Dual Selective Pressure and Intelligent Screening of Mutagenized <i>Bacillus subtilis</i> for Enhanced Riboflavin Production

Riboflavin is an essential nutrient widely used in food, feed, and pharmaceutical industries, yet efficient screening of high-yield microbial strains remains a major bottleneck in industrial production. Here, we developed an integrated strategy combining dual selective pressure, optimized microplate fermentation, and stepwise high-throughput screening to accelerate the isolation of riboflavin-overproducing Bacillus subtilis mutants. A dual selective pressure system employing phosphonoarginine-5-amino-6-( d -ribosylamino)uracil (Arp) and the GTP analog 8-azaguanine (8-AZG) enhanced the positive mutation rate. Optimization of microplate culture enabled strong correlation across 96-well, 24-well, and shake-flask fermentations. Stepwise screening further enriched stable high producers, achieving a final positive rate of 21.4%. The best mutant, Hs-G87, produced 4.1 g/L riboflavin in shake flasks and 32.1 g/L in a 5 L bioreactor (yield of 0.11 g/g glucose; productivity of 0.594 g/L/h), demonstrating strong industrial potential.