Tracking Colanic Acid Repeat Unit Formation from Stepwise Biosynthesis Inactivation in Escherichia coli

Abstract

Colanic acid is a glycopolymer loosely associated with the outer membrane of Escherichia coli 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 E. coli. 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 rcsA 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.

Metabolic Engineering for Overproduction of Colanic Acid in Escherichia coli Mutant with Short Lipopolysaccharide

“…The first step of CA repeat unit assembly transferred Glc-P to Und-P to form Und-PP-glucose, initiated by WcaJ. Bacteria with the overexpression of uppS and rcsA could accumulate a detectable intermediate of CA repeat unit . Therefore, pTrcS vector harboring uppS under P trc promoter was constructed to provide more und-P. RfaH, a transcriptional antiterminator, was demonstrated to have the potential to up-regulate the transcription of wca gene cluster for CA biosynthesis at low temperatures .…”

“…Bacteria with the overexpression of uppS and rcsA could accumulate a detectable intermediate of CA repeat unit. 16 Therefore, pTrcS vector harboring uppS under P trc promoter was constructed to provide more und-P. RfaH, a transcriptional antiterminator, was demonstrated to have the potential to up-regulate the transcription of wca gene cluster for CA biosynthesis at low temperatures. 43 The pTrcH vector was constructed to overexpress rfaH under P trc promoter to upregulate the wca operon.…”

“…15 WcaK transfers a pyruvate moiety to the second galactose to form a pyruvylated hexasaccharide. 16 CA is assembled by a Wzy-dependent polymerization system. WzxC translocates the repeating unit of CA from the cytoplasm to the periplasm, and WcaD polymerizes a CA repeating unit to form a long-chain CA polymer.…”

“…WcaF and WcaB have been characterized biochemically as acetyltransferases, which transfer an acetyl group to the first fucose or galactose, respectively . WcaK transfers a pyruvate moiety to the second galactose to form a pyruvylated hexasaccharide . CA is assembled by a Wzy-dependent polymerization system.…”

Discovery of 3′-O-β-Glucosyltubercidin and the Nucleoside Specific Glycosyltransferase AvpGT through Genome Mining

“…Cluster A contains Escherichia coli WcaA (accession: P77414), a UDP-glucuronic acid-dependent GT involved in the biosynthesis of colonic acid. 26 Cluster B contains Bacillus subtilis YwdF (accession: P39614), a GT that has been implicated in spore maturation. 27 Cluster C contains Campylobacter jejuni PglI (accession: Q0P9C6), a GalNAc(5)-diNAcBac-PP-undecaprenol β-1,3-glucosyltransferase.…”