枯草芽孢杆菌ccpA基因敲除及对其核黄素产量的影响

CcpA蛋白是介导枯草芽孢杆菌碳分解代谢物阻遏(CCR)的全局调控因子,由ccpA基因编码。CCR效应的存在影响B.subtilis对葡萄糖的利用,降低B.subtilis生产发酵产品的效率。采用基因重组技术敲除了核黄素发酵菌株B.subtilis24/pMX45的ccpA基因,构建了CcpA缺陷株B.subtilis24A1/pMX45。发酵结果显示:B.subtilis24A1/pMX45能够在70h内基本耗尽10%的葡萄糖,生物量达到1.5×109个细胞/mL,溢流代谢产物积累量减少,在8%和10%葡萄糖浓度下,B.subtilis24A1/pMX45核黄素产量分别比B.subtilis24/pMX45提高了62%和95%。CcpA的缺陷,可以缓解葡萄糖引起的CCR效应,显著提高菌株的核黄素产量。

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.