Knockout Of CcpA Gene And Characterization Of Fermentation Of Riboflavin-Producing Bacillus Subtilis

B.subtilis24/pMX45 is an over-producing riboflavin strain in fermentation industry. But carbon catabolite repression (CCR) will be induced intensively by glucose in culture medium, and excess glucose would weaken the riboflavin fermentation ability of B.subtilis24/pMX45. CCR in Bacillus subtilis is mainly mediated by the global regulator protein CcpA. Here, adopt deletion-insertion to disrupt ccpA gene of B.subtilis24/pMX45 and construct ccpA knockout mutant——B.subtilis24A1/pMX45. The comparison of shake-flask fermentation indicates B.subtilis24A1/pMX45 can grow normally in culture medium containing 8% glucose; The ratio of glucose consumption increases remarkably; the accumulation amount of overflow metabolite decreases distinctly and riboflavin synthesis ability is released entirely. On the other hand, amount and fashion of accumulation of glucose and fructose alter in fermentation culture medium containing 10% sucrose. These results indicate knockout of ccpA gene can relieve partially CCR induced by glucose and lead to the variety of central metabolic pathways in favor of riboflavin synthesis, at the same time, affect sugar membrane transport systems. Orthogonal testing method is used to optimize nitrogen source in fermentation culture medium of B.subtilis24A1/pMX45, and engineering average estimation method is used to estimate the range of riboflavin production. Under the optimum culture medium with 8% glucose, it has been fermenting for 60 hours, production and yield of riboflavin get 5.0g/L and 0.067molRF/molGlu respectively.