Abstract
ABSTRACT The global rise of drug‐resistant bacterial infections underscores an urgent demand for novel antimicrobial agents. Roseoflavin, a naturally occurring riboflavin analog, has emerged as a promising candidate for drug development. In this study, the industrial workhorse Corynebacterium glutamicum was metabolically engineered towards roseoflavin production. Overexpression of the roseoflavin biosynthetic genes rosABC and the flavin transport gene ribM from the native roseoflavin producer Streptomyces davaonensis was evaluated in C. glutamicum . To further link roseoflavin biosynthesis to its riboflavin precursor, the riboflavin kinase gene ribF from both S. davaonensis and C. glutamicum was evaluated, revealing that its overexpression is essential for enhancing roseoflavin production. The final engineered strain CgRose6 achieved a roseoflavin titer of 17.4 ± 1.5 mg/L and a volumetric productivity of 0.36 ± 0.03 mg/L·h when cultivated in glucose minimal medium supplemented with thiamine, a relevant coenzyme for roseoflavin biosynthesis. These production values are the highest reported by a non‐native RoF producer to date and demonstrate the feasibility of using C. glutamicum as a platform for sustainable roseoflavin production, opening avenues for scalable biosynthesis of this valuable antimicrobial compound.