Enhancing Vitamin B12 Production in Engineered Escherichia coli through Cofactor Engineering and Fermentation Media Optimization

Vitamin B₁₂ has garnered increasing interest in biotechnological applications due to its critical role in human health and economic benefits. In this study, heterologous vitamin B₁₂ biosynthetic pathway genes were integrated into the chromosome of Escherichia coli. Cofactor engineering significantly enhanced production levels by expressing the hemoglobin gene vgb from Vitreoscilla for improved oxygen transfer and substituting the Embden–Meyerhof–Parnas pathway with the Entner–Doudoroff pathway to increase ATP and NADPH availability. The optimal strain produced 3.54 mg/L of vitamin B₁₂ in a respiration activity monitoring system. Further optimization of fermentation media through single-factor and Taguchi methods resulted in a significant increase in production, achieving a remarkable 21.09 mg/L in a 5 L fermenter containing a low-cost defined medium. These findings demonstrate the potential of engineered E. coli strains for industrial vitamin B₁₂ production, offering a cost-effective and efficient biotechnological solution.