【作者】 应明；

【导师】 班睿；

【作者基本信息】 天津大学 ， 生物化工， 2005， 硕士

【摘要】 B.subtilis24/pMX45是核黄素工业发酵菌株,但葡萄糖的存在引起强烈的碳分解代谢物阻遏作用(CCR),高浓度的葡萄糖严重消弱菌株生产核黄素的能力。CcpA蛋白作为全局调控因子介导由葡萄糖引起的CCR作用。采用插入失活的方法破坏B.subtilis24/pMX45的ccpA基因,得到ccpA基因敲除菌株B.subtilis24A1/pMX45。摇瓶发酵比较显示:B.subtilis24A1/pMX45能够在8%葡萄糖的培养基中正常生长,糖耗速率显著提高,溢流代谢产物积累量明显降低,核黄素合成能力完全恢复。在10%蔗糖发酵培养基中,发酵液中葡萄糖和果糖的积累量和积累方式发生改变。这一结果表明ccpA基因的缺失,在一定程度上解除了葡萄糖引起的CCR效应,使中心代谢发生了有利于核黄素合成的改变; 同时影响了糖的跨膜运输。采用正交实验,对B.subtilis24A1/pMX45发酵培养基的氮源进行了优化; 并采用工程平均估计法,估算了优化条件下核黄素的最大产量范围。在8%葡萄糖为碳源的优化条件下,摇瓶发酵60小时,核黄素产量达到5.0g/L,收率为0.067molRF/molGlu。更多还原

【Abstract】 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.更多还原