LAUSR

Heparosan is a crucial‐polysaccharide precursor for the chemoenzymatic synthesis of heparin, a widely used anticoagulant drug. Presently, heparosan is mainly extracted with the potential risk of contamination from Escherichia coli strain K5, a pathogenic bacterium causing urinary tract infection. Here, a nonpathogenic probiotic, E. coli strain Nissle 1917 (EcN), was metabolically engineered to carry multiple copies of the 19‐kb kps locus and produce heparosan to 9.1 g/L in fed‐batch fermentation. Chromosome evolution driven by antibiotics was employed to amplify the kps locus, which governed the synthesis and export of heparosan from EcN at 21 mg L−1 OD−1. The average copy number of kps locus increased from 1 to 24 copies per cell, which produced up to 104 mg L‐1 OD−1 of heparosan in the shaking flask cultures of engineered strains. The following in‐frame deletion of recA stabilized the recombinant duplicates of chromosomal kps locus and the productivity of heparosan in continuous culture for at least 56 generations. Fed‐batch fermentation of the engineered strain EcN8 was carried out to bring the yield of heparosan up to 9.1 g/L. Heparosan from the fermentation culture was further purified at a 75% overall recovery. The structure of purified heparosan was characterized and further modified by N‐sulfotransferase with 3′‐phosphoadenosine‐5′‐phosphosulfate as the sulfo‐donor. The analysis of element composition showed that heparosan was N‐sulfated by over 80%. These results indicated that duplicating large DNA cassettes up to 19‐kb, followed by high‐cell‐density fermentation, was promising in the large‐scale preparation of chemicals and could be adapted to engineer other industrial‐interest bacteria metabolically.