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Abstract：
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.
摘要：
维生素B12是由微生物合成的复杂化合物。维生素B12的工业生产依赖于特定的微生物发酵过程。大肠杆菌已被用作从头生物合成维生素B12的宿主,掺入了大约30个异源基因。然而,复杂途径中的代谢失衡显著限制了维生素B12的产生。在这项研究中,我们采用多变量模块化代谢工程,通过操纵两个模块,在维生素B12生物合成途径中包含总共10个基因,来提高大肠杆菌中维生素B12的产量.这两个模块被整合到底盘细胞的染色体中，由T7、J23119和J23106启动子调控，实现组合途径优化。通过对由J23119和T7启动子控制的两个模块进行工程改造来获得最高的维生素B12滴度。在发酵培养基中掺入酵母粉可使维生素B12滴度提高至1.52mg/L。这种增强归因于酵母粉对提高氧转移速率和增强菌株的异丙基-β-d-1-硫代半乳糖苷（IPTG）耐受性的影响。最终,通过在5升发酵罐中放大发酵，维生素B12滴度达到2.89mg/L。本文报道的策略将加速利用大肠杆菌的工业规模维生素B12生产的发展。
