Dual Selective Pressure and Intelligent Screening of Mutagenized Bacillus subtilis 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.