Abstract
Vitamin B(12) is a complex compound synthesized by microorganisms. The industrial production of vitamin B(12) relies on specific microbial fermentation processes. E. coli has been utilized as a host for the de novo biosynthesis of vitamin B(12), incorporating approximately 30 heterologous genes. However, a metabolic imbalance in the intricate pathway significantly limits vitamin B(12) production. In this study, we employed multivariate modular metabolic engineering to enhance vitamin B(12) production in E. coli by manipulating two modules comprising a total of 10 genes within the vitamin B(12) 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 B(12) 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 B(12) 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 B(12) 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 B(12) production utilizing E. coli.