Development of Sucrose-Utilizing Escherichia coli Nissle 1917 for Efficient Heparosan Biosynthesis

<b>Background/Objectives</b>: Heparosan is a component of the capsular polysaccharide in <i>Escherichia coli</i> K5 and <i>Pasteurella multocida</i> Type D. It shares a similar glycan structure with heparin and can be enzymatically modified to produce bioactive heparin. <b>Methods</b>: In this study, the probiotic strain <i>E. coli</i> Nissle 1917 (EcN), which naturally produces heparosan, was genetically engineered to utilize sucrose as a carbon source for growth while achieving high-yield heparosan biosynthesis. <b>Results</b>: By expressing the sucrose hydrolase genes <i>sacA</i> (from <i>Bacillus subtilis</i>) or <i>spI</i> (from <i>Bifidobacterium adolescentis</i>), EcN was enabled to utilize sucrose, achieving heparosan titers of 131 mg/L and 179 mg/L, respectively. Further metabolic engineering was performed to block the glycolytic and pentose phosphate pathways, thereby redirecting sucrose-derived glucose-6-phosphate and fructose-6-phosphate toward heparosan biosynthesis, while glycerol was supplemented as an auxiliary carbon source to support cell growth. Finally, the key biosynthesis genes <i>galU</i>, <i>kfiD</i>, and <i>glmM</i> were overexpressed, resulting in an engineered strain with a heparosan titer of 622 mg/L. <b>Conclusions</b>: This study represents the first successful engineering of EcN to utilize sucrose as the carbon source for growth, while achieving enhanced heparosan production through synergistic carbon source utilization. These findings establish a foundational strategy for employing this strain in the sucrose-based biosynthesis of other glycosaminoglycans.