[Knockout of the ccpA gene in Bacillus subtilis and influence on riboflavin production]

Bacillus subtilis uses glucose as preferred source of carbon and energy, but expression of many genes involved in central pathways is often repressed or activated in the presence of glucose. This phenomenon is called carbon catabolite repression (CCR) in B. subtilis, which is mainly mediated by the global regulator CcpA encoded by ccpA gene. In the present of glucose, the genes encoding glycolytic enzymes are induced; the genes responsible for the production of acetate from pyruvate are induced also, however, the genes required for Krebs cycle are repressed. Sometimes, this leads to lower production in fed-batch fermentation. In this study, ccpA gene of the riboflavin producing train B. subtilis24/ pMX45 was disrupted using homologous recombination technology and a ccpA mutant B. subtilis24A1/pMX45 was constructed. The comparison of shake-flask fermentation indicates B. subtilis24A1/pMX45 could grow normally in culture medium containing excess glucose; and the ratio of glucose consumption increased remarkably, for example, 10% glucose can be consumed within 70 hours, biomass increased to 1.5 x 10(9)cell/mL; at the same time the accumulation amount of overflow metabolite decreases. Especially, when fermenting with culture medium containing 8% and 10% glucose respectively, the yields of riboflavin increase 62% and 95% than B. subtilis24/pMX45. These results demonstrate knockout of ccpA gene can relieve carbon catabolite repression and lead to the alteration of central catabolite flux in favor of synthesis of riboflavin, so the yield of riboflavin can be enhanced significantly.