Multivariate modular metabolic engineering and medium optimization for vitamin B₁₂ production by Escherichia coli

Vitamin B₁₂ is a complex compound synthesized by microorganisms. The industrial production of vitamin B₁₂ relies on specific microbial fermentation processes. E. coli has been utilized as a host for the de novo biosynthesis of vitamin B₁₂, incorporating approximately 30 heterologous genes. However, a metabolic imbalance in the intricate pathway significantly limits vitamin B₁₂ production. In this study, we employed multivariate modular metabolic engineering to enhance vitamin B₁₂ production in E. coli by manipulating two modules comprising a total of 10 genes within the vitamin B₁₂ biosynthetic pathway. These two modules were integrated into the chromosome of a chassis cell, regulated by T7, J23119, and J23106 promoters to achieve combinatorial pathway optimization. The highest vitamin B₁₂ titer was attained by engineering the two modules controlled by J23119 and T7 promoters. The inclusion of yeast powder to the fermentation medium increased the vitamin B₁₂ titer to 1.52 mg/L. This enhancement was attributed to the effect of yeast powder on elevating the oxygen transfer rate and augmenting the strain's isopropyl-β-d-1-thiogalactopyranoside (IPTG) tolerance. Ultimately, vitamin B₁₂ titer of 2.89 mg/L was achieved through scaled-up fermentation in a 5-liter fermenter. The strategies reported herein will expedite the development of industry-scale vitamin B₁₂ production utilizing E. coli.