Auxotrophic Selection Strategy for Improved Production of Coenzyme B₁₂ in Escherichia coli

The production of coenzyme B₁₂ using well-characterized microorganisms, such as Escherichia coli, has recently attracted considerable attention to meet growing demands of coenzyme B₁₂ in various applications. In the present study, we designed an auxotrophic selection strategy and demonstrated the enhanced production of coenzyme B₁₂ using a previously engineered coenzyme B₁₂-producing E. coli strain. To select a high producer, the coenzyme B₁₂-independent methionine synthase (metE) gene was deleted in E. coli, thus limiting its methionine synthesis to only that via coenzyme B₁₂-dependent synthase (encoded by metH). Following the deletion of metE, significantly enhanced production of the specific coenzyme B₁₂ validated the coenzyme B₁₂-dependent auxotrophic growth. Further precise tuning of the auxotrophic system by varying the expression of metH substantially increased the cell biomass and coenzyme B₁₂ production, suggesting that our strategy could be effectively applied to E. coli and other coenzyme B₁₂-producing strains.