Elucidating the metabolic network and pH-dependent biosynthesis of bioactive exopolysaccharides XY1-EPS in Bacillus...

Compared to plant-derived polysaccharides, which require lengthy agricultural growth and post-harvest extraction processes, microbial exopolysaccharides benefit from rapid microbial growth, enabling shorter overall production cycles, along with greater process controllability and batch-to-batch consistency. This study centers on the exopolysaccharide produced by Bacillus smithii XY1 (XY1-EPS), with the aim of elucidating its biosynthetic mechanisms and functional characteristics. XY1-EPS exhibited pronounced concentration-dependent antioxidant activity through free radical scavenging and demonstrated significant protective effects in a zebrafish model of enteritis, markedly increasing survival rates. Using response surface methodology, pH was identified as the most critical factor influencing XY1-EPS yield. Subsequent comparative transcriptomic analysis under varying pH conditions uncovered essential regulatory networks involved in its biosynthesis. Furthermore, the complete metabolic network of B. smithii XY1 was mapped for the first time, detailing pathways for six sugar nucleotide precursors and identifying gene clusters responsible for exopolysaccharide synthesis. A key glycosyltransferase gene, gtrB, critical for XY1-EPS assembly and export, was also characterized. HPLC analysis revealed that XY1-EPS is composed of glucose, galactose, glucuronic acid, and N-acetylglucosamine. A purified fraction, XY1-EPS2, consisting exclusively of N-acetylglucosamine, displayed considerable beneficial potential. This study provides fundamental insights into the targeted biosynthesis and functional exploitation of microbial exopolysaccharides.