Biosynthesis, Regulation, and Biotechnological Production Strategies of Riboflavin (Vitamin B₂) and Its Derivatives:...

Riboflavin (RF; vitamin B₂) is an essential micronutrient with broad applications in the food, feed, pharmaceutical, and cosmetic industries and is increasingly relevant in bioelectrochemical systems and environmental biotechnology. Microbial fermentation has replaced chemical synthesis as the dominant industrial production route due to its superior sustainability and scalability. However, despite substantial progress, RF biosynthesis remains constrained by imbalances in precursor supply, complex redox regulation, and regulatory feedback mechanisms that limit metabolic flux toward guanosine triphosphate and ribulose-5-phosphate. This review provides an updated, integrative analysis of RF biotechnology, encompassing biosynthetic pathways, transcriptional and redox-regulation, strain improvement strategies, and fermentation process optimization. Representative industrial producers-including Bacillus subtilis, Ashbya gossypii, and Candida famata-are critically evaluated for productivity, yield, and metabolic robustness, with reported titers reaching up to 29 g L^-1 in engineered systems. Emerging microbial platforms, including lactic acid bacteria, thermotolerant and methylotrophic microorganisms, and electroactive bacteria, are discussed in the context of niche applications such as food biofortification and microbial fuel cells. Special emphasis is placed on oxidative stress as a regulatory signal influencing RF overproduction, metabolic rewiring strategies to alleviate precursor bottlenecks, and the biosynthesis of RF derivatives (FMN, FAD, roseoflavin, and 8-aminoriboflavin). In addition, biosafety, regulatory constraints, concerns about genome stability, and antibiotic-free engineering approaches are examined as critical determinants of future industrial competitiveness. By integrating molecular regulation, metabolic engineering, fermentation design, emerging applications, and regulatory perspectives within a unified framework, this review outlines current bottlenecks and future directions for developing safer, more robust, and economically competitive RF-producing microbial platforms.