Tracking Colanic Acid Repeat Unit Formation from Stepwise Biosynthesis Inactivation in .

Colanic acid is a glycopolymer loosely associated with the outer membrane of that plays a role in pathogen survival. For nearly six decades since its discovery, the functional identities of the enzymes necessary to synthesize colanic acid have yet to be assessed in full. Herein, we developed a method for detecting the lipid-linked intermediates from each step of colanic acid biosynthesis in . The accumulation of each enzyme product was made possible by inactivating sequential genes involved in colanic acid biosynthesis and upregulating the colanic acid operon by inducing transcription. LC-MS analysis revealed that these accumulated materials were consistent with the well-documented composition analysis. Recapitulating the native bioassembly of colanic acid enabled us to identify the functional roles of the last two enzymes, WcaL and WcaK, associated with the formation of the lipid-linked oligosaccharide repeating unit of colanic acid. Importantly, biochemical evidence is provided for the formation of the final glycosylation hexasaccharide product formed by WcaL and the addition of a pyruvate moiety to form a pyruvylated hexasaccharide by WcaK. These findings provide insight into the development of methods for the identification of enzyme functions during cell envelope synthesis.