Enhancing Yields and Extending Production Cycles of Bacteriocin from the Bacillus cereus Group Through the Optimization of Culture Conditions and Removal of Proteolytic Digestion

Bacillus cereus group strains are prolific producers of diverse bacteriocins with significant application potentials; however, their industrial utilization is often hampered by short production cycles and low yields. Using the leaderless bacteriocin toyoncin as a model, we systematically evaluated the impact of culture medium, temperature, and initial pH on its production. Our findings demonstrate that these factors critically affected yield, with no production under oligotrophic, acidic, or high-temperature conditions. Optimal production was achieved in MH medium (initial pH 8.5, 25 °C), significantly enhancing fermentation duration and yield compared to original conditions (LB medium, 30 °C, pH 7.0). Transcriptional analyses revealed that these improvements were attributable to extended transcription periods and increased transcript levels of the toyoncin gene cluster. Furthermore, we demonstrated that toyoncin disappearance in the supernatant is caused by transcriptional cessation and degradation by membrane-associated proteases. By combining optimized fermentation with protease inhibition, a high and stable toyoncin yield of 53.86 mg/L was achieved, representing a 3.07-fold increase over the initial yield (17.52 mg/L). This study establishes an integrated strategy to enhance bacteriocin production through simultaneous optimization of culture conditions and inhibition of enzymatic degradation, providing important insights for advancing bacteriocin development in the Bacillus cereus group.