大肠杆菌Nissle 1917菌株荚膜多糖合成基因kfiA的功能研究

摘要

[Objective] The aim was to analyze genes in heparosan synthesis of EcN heparosan,and find out alternative sources of heparin precursor polysaccharide for E.coli K5 strain.[Method] We studied the key role of kfiA gene in heparosan synthesis by using the techniques,such as gene knockout,gene knock-in,NMR,and so on.[Result] The kfiA was knock out with the λ-red recombination system.The NMR analysis showed that disappeared the specific signal of heparosan from the kfiA mutant.Furthermore,the complementary strain with a kfiA gene inserted into the EcN chromosome was constructed with a phage helped recombination system.The insertion of kfiA restored the synthesis of heparosan and the specific signal of heparosan in the NMR spectrum.[Conclusion] The kfiA plays a critical role in the synthesis of EcN heparosan.Furthermore,both the λ-red recombination system and the phage helped recombination system are useful as a tool for strain engineering in EcN.%[目的]分析益生菌EcN的荚膜多糖合成基因簇,寻找致病性大肠杆菌K5菌株的肝素前体多糖替代来源.[方法]通过基因敲除、基因回补、1H核磁共振(NMR)检测等技术手段,研究了kfiA基因在EcN荚膜多糖合成过程中的功能.[结果]利用λ-red重组系统成功构建了kfiA突变株,利用NMR检测发现kfiA缺失突变株的荚膜多糖特征峰消失,又通过染色体重组技术,把kfiA基因插入了EcN突变株的基因组中,核磁共振检测重新发现了荚膜多糖特征峰.[结论]kfiA基因是EcN菌株荚膜多糖合成途径的一个关键基因,参与了荚膜多糖合成.λ-red重组系统与染色体重组技术同样适用于EcN菌株,可为后续的菌株改造提供参考.