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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