Dual Selective Pressure and Intelligent Screening of Mutagenized Bacillus subtilis for Enhanced Riboflavin Production

Area of Science:

• Biotechnology
• Microbial Engineering
• Industrial Microbiology

Background:

• Riboflavin (vitamin B2) is vital in food, feed, and pharmaceuticals.
• Efficiently screening high-yield microbial strains for riboflavin production is a significant industrial challenge.

Purpose of the Study:

• To develop an integrated strategy for accelerating the isolation of riboflavin-overproducing Bacillus subtilis mutants.
• To enhance the efficiency and success rate of identifying superior microbial strains for industrial riboflavin synthesis.

Main Methods:

• Implemented a dual selective pressure system using phosphonoarginine-5-amino-6-(d-ribosylamino)uracil (Arp) and 8-azaguanine (8-AZG).
• Optimized microplate fermentation techniques for scalability and correlation with larger fermentation formats.
• Employed stepwise high-throughput screening to enrich for stable, high-producing mutants.

Main Results:

• The dual selective pressure system increased the positive mutation rate.
• Optimized microplate cultures showed strong correlation with 96-well, 24-well, and shake-flask fermentations.
• Stepwise screening achieved a final positive rate of 21.4% for high producers.
• The best mutant (Hs-G87) yielded 4.1 g/L in shake flasks and 32.1 g/L in a 5 L bioreactor.

Conclusions:

• The integrated screening strategy effectively accelerates the isolation of high-yield riboflavin-producing Bacillus subtilis.
• The developed methods demonstrate strong industrial potential for efficient riboflavin production.
• Mutant Hs-G87 shows significant promise for large-scale riboflavin manufacturing.