Enhancing Vitamin B₁₂ Production in Engineered <i>Escherichia coli</i> 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 <i>Escherichia coli</i>. Cofactor engineering significantly enhanced production levels by expressing the hemoglobin gene <i>vgb</i> from <i>Vitreoscilla</i> 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 <i>E. coli</i> strains for industrial vitamin B₁₂ production, offering a cost-effective and efficient biotechnological solution.