerase (Wzy) based on homology tools. A route for the complete biosynthesis of CPSA has been elucidated. The initiating sugar transferase, WcfS has been previously identified and characterized. An *in vitro* method was used to enzymatically synthesize CPSA, which was assembled on a fluorescent analogue of the native bactoprenyl diphosphate anchor one sugar at a time. Function of the hypothesized pyruvyltransferase WcfO was also determined. This is the first study to characterize a pyruvyltransferase involved in polysaccharide biosynthesis from a prokaryote. The biosynthesis of the polysaccharide was achieved in a single pot, compared to multiple steps involved in chemical synthesis, displaying an enormous leap in the biosynthesis of complex molecules like CPSA.

**Keywords:** *Bacteroides fragilis*, pyruvyltransferase, glycosyltransferase, capsular polysaccharide A, biosynthesis
