Metabolic engineering of Bacillus subtilis for de novo synthesis of 6′-sialyllactose

6′-sialyllactose (6′-SL) is an important component of human milk oligosaccharides (HMOs) and has numerous infant health benefits. The construction of efficient and food-safe microbial cell factories to produce 6′-SL has attracted increasing attention. In this study, a Bacillus subtilis strain was metabolically engineered for 6′-SL production. First, a de novo synthesis pathway for 6′-SL was constructed by heterologous expression of neuC, neuB, neuA, and pst6, enabling 6′-SL synthesis at a titer of 135.17 mg/L. Subsequently, bioinformatics-guided enzyme modification and promoter substitution strategies were used to fine-tune the pathway strength. Moreover, inhibition of competing pathways and copy number optimization of synthetic modules were used to increase the precursor concentration, raising 6′-SL titer to 621.8 mg/L. Furthermore, a strategy to overcome carbon catabolite repression (CCR) was developed for B. subtilis to improve lactose utilization and increase 6′-SL titers, reaching 3.55 g/L in shake flasks and 15.0 g/L in 3-L fermenters. This study established a foundation for efficient 6′-SL production.