基因组改组及代谢通量分析在产核黄素Bacillus subtilis性能改进中的应用

TRAIT IMPROVEMENT OF RIBOFLAVIN-PRODUCING Bacillus subtilis BY GENOME SHUFFLING AND METABOLIC FLUX ANALYSIS

综合应用基因组改组和DNA重组技术改进了核黄素生产菌B subtilis 2 4 /pMX4 5的性状 ,在B subtilis染色体上整合和扩增了多个B subtilis 2 4的核黄素操纵子拷贝 ,并经过两轮的基因组改组后筛得菌株B subtilisRH33/ pMX4 5 ,在以 12 %葡萄糖为碳源的分批发酵中 ,其核黄素产量约为B subtilis 2 4 / pMX4 5的 2倍 ,并具有快速同化利用葡萄糖、生长迅速、可形成感受态和进行基因整合、扩增等优良性状 .随后比较分析了三株产核黄素B subtilis在间歇培养条件下的代谢通量分布 ,通量分析和实验结果表明 ,B subtilisRH33/pMX4 5中核黄素合成的主要通量限制因素存在于从 5 磷酸核酮糖到GTP的一系列反应中 .

Traits of riboflavin-producing B. subtilis 24/pMX45 were improved by genome shuffling a DNA recombination. Riboflavin operon of B. subtilis 24 was integrated within chromosome of B. subtilis and was amplified to multi-copies, and B. subtilis RH33/pMX'45 were obtained after two rounds of genome' shuffling. The strain produced about 100% more riboflavin than the original strain B. subtilis 24/pMX' in batch culture with 12% glucose as carbon source. In addition, the strain showed some promising trail such as quick assimilation of glucose, rapid growth, competence development and capability of gene integration and amplification. Subsequently, the metabolic flux distribution in batch culture of three riboflavin-producing B. subtilis was compared and analyzed. The results of flux analysis and experiment indicate that the primary limiting factor for improvement of riboflavin production in B. subtilis RH33/pMX45 lies in the reactions between 5-P-ribulose and GTP.