Abstract
Riboflavin, an important vitamin utilized in pharmaceutical products and as a feed additive, is mainly produced by metabolically engineered bacterial fermentation. However, the reliance on antibiotics in the production process leads to increased costs and safety risks. To address these challenges, an antibiotic-free Escherichia coli riboflavin producer was constructed using metabolic engineering approaches coupled with a novel plasmid stabilization system. Initially, competitive pathways and feedback inhibition were attenuated to enhance the metabolic flux towards riboflavin. Key genes in the purine pathway were overexpressed to boost the availability of riboflavin precursors. Subsequently, a plasmid stabilization system based on toxin was screened and characterized, achieving a plasmid retention rate of 84.9% after 10 days of passaging. Finally, transcriptomic analysis at the genome-wide level revealed several rate-limiting genes, including pgl, gnd, and yigB, which were subsequently upregulated, leading to a 26% improvement in riboflavin production. With optimization of the culture medium, the final strain allowed the production of 11.5 g/L of riboflavin with a yield of 90.4 mg/g glucose in 5 L bioreactors without antibiotics. These strategies can be extended to other plasmid-based riboflavin derivative production systems.