Enhanced heparosan biosynthesis in Escherichia coli Nissle 1917 through carbon flux redirection

Heparosan, a critical precursor for heparin production, is naturally biosynthesized as capsular polysaccharides by the probiotic strain Escherichia coli Nissle 1917 (EcN). This study presents a systematic metabolic engineering strategy to enhance heparosan biosynthesis through coordinated pathway engineering and carbon flux redirection. By disrupting glucose catabolism via deletion of zwf and pfkAB, we decoupled cell growth from heparosan synthesis while maintaining precursor availability, elevating titers from 137.68 mg/L to 422.11-486.13 mg/L in mixed carbon source cultures. Subsequent overexpression of UDP-glucose dehydrogenase, a key enzyme in UDP-glucuronic acid biosynthesis, achieved 1.04 g/L heparosan in shake-flask cultivations. Scale-up in a 5-L bioreactor demonstrated industrial scalability, yielding 4.34 g/L heparosan. Our work establishes EcN as a microbial chassis for glycosaminoglycan production and provides a generalizable framework for engineering complex polysaccharide biosynthesis through rational metabolic partitioning.