The Regulation Of EcN Heparosan Synthesis

Heparin is a widely used clinical anticoagulant drug. People concern more about the heparin safety from the animal source since the breakout of heparin contaminate incident. Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, which provides another heparin source. Heparosan is the capsule polysaccharides in Escherichia coli strain Nissle 1917. It can inhibit the adhesion of pathogenic bacteria to intestinal tract, and regulate the composition of intestinal microbes. EcN colonizes to intestine by capsule polysaccharides, and competes with foodborne pathogenic bacteria according to severe systems. Recent studies have indicated that carbon sources have different impacts on heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we study the regulation mechanism of carbon source on the synthesis of heparosan by promoter expression analysis, EMSA and NMR analysis. And we find that glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. According to gene study,we demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription.1H nuclear magnetic resonance (NMR) analysis was further performed to determine the structure of EcN heparosan and quantify extracellular heparosan production. We obtained 5 mg/L heparosan by addition of glucose, a level which was lower than those with fructose (13 mgL) and mannose (10 mg/L). The results add to the understanding of the regulation of heparosan biosynthesis, and may contribute the biosynthsis and preparation of heparosan.