Enhanced vitamin B6 production in engineered Escherichia coli via restricted mix-carbon feeding and pressure-controlled fermentation.

Pyridoxine (PN), a major commercial form of vitamin B6, is mainly produced via chemical synthesis, raising environmental concerns. Microbial fermentation offers a greener alternative, but low biomass and titer limit industrial application. In this study, we developed an integrated strategy combining DO-stat restricted mix-carbon feeding with two-stage pressure-controlled fermentation conditions, and medium optimization to enhance cell growth and PN production in Escherichia coli. DO-stat feeding enabled dynamic regulation of oxygen and carbon sources, while a two-stage pressure-controlled (0.1 bar for 0-24 h, 0.2 bar thereafter) improved both growth and PN biosynthesis. Meanwhile, a novel medium (CRS-67), optimized via Taguchi design, increased shake flask PN titer by 275 mg/L, 75.5 % over CS medium. Under systematic fermentation optimization in a 5-L bioreactor, OD600 reached 142.8 and PN titer reached 3.33 g/L-the highest reported level to date. This study presents a robust and scalable process for microbial PN production and offers insights for industrial biomanufacturing of vitamin B6.