3型荚膜多糖(type 3 capsular polysaccharide,Type 3 CPS)作为肺炎链球菌重要的毒力因子,在疫苗开发、多糖材料等方面具有重要价值。该文旨在构建合成Type 3 CPS的谷氨酸棒杆菌和大肠杆菌细胞工厂,并探索其发酵合成方法。首先对Type 3 CPS的基因簇、合酶结构和糖链结构进行分析,在此基础上过表达Type 3 CPS合酶Cps3B和Type 3 CPS单糖前体的合成途径相关酶,并分析辅助膜蛋白CpsC对Type 3 CPS合成的影响。谷氨酸棒杆菌细胞工厂的合成水平达到69 mg/L,随后构建大肠杆菌细胞工厂合成水平达到79 mg/L。为进一步提高Type 3 CPS合成,引入Type 3 CPS的辅助膜蛋白CpsC,Type 3 CPS合成水平提高至105 mg/L。最后通过结构模拟和分子动力学模拟,发现辅助蛋白CpsC通过与Type 3 CPS合酶Cps3B形成氢键作用参与Type 3 CPS糖链合成与跨膜转运过程。研究实现Type 3 CPS细胞工厂的构建和优化,为Type 3 CPS的发酵合成奠定了初步基础。
Type 3 capsular polysaccharide (Type 3 CPS), a crucial virulence factor of Streptococcus pneumoniae, holds significant value in vaccine development and polysaccharide materials.This study aims to construct microbial cell factories based on Corynebacterium glutamicum and Escherichia coli for the synthesis of Type 3 CPS and to explore its fermentation synthesis methods.Initially, an analysis was conducted on the gene cluster, synthase structure, and sugar chain architecture of Type 3 CPS.Based on this analysis, the overexpression of the Type 3 CPS synthase Cps3B and the enzymes involved in the synthesis pathway of Type 3 CPS monosaccharide precursors was carried out, and the influence of the auxiliary membrane protein CpsC on Type 3 CPS synthesis was evaluated.The microbial cell factory based on C.glutamicum achieved a synthesis level of 69 mg/L.Subsequently, E.coli microbial cell factory was constructed, reaching a synthesis level of 79 mg/L.To further enhance the synthesis of Type 3 CPS, the auxiliary membrane protein CpsC was introduced, which increased the synthesis level to 105 mg/L.Finally, through structural modeling and molecular dynamics simulation, it was discovered that the auxiliary protein CpsC participates in the sugar chain synthesis and transmembrane transport process of Type 3 CPS by forming hydrogen bonds with the Type 3 CPS synthase Cps3B.This study represents the successful construction and optimization of a Type 3 CPS microbial cell factory, thereby laying a preliminary foundation for the fermentation synthesis of Type 3 CPS.
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