Engineering of Riboflavin Transporter and Enzymes in Bacillus subtilis Facilitates High-Efficiency FAD Synthesis from RF

The biosynthesis of flavin adenine dinucleotide (FAD), an essential redox coenzyme, is limited by inefficient substrate utilization and low catalytic activity. To address this, an “Uptake-Catalysis” system was constructed in Bacillus subtilis for FAD synthesis from riboflavin (RF). First, the Streptomyces davawensis transporter (RibM) was coexpressed with native FAD synthase (BsFADs) at a 2:1 ratio, which increased RF consumption to 51%. Subsequently, the isothermal compressibility perturbation engineering (ICPE) strategy was adopted, generating the optimum mutant BsFADsP61W+R202Y, which increased catalytic efficiency (kcat/Km) toward RF and FMN by 83% and 73%, respectively. Mechanistic analysis revealed that this improvement was due to a 39% shortening of RF substrate channel. Finally, FAD titer peaked at 563 mg/L with a conversion rate (cr-FAD) of 92% in shaking-flask fermentation optimization. This work achieved a 225-fold increase in FAD production, validating the effectiveness of the uptake-catalysis synergistic strategy, thereby establishing an efficient and scalable synthesis paradigm.