合成软骨素的益生菌的构建

硫酸软骨素(Chondroitin sulfate, CS)是一种具有多种临床治疗作用的糖胺聚糖。目前,软骨素主要是从能引起尿路感染的病原菌E. coli O5: K4: H4(E. coli K4)中提取的,但该提取方式存在碳链骨架完整性受损和潜在的污染风险。为了解决该问题,文章以非致病性益生菌大肠杆菌Nissle 1917(EcN)为出发菌株,将UDP-葡萄糖胺异构酶基因(kfoA)和软骨素聚合酶基因(kfoC)转入至EcN,并敲除了其kfiA和kfiC基因,从而获得工程益生菌EcN-kfoAC。经过分批补料发酵后,对EcN-kfoAC发酵液上清使用化学酶法定量分析软骨素,结果显示其质量浓度达到650 mg/L。该工程型菌株不仅成功实现了软骨素的合成与分泌,同时也为后续软骨素多糖的大规模纯化和硫酸化修饰奠定了基础。

Chondroitin sulfate (CS) is a glycosaminoglycan known for its diverse clinical therapeutic effects. Currently, chondroitin is primarily sourced from $ E.\ coli $ O5:K4:H4 (E. coli K4), a pathogenic bacterium implicated in urinary tract infections. However, this extraction method is associated with damage to the carbon chain skeleton and potential contamination risks. To address these challenges, this study employs the non-pathogenic probiotic strain $ E.\ coli $ Nissle 1917 (EcN) as the foundational organism. The UDP-glucosamine isomerase gene (kfoA) and chondroitin polymerase gene (kfoC) were introduced into EcN, while its kfiA and kfiC genes were knocked out, resulting in the creation of the engineered probiotic EcN-kfoAC. Following fed-batch fermentation, chondroitin was quantitatively assessed using a chemoenzymatic method applied to the supernatant of the EcN-kfoAC fermentation broth, revealing a concentration of 650 mg/L. This engineered strain not only successfully synthesizes and secretes chondroitin but also paves the way for subsequent large-scale purification and sulfation modification of chondroitin polysaccharides.