Enhanced microbial production of pyridoxine (Vitamin B₆) in Bacillus subtilis via pathway and process optimization

Vitamin B₆ refers to a family of water-soluble B vitamin, which is essential for various physiological functions, including amino acid metabolism, neurotransmitter synthesis, and hemoglobin synthesis. The biosynthesis of pyridoxine (PN), a commercial form of vitamin B₆, through microbial fermentation has garnered widespread attention owing to its environmentally friendly and safe production methods, as well as its mild reaction conditions. However, the low yield of natural strains limits their application. This study focused on constructing a high-yielding strain of PN through pathway engineering and process optimization. Firstly, five key deoxyxylulose-5-phosphate-dependent pathway genes (epd, pdxB, serC, pdxA, and pdxJ) were overexpressed in Bacillus subtilis ARTP, which improved the PN titer by 3.2-fold to 2.9 mg/L. Subsequently, pdxST genes involved in the DXP-independent pathway were screened from various strains. Ribosome binding site (RBS) sequences were optimized to regulate their expression, which further increased the PN titer to 24.6 mg/L. Finally, systematic medium optimization was identified as a critical strategy for enhancing PN biosynthesis, leading to a remarkable 1.8-fold improvement in PN production. Under optimized fermentation conditions, the engineered strain achieved a PN titer of 174.6 mg/L in fed-batch fermentation, which represents the highest level reported to date in B. subtilis. Overall, this study presents an effective strategy combining pathway engineering and medium optimization for significantly improving PN production, offering valuable insights for the industrial development of PN biosynthesis.