Enhancing riboflavin production by modulating the expression of the rib operon in Bacillus subtilis

Abstract

Riboflavin is an essential vitamin for humans and animals, with its annual demand experiencing a consistent upward trajectory. In recent years, newly constructed riboflavin producing strains are difficult to reach the level of industrial strains. Therefore, developing competitive strains is crucial for advancing the riboflavin industry. The expression level of the rib operon is strongly correlated with riboflavin biosynthesis. The strain S12, which overexpressed the rib operon of B. subtilis using plasmids with approximately 8 copies, achieved a 53.9 % increase in riboflavin biosynthesis during shake-flask fermentation. However, the strain cannot grow normally in a 7.5-L bioreactor. The expression of rib operon may have competed with cell growth for GTP or produced toxic intermediate ArP. Therefore, the strain S16 replaced ribA in rib operon with a monofunctional DHBP synthase, increased 20 % of riboflavicn production in a 7.5-L bioreactor. Furthermore, the strain S24, which overexpressed the rib operon using the phase-dependent promoter P_yqgZ, attained a riboflavin production of 29 g/L in a 7.5-L bioreactor, achieving the highest reported riboflavin production to date. This study reports for the first time using the phase-dependent promoter for overexpression of rib operon, effectively regulated the relationship between cell growth and riboflavin production.